get Textbook MV401 (.pdf)

Scholar year 2012/2013

MASTER IN LIFE SCIENCES AND TECHNOLOGIES

MAJOR IN MOLECULAR AND CELLULAR BIOLOGY

OBLIGATORY COURSE BMC401

"METHODS IN MOLECULAR AND CELLULAR BIOLOGY"

GENERAL INFORMATION

CONTENT:

Organization of the course 2

Security instructions 3

Practical information 5

Instructions for writing a laboratory notebook 6

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ORGANISATION OF THE COURSE

PEDAGOGIC RESPONSIBILITY

Organisation: Agnès Audibert and Sophie Louvet. Practical course « Purification and analysis of a recombinant protein » : Rozenn Bernard and Sandrine Castella. Practical course « Methods of cellular analysis »: Florence Bourgain and Anthi Karaiskou. Practical course « Construction and functional screening of a bacteriophage lambda genome library »: Laure Bidou and Mathilde Garcia. Secretariat: Carine Joseph; tel : 01-44-27-35-35; email: [email protected].

LOCATION

Practical course « Purification and analysis of a recombinant protein » : classroom « TP », building D, 2nd floor, Jussieu. Practical course « Methods of cellular analysis »: classroom « Ateliers de Biotechnologie », Atrium, 3rd floor, Jussieu. Practical course « Construction and functional screening of a bacteriophage lambda genome library » : classroom « TP », Building B, 3rd floor, Jussieu. The course is composed of three sessions. Each session lasts the whole week from 9 am to 6 pm approximately. Each student is working within a pair. You will need a pocket calculator, graph paper and two permanent markers (fine and medium). Don’t forget your laboratory coat and a notebook!

EVALUATION OF YOUR KNOWLEDGE/NOTATION Final mark (out of 100) = Writing exam (out of 50) + Oral exam (out of 30) + Participation (out of 20).

PROGRAM

from 10/09 to 14/09 Purification and analysis of a recombinant protein

from 17/09 to 21/09 Methods of cellular analysis

from 24/09 to 28/09 Construction and functional screening of a bacteriophage lambda genome library

Week of December 3rd Oral exam

SECURITY INSTRUCTIONS

(for your personal and surroundings safety during practical courses) It is forbidden to smoke, to drink and to eat in classrooms! Use a space provided for this purpose. It is mandatory to wear a lab coat buttoned completely!

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Respect security instructions:

- when manipulating electrophoresis apparatus (electric current); - when manipulating dangerous products (acrylamide, EtBr, etc.); - when working in sterile conditions near the flame (attach your hairs and remove gloves).

NEVER USE YOUR MOUTH TO PUT LIQUID INTO A PIPETTE! Dangerous products

1) Ethidium bromide (EtBr) and propidium iodide (PI): Both, EtBr and PI are strong cancerogenic chemicals once in contact with your skin. Wear gloves and a lab coat to protect yourself as well as the surrounding environment (bench, pipettes, other objects) from contamination. Work on a special bench provided for EtBr/PI manipulations and discard contaminated consumables/products into a special bin labelled «EtBr / BET» et «IP / PI».

2) Acrylamide: Acrylamide is a strong neurotoxin and carcinogen in contact with skin and if swallowed. Wear gloves and discard acrylamide products in a special bin « ACRY ».

3) Enzymatic substrates: Hydrogen peroxide at high concentrations provokes skin burns. Handle carefully.

4) Fixators: Methanol is neurotoxic in contact with skin and if swallowed. The optic nerve is particularly sensible to methanol. Manipulate in gloves and under a fume hood.

5) Electrophoresis: Be aware of the electric current.

All dangerous products are labelled with pictograms in accordance with regulations in force.

Examples of pictograms: Product labelling:

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ETHIDIUM BROMIDE 1. IDENTIFICATION OF THE SUBSTANCE/MIXTURE AND THE SOCIETY/COMPANY 1.1. Identification of the product

Name of the product: Ethidium bromide solution Code: E1510 Society: Sigma CAS Number: 1239-‐45-‐8 1.2. Suitability of the product and dissuadable usage Suitability: Chemical substance for laboratory use. Production of the substance. 2. DANGER IDENTIFICATION 2.1. Classification of the substance or mixture Classification in accordance with regulations in force (EC) No 1272/2008 (EU-‐GHS/CLP) Highly toxic, inhalation (Category 3) Mutagenic on germ cells (Category 2) Classification according to Directives UE 67/548/EEC or 1999/45/EC Phrase(s) R R23 Toxic if inhaled. R68 Possible risk of irreversible effects. Phrase(s) S S36/37 Wear suitable protective clothing and gloves. S45 In case of accident or if you feel unwell, seek medical advice immediately (show label where possible). 2.2. Label content

Labelling in accordance with regulations (CE) No 1272/2008 (EU-‐GHS/CLP) Pictogram:

Signal word: Danger. Hazard statements:

H331 Toxic if inhaled. H341 Suspected of causing genetic defects.

Precautions statements: P261 Avoid breathing dust/fume/gas/mist/vapours/spray. P281 Use personal protective equipment as required. P311 Call a POISON CENTER or doctor/physician.

ACRYLAMIDE Classification according to Directives UE 67/548/EEC or 1999/45/EC Harmful by inhalation and if swallowed. Irritating to the eyes. Harmful in contact with skin. May cause sensitization by skin contact. Danger of serious damage to health by prolonged exposure. May cause cancer. May cause heritable genetic damage. Possible risk of impaired fertility.

Figure 1: Hazards and precautions to take into account during manipulations.

Biological material (nucleic acids, proteins, cell culture) Nucleic acids are easily degraded by nucleases present all over the environment and in particular on your fingers. Proteins are easily degraded by proteases. Avoid direct contact of your samples with the skin and all objects that are in contact with the skin (pipettes, tips, tubes). All supplied consumables and solutions are sterile. Do not soil and use sterile distilled water when set up enzymatic reactions.

During the practical course «Construction and functional screening of a bacteriophage lambda genome library» manipulation with bacteria is done near the flame of the Bunsen burner to create a sterility cone and avoid contamination by microorganisms. Pay attention to the flame. Do not manipulate inflammable chemicals (alcohol) nearby. Attach your hairs, put on your lab coat and remove your gloves.

During the practical course « Methods of cellular analysis » you will do a culture of a cell line in sterile conditions. Please, respect sterility rules given by your supervisors and read attentively a chapter dedicated to this issue in your practical manual.

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PRACTICAL INFORMATION

B) Manipulation of small quantity and volumes You will manipulate a very tiny quantity of substances. One milligram (10-3 g) of DNA represents a huge quantity of the matter that is enough for several years of experiments in the laboratory scale. In general, you will work with 10 to 100 nanograms (10-9) of DNA/protein. To give you an idea of quantity: one bacterial cell contains 17 femptograms of DNA (17x10-15 g) that represents 4,7x106 base pairs of the haploid genome. In the stationary phase, 1 milliliter of bacterial culture contains 109 cells or 1,7 x 10-5 g (17 µg) of DNA that allows you to perform hundreds of experiments, such as PCR (several pg of DNA per reaction).

You will work with very tiny solution volumes, i.e. microlitres (μL) or 10-6 litres (L). Confusion between µL and mL will have dramatic consequences for your experiments and results interpretation. The volumes to use will often not exceed 20 µL (visually, an equivalent of one drop of water in the air).

To ensure the precision of pipetting always eject all liquid onto a tube wall. When you finish pipetting and all solutions are added into the tube, don’t forget to centrifuge it to let all drops collecting in the bottom. In some cases you can just slightly tap your tube against a bench.

C) Micropipettes

Micropipettes are very expensive and fragile material. They allow manipulating very small volumes of liquid with a great accuracy. Avoid dropping them! When taking out a solution, raise a piston slowly to ensure precision of pipetting and to avoid any liquid to go into the pipette. Pay attention to choose a good micropipette and a suitable tip for the volume you should take out!!! Don’t forget to change a tip after each manipulation to avoid any contamination of your stock and reaction solutions. To avoid touching contaminated tips, hold the pipette over the trash bin and press the tip ejector push-button.

Pipette Name Volumes to take out Tips sutable for the pipette P20 from 2 µL to 20 µL Yellow or white tips P200 from 20 µL to 200 µL Yellow or white tips P1000 from 200 µL to 1000 µL (1 mL) Bleu or white tips

C) Conservation of solutions, samples, etc. DNA, protein extracts and the majority of solutions are kept at -20°C. To ensure good conservation of solutions keep them on ice during manipulations (to limit side reactions, nucleases action, etc.).

Restriction enzymes, polymerases, antibodies are very expensive and fragile, they are stored at -20°C, are kept on ice only during sampling and are put back at -20°C immediately after use. Don’t forget to change a tip between sampling to avoid contamination of stock solutions.

Fluorescent substances (EtBr, PI or fluorochromes coupled to antibodies) are sensible to light and should be kept in obscurity.

D) Accuracy of pipetting Don’t try to put more volume than required when set up your enzymatic reactions. Enzymes are stored in 50% glycerol buffers and high concentrations of glycerol can affect the reaction efficacy. All volumes/concentrations are adjusted to your experimental conditions and are optimal for maximal efficacy. Respect the indicated volumes with a one µL precision!

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E) Organisation To finish your experimental work in time, it is essential to follow all written and oral instructions. Don’t hesitate to report any problem or ask for additional explanations in case of confusion or mismanipulation. The success and the speed of manipulations depend on your organisation: well-organised and clean bench, clearly labelled samples, anticipated results, etc. The cleanness of a working space (your own bench and common benches) is primordial: consider your work finished only when everything is cleaned and put in order.

INSTRUCTIONS FOR WRITTING A LABORATORY NOTEBOOK During each practical course you should fill your laboratory notebook (lab book) where day-to-day you will mark all realised experiments, analysis of your results, etc. Do not note theoretical aspects and supervisor’s explanations in this part of the lab book (do it somewhere else, for example in the back).

The lab book will allow you to record all manipulations and obtained results. It should be written during manipulations in the classroom and in chronological order. Leave it on your bench after the work accessible to supervisors for reading.

Your lab book should be well structured, synthetic and explicit to demonstrate your scientific and analytical skills. Your notes should allow reproduction of the experimental procedure by yourself and by others and in case of problems to find an eventual error. For each experiment you should mark: - the date of the experience, the time if important; - the aim of the experiment (in form of a title); - the method employed; - manipulations: clearly indicate all manipulations, including mismanipulations (solutions, composition,

quantity/volume, concentrations), parameters (time, T°C, voltage, etc.); - observations during manipulations, if important; - Results: figures, photos, schemes should be correctly annotated with a title, legends, etc. and

attached to your lab book; - Observation of the results; - Interpretation of the results in accordance with a given experimental procedure (observed results); - Conclusions: critical analysis and summary of results in respect to all questions you addressed in this

study. Critical view of your own results and results of others, if necessary, comparison with expected results;

- Hypothesis: propose additional experiments that might allow you to ameliorate your approach and to help in confirmation of working hypothesis.

The quality of your lab book will be taken into account during your oral exam and in the classroom. It is absolutely useless to copy your practical manual into the lab book!

MasterdeSciencesetTechnologies/MasterofScienceandTechnology

Mention:BiologieMoléculaireetCellulaire/Option:MolecularandCellularBiology

UEfondamentaleMV401/FundamentalmoduleMV401

MéthodologiesenBiologieMoléculaireetCellulaireMethodologiesinMolecularandCellularBiology

!ear &'(& ) &'(*

WorkshopPurificationandAnalysisofaRecombinantProtein

‘TravauxPratiques’Room:UniversitéPierreetMarieCurie

PlateformedeTPs,Bldg.B,2ndfloor7,quaiSaint–Bernard,75005Paris

Faculty members in charge of the ‘Unité d’Enseignement’ : Agnès Audibert and Sophie Louvet-Vallée Faculty members in charge of the Workshop : Rozenn Bernard and Sandrine Castella

Secretary : Carine Joseph

Bldg. K, Rm. 129, Tel. : 01 44 27 35 35e-mail : [email protected]

Teaching and technical teams: UPMC Biochemistry faculty members,Marie-Christine Chauvière, Bldg. B, Rm. 222, Tel. : 01 44 27 39 54

Workshop:PurificationandAnalysisofarecombinantprotein

SchematicoftherecombinantpGEXvectorencodingtheGST‐GRDBDfusionprotein

TABLEOFCONTENTS

PracticalCoursePlanning‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐1

LaboratoryNotebook ‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐ 2

Introduction ‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐4

Cloningstrategy ‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐ 5

BacterialcloneconfirmationusingPCR ‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐ 7

AffinitypurificationofGST‐proteins ‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐8

Purificationanalysis

Quantitativeanalysis ‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐ 11

Qualitativeanalysis‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐12

Specificationsheets

Specificationsheet1:Nativeagarosegelelectrophoresis‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐13

Specificationsheet2:Denaturingpolyacrylamidegelelectrophoresis ‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐ 14

Specificationsheet3:Bradfordproteinassay ‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐ 16

Specificationsheet4:Transferaseenzymaticactivityassay‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐17

Appendixes

AppendixI ‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐ 18

AppendixII‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐ 19

AppendixIII‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐20

Structuralstudy‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐21

M, -'( ) Purification and Analysis of a Recombinant Protein PR0S023A3IO2

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PRACTICALCOURSEORGANIZATION

StudyDirectors:

MV401:AgnèsAudibertandSophieLouvet‐Vallée

Workshop:RozennBernardandSandrineCastella

These MV 401 practical courses are held in three sessions. Each session lasts one weekcontinuously,from9a.m.toapproximately6p.m.Youwillbeworkinginpairs.Don’tforgetyourlabcoatandlabnotebook!Acalculator,graphpaperandtwopermanentmarkers(thinandthick)willalsobeuseful.


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LABNOTEBOOK

DuringthisPracticalCourse,youwillkeepalabnotebookdailyinwhichyouwillcarefullywritedownyourexperiments.

Thisnotebookwillbe usedasabasis forquestionsduring theoral test.Youshould not copy thepresenthandout.

Notesshouldbetakenduringtheexperiments,inchronologicalorder.Thislabnotebookallowsyoutokeeptrack of your work, to communicate your data and to understand how these data are gathered frommeasurementsandobservations.

Foreachandallexperiment,youmustwritedown:‐thedate,‐theaim,‐theunderlyingprincipleofthemethodsusedtoreachthisaim(afewlines)‐theexperimentitself:clearlydescribeallstepsastheyaredone,eventhosethathavefailed.Writedownallparameters(eventhosethatyoumightthinkunnecessary)andallyourobservations,includingvariationsfromtheprotocol(suchasmistakes,substitutions).‐theresults:describetheresultsthatarepresentedasfiguresordiagrams,withaccuratetitlesand legends,andclearlyinterpret.‐theconclusion:analyzeyourresultscriticallyandcomparethemwiththoseofotherstudentpairs,integratetheoryandexperiment.Lastly,proposefurtherexperimentstoimproveandexpoundontheresults.

ConcerningthisPracticalCourse,youmustcompletethefollowing11items(forabbreviations,seethe“Introduction”):

Cloningandscreeningofbacterialclones:Documentspresentingallcloningstepsandtheirrationale1‐TheGRsequenceshowingtheATGandthepositionoftheDBDupandDBDdownprimers,theamplifiedGRDBDsequence,showingtherestrictionsitesused.

2‐ThepGEXplasmidsequenceused,showingtheATGforGST,therestrictionsitesused,theSTOPcodonandthepositionsoftheup‐GSTanddown‐GSToligosusedtotestthebacterialclones.

3‐ The recombinant pGEX plasmid sequence with the same comments and highlighted GRDBD. Give theinsertsize.

4‐ Calculate the theoretical molecular weight of each of these proteins (in Daltons) and compare withthoseobtainedwiththeDNAstridersoftware.5‐RationaleforthechoiceoftheprimerpairsforthebacterialclonePCRscreening(andfortheexclusionoftheotherpairs),withsupportingdiagrams.6‐AnnealingregionofthechosenPCRprimersonthepGEXplasmidsequenceandestimatesoftheirTmandTa.7‐ Calculate the theoretical sizes of the fragments amplified from the native and recombinant pGEXplasmids.8‐ Labeled photograph of the PCR product analytical gel, including rationale for the chosen agarosepercentageandananalysisofthePCRresults.

Note:thesectiononPCRbacterialclonestestingwillbecorrectedandgraded(marked)


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GSTandfusionproteinpurification

9‐Quantitative analysis: calibration curve,detailed table of the resultswith theprotein concentrationofeach fraction, including purified GST andGST‐GRDBD, enzymatic activities, yields and purification factors,lossestimation.10‐Qualitativeanalysis:SDS‐PAGE,GSTandGST‐GRDBDsizecalculation.

FunctionalanalysisoftheglucocorticoidreceptorDNAbindingdomain11‐Proposeoneorseveralmethodsinorderto:

a–Solvethethree‐dimensionalstructureoftheDBDdomain. b–DemonstratetheinteractionoftheDBDdomainwithitsDNAtargetsequence.Note:although itwillnotbeexperimentally studied, theGRDBD functionalanalysiswillbediscussedduringtheweekandmaybecoveredonthewrittentest.

M, -'( ) Purification and Analysis of a Recombinant Protein I23ROD893IO2

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INTRODUCTION

Objective:PurificationandanalysisoftheDNA‐bindingdomainoftherainbowtrout(Oncorhynchusmykiss)GlucocorticoidReceptor(GR).

Thenuclearreceptorsuperfamilycontainssteroidhormonesreceptors,whichplayanimportantroleintheregulationofreproduction‐associatedfunctions.Molecularstudiesofsuchfactorsareofgreatscientificinterestandaregermanetomedicalandagronomicapplications.

The Glucocorticoid Receptor, once bound to ligand, specifically interacts with its cognate DNAsequence,termeda‘responseelement’.Thisreceptorproteincanformdimersandcontainsseveraldomains,includingtheDNAbindingdomainthatisbeingstudiedinthisworkshop.

Productionandpurificationofthedesignedprotein

Inordertoobtainsufficientquantitiesofthisproteindomain,wechosetoexpressitinbacteriaasafusionprotein.Thenucleotidesequence(cDNA)correspondingtotheDNAbindingdomain(DBD),GRDBD, was cloned in a pGEX vector. This cloning generates a plasmid encoding a recombinantprotein bearing an in‐frame Glutathion‐S‐Transferase (GST) N‐terminal to the GRDBD domain(GST‐GRDBD).

BL21 bacteria were transformed, either with the control pGEX‐3X plasmid or with the plasmidencodingtherecombinantproteinGST‐GRDBD.Afterproteinexpressioninductionby IPTGaddition,bacteriaaregrownonaselectivemedium,thenharvestedbycentrifugationandkeptfrozenasdrypellets.

Find the cloning strategy that was used, and calculate the theoretical size of the fusionprotein.

UsingPCR,assess,usingtheadequateprimerpairs thatwillhybridizetotheDNAsequenceupstream and downstream the plasmidMultiple Cloning Site,whether the bacterial clonescontainthecontrol(withoutinsert)orrecombinantpGEXplasmids.

Purifythefusionprotein,anddothenecessarycontrolsinordertoanalyzethepurificationstepsandtomeasurethequantityoftheresultingprotein.

M, -'( ) Purification and Analysis of a Recombinant Protein 9:O2I2; S3RA30;!

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CLONINGSTRATEGY

Material

Internetconnections: «National Center for Biotechnology Information», NCBI,http://www.ncbi.nlm.nih.gov/.

This site is linked to themain sequencedatabases (nucleicacids,proteins) and containsaccess tosequencealignments(liketheBLASTprogram,BasicLocalAlignmentSearchTool).

Othersoftware:DNAStriderallowssequenceanalysis,restrictionmapping,ORFidentification;Wordenablespastingandformattingofcontent.

DATA

The cDNA encoding the Glucocorticoid Receptor DNA binding domain (GRDBD) was PCR‐amplifiedwiththeprimers(“oligos”)below,theninsertedinthepGEX‐3Xplasmidmultiplecloningsite(MCS),in frame with the GST‐coding sequence. Knowing that DNA polymerases, even those with high‐fidelity, have a detectable error rate, the PCR GRDBD‐fragment inserted in the plasmid must beverifiedpriortoexpression.

PrimerDBD‐up(18‐mer)=5’‐CATAAGATCTGCCTGGTG‐3’PrimerDBD‐down(25‐mer)=5’‐GCGAATTCCCAGCTGGGGCATGGAC‐3’

Sequencesaccesscodes:

GlucocorticoidreceptorO.mykissorZ54210

CloningvectorpGEX‐3XorU13852.

Indications

GototheNCBIwebsite.Select«nucleotide»inthe«search»windowandentertheaccesscodesforthesequencesofinterest.Copy/pastethesequencesandsavetheminDNAStriderformat.

UsingBLAST,fromNCBI,orDNAStrider,findoutwheretheaforementionedPCR‐primershybridize,andindicatethePCR‐amplifiedGRDBDsequence.

FindtheGRDBDsequencereadingframe.

UsingDNAStrider,gettheGRDBDsequencerestrictionmap,andspecifywhichenzymeswereusedtoinsertitintothepGEX‐3Xvector.

ReconstructthecompletesequenceencodingtherecombinantproteinusingDNAStrider.

M, -'( ) Purification and Analysis of a Recombinant Protein 9:O2I2; S3RA30;!

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MV401–Purification and Analysis of a Recombinant Protein BACTERIALCLONESVERIFICATION

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PCRVERIFICATIONOFTHEBACTERIALCLONESReminder:youareresponsibleforthisentireexperiment,whichwillbegraded(cf«Labnotebook»p2,points

5‐7)!

Allyourreactiontubesmustbelabeledandidentifiedwithyourroomcode

Material

‐PCRtubes(0,5mL)forthePCRmachine(Warning,theyaresmallerthantheusual1,5mLEppendorf)‐PCRH2O‐10XPCRbuffer:Tris‐HCl100mM,pH8,8,KCl500mM,MgCl215mM,TritonX‐1001%.‐FreedXTPmix:10mM‐Primers:10µM,choose1primerpairamongthefollowingfour: pairn°1: Primera(24mer)=5’‐TATAGCATGGCCTTTGCAGGGCTG‐3’ Primerb(20mer)=5’‐CCGGGAGCTGCATGTGTCAG‐3’ pairn°2: Primera(24mer)=5’‐CAGCCCTGCAAAGGCCATGCTATA‐3’ Primerb(20mer)=5’‐CTGACACATGCAGCTCCCGG‐3 pairn°3: Primera(24mer)=5’‐GTCGGGACGTTTCCGGTACGATAT‐3’ Primerb(20mer)=5’‐GACTGTGTACGTCGAGGGCC‐3 pairn°4: Primera(24mer)=5’‐CAGCCCTGCAAAGGCCATGCTATA‐3’ Primerb(20mer)=5’‐CCGGGAGCTGCATGTGTCAG‐3‐Taqpolymerase:1unit/µL‐Samples:transformedbacteriafromtheGSTandGST‐GRDBDclones,untransformedbacteria.

Protocol

1‐Reactionmix,finalvolume25µLH2Oqsp(“quantitésuffisantepour”=QS,quantumsatis)25µL1XfinalPCRbufferFreedXTP:200µMfinalPrimera:0,2µMPrimerb:0,2µMTaqpolymerase:1UBacterialculture:2,5µLPrepareamastermixcontainingthereactantscommontoalltests:thatincludesallsamplesplusthenegativecontroltest(H2O)andthepositivecontroltest(primersTa+Tb+transformedbacteria).Allowapre‐mixfor7tests.Homogenizewell.

‐ For samples and negative control tests: take enough pre‐mix for 5 tests, add the chosen primers(homogenize!)andsplitinto4previouslylabeledPCRtubes.Add2,5µlofeachsample.

‐ Forthepositivecontrol:take21,5µLpre‐mix,addcontrolprimersand2,5µlofsample.Homogenizewellandcentrifugebriefly.PlacethetubesinthePCRmachineandnotetheirposition.2‐Amplificationcyclesparameters.Annealingtemperaturecalculation:Ta=Tm–5°C (tobediscussed!)withTm=2°Cx(A+T)+4°Cx(G+C) forprimerslessthan30baseslong.Note:thisapproximateformulatendstooverestimatetheTmvalue!Initial denaturation 95°C, 5 min then 25 amplification cycles (Denaturationat 94°C, 30 sec;Hybridization/annealing at Ta, 30 sec; Elongation/extension at 72°C, 40 sec then final elongation at 72°C, 5min).3‐AgarosegelelectrophoresisofPCRproducts(seespecificationsheet1)

MV401–Purification and Analysis of a Recombinant Protein GSTPROTEINPURIFICATION

8

AFFINITYPURIFICATIONOFGSTPROTEINSMaterial

‐Grinder,cooledcentrifuges,glassbeads‐Proteaseinhibitorsmix:AminoEthylBenzeneSulfonylFluoride(AEBSF),Aprotinin,EDTA,Leupeptin,Pepstatin(seeactionmechanismonapaperpostedinthe‘TP’,PracticalCourseroom)‐ReducedGlutathione20mMinTris‐HCl100mM,pH8,NaCl120mM,‐Lysozyme10X:10mg/mL‐Glutathione‐Agaroseresin‐Nativebuffer10X:Tris‐HCl100mM,pH8,NaCl1.5M,Na2HPO4160mM,NaH2PO440mM‐Phosphate‐BufferedSaline(PBS)10X:NaCl1.5M,Na2HPO450mM,KH2PO417mM;pH7.4‐TE±SDSBufferTris‐EDTA±SDS:Tris‐HCl10mM,pH7.4,EDTA1mM,±SDS1%‐UreaBuffer:Urea8M,Tris‐HCl10mM,pH8,Na2HPO416mM,NaH2PO44mM

Protocol

WARNING!Unlessotherwisespecified,workat4°Ctominimizeproteindegradationanddenaturation

1‐Proteinextraction

Prepare10mLNativeBuffer1XwithProteaseinhibitormix,keepat4°C.Bothculturepelletswillbeidenticallyprocessed.However,fortheGSTprotein,onlytakefraction1andkeepfraction6.1.

Sampleswillberemovedatallstepsforthefusionproteinonly,inordertoanalyzeandquantifythepurificationprocess (see table 1: purification of the fusion protein).Atall steps,note the volumesobtainedandthevolumesremovedinordertocalculateyieldsandpurificationfactors.

Inducedbacteriagrinding:

Homogenizebacteriapellets in500µLLysisBuffer (NativeBuffer 1X, lysozyme1mg/mL).Measurefinalvolumeafterpelletdissolution.Addpre‐cooledglassbeadstoeachbacteriasolution.Grindbacteriafor10minatmaximalspeed.

PreparationoftheGlutathion‐Agaroseresin:(donebytheteachers)

Swellthelyophilizedglutathion/agarosepowderinPBS1X.Centrifuge4min,2000rpm,at4°C.Resuspendpelletin1mLNativeBuffer1X,splitby2x500µlintransparent1,5mLEppendorftubes)giventothestudents.

2‐Proteinpurification

Crudeextractstreatment(step1):

Centrifugegroundbacteria,10min,12000rpm,at4°C.Separatepellet1fromsupernatant1.Transfersupernatantsinto1.5mLtubes.Rinsebeadsinpelletsbypipetting500µLofNativeBufferandspinoncemorefor10minat12000rpmat4°C.Transfertheresultingsupernatants(1bis)intothetubescontainingsupernatant1.Notethevolumeoftotalsupernatant(1+1bis),collecta60‐µLaliquot. ⇒fraction1(solubleproteins)


9

Keeppellet 1 from«GST‐X»cultureon ice, tobetreated later.Discard«GST»culturepellet.Treatsupernatants(1+1bis)priortoanythingelse.

Proteinsadsorptiononresin,andrinses(steps3,4et5):

PreparetheGlutathione‐Agaroseresinsolution:centrifuge(1 to2min) the0,5mLandrecover thesupernatantwithoutdisturbingtheresin!Add supernatants (1+1bis) to the resin.Homogenizeandput the tubeson therotatorat4°C for 1hourminimum.Centrifugethetubes,3min,1500rpmat4°C.Removethesupernatant(3)withoutresin. ⇒fraction3(solubleproteinsunboundtotheresin)Resuspendpellet3with900µLNativeBuffer,homogenizeandletagitate5min,at4°C.Centrifugethetubes,3min,1500rpm,at4°C.Recoverthesupernatant4(fraction4.1)withoutresin.Washpelletswith500µLNativeBuffer,homogenizeandputthetubesonthewheelfor5minat4°C,andcentrifugeasabove(fraction4.2).Keepthesupernatantsfromallwashesonice. ⇒fractions4.1et4.2(solubleproteinsunboundtotheresin)Resuspendpellets4with200µLNativeBuffer.Takeup30µLoftheresinsolution. ⇒ fraction 4.3(resinwithGST‐Xproteins)Centrifugethetubesforthelasttime,3min,1500rpm,at4°Candkeeppellet5.

Proteinelution(step6):

Add100µLoffreshlyprepared20mMreducedglutathionesolution.Homogenizeandputthetubesontherotatorat4°Cfor30min.Centrifugethetubes,5min,2000rpm,at4°C.Recoverthesupernatant6.Resumetheelutionprotocolwith100µLofreducedglutathionesolutionandpoolwiththeprevioussupernatants.Keepthetubesonice,astheycontainthepurifiedproteins!

⇒fraction6.1(purifiedGST‐Xproteins)

Resuspendpellet6with200µLTE‐SDSbuffer. ⇒fraction6.2(resinafterGST‐Xproteinselution)

Pellet1treatment(step2)

Cut the tip of a blue pipette tip (with scissors), and use it to resuspend pellet 1, with successivepipetting,in1mLTEbuffer(WARNING:setpipetteto500µL).Centrifuge2min,12000rpm,at4°C.Recoversupernatant2,carefullyavoidingglassbeads.⇒fraction2.2 (solubleproteins lost inbeadsdeadvolume).

Resuspendpellet2with500µLUreabufferbystrongvortexingorsuccessivepipetting.Incubate20minat room temperature.Centrifuge 10min, 12000 rpm.Remove supernatant, carefully avoidingglassbeads ⇒fraction2.1(insolubleproteins)


8

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MV401–PurificationandAnalysisofaRecombinantProteinPURIFICATIONANALYSIS

11

ANALYSESDELAPURIFICATION

QuantitativeAnalysis1‐ColorimetricassayoftheproteinsinallthefractionskeptduringthepurificationprocessusingtheBradfordmethod(seespecificationsheet3).Thevolumesofallfractionsnecessaryfortheassayareindicatedinthetablebelow:

Tableau2:Bradfordassayoftheproteinfractions

GST‐GRDBDFractionsn°volume(µL)

Abs595nm

Proteinconcentration

(µg/µL)

Totalvolumeofanalyzedproteinsolutions(µL)

Totalproteinquantity(µg)

1 5

2.1 5

2.2 5

3 5

4.1 20

4.2 20

4.3 15

6.1 10

6.2 20

GSTFractions

Abs595nm

Proteinconcentration

(µg/µL)

Totalvolumeofanalyzedproteinsolutions(µL)

Totalproteinquantity(µg)

1 5

6.1 10

Using table 1, calculate the total protein quantity at each step of GST‐GRDBD purification, and offractions1and6.1only,forGSTpurification.

2‐EnzymaticactivityassayofGlutathione‐S‐Transferase(seespecificationsheet4)For each protein, GST and GST‐GRDBD, assay the enzymatic activity of 5µl of fraction1 (pure anddiluted1/10)andfraction6.1.

Ifnecessary,repeattheassaywithadifferentfractionvolume.

3‐YieldandpurificationfactorCalculate the total and specific enzymatic activities, taking into account the total volumes of thecrudeextractsandpurifiedproteins,aswellastheproteinconcentrations.Calculatetheyieldsandpurificationfactorsofyourpreparations.

MV401–Purification and Analysis of a Recombinant ProteinPURIFICATIONANALYSIS

12

QualitativeAnalysisSDS‐PAGE(seespecificationsheet2)ofallthefractionstakenduringthepurificationprocess.Addtheindicatedvolumeof3XLaemmlisolutiontoeachofthetestedfractions,anddenaturebyincubatingat95°Cinaheatingblockfor5min.Toanalyzethetotalproteininthecontrols(IPTG‐inducedandnon‐inducedbacteria),resuspendthebacteriapellets in 100 µL 1X Laemmli solutionand lyse byheating at95°C, 10min.Centrifuge20min, 12000 rpm, atroom temperature. Immediately recover the indicated volume, always keeping the tip just beneath thesurfaceofthesupernatant.

Table3:Samplepreparation

SampleVolume

µLLaemmli3X,µL well

Solutionoftotalproteinfromnon‐induced

pGEX‐transformedbacteria15 Alreadyadded 1

SolutionoftotalproteinfromIPTG‐induced

pGEX‐transformedbacteria15 Alreadyadded 2

GST

SolutionofpurifiedGST 10 5 3

Sizemarkers 5 Alreadyadded 4

SolutionofpurifiedGST‐GRDBDfraction6.1

10 5 5

Solutionofnon‐elutedGST‐GRDBDfraction6.2

10 5 6

Solutionofglutathion‐agarose/GST‐GRDBD

fraction4.310 5 7

1stwashfraction4.1

10 5 8

Solutionofunboundproteinfraction3

10 5 9

Solutionoflostsolubleproteinfraction2.2

10 5 10

Fractionofunsolubleproteinfraction2.1

10 5 11

Fractionofsolubleproteinfraction1

10 5 12

Solutionoftotalproteinfromnon‐induced

pGEX‐GRDB‐transformedbacteria15 Alreadyadded 13

FusionProteinGST‐GRD

B

D

SolutionoftotalproteinfromIPTG‐induced

pGEX‐GRDB‐transformedbacteria15 Alreadyadded 14

Load15µLofeachsampleand5µLofsizemarkerssolution.Runasindicatedinspecificationsheet2SDS‐PAGE

MV401–Purification and Analysis of a Recombinant ProteinSPECIFICATIONSHEET1:AGAROSEGEL

13

ELECTROPHORESIS

AgarosegelelectrophoresisMaterial

‐Electrophoresissystem«MUPID‐One»,tank,gelcasts,13‐wellcomb.‐Balance,testtube,heatingblock,microwaveoven‐Agarose‐GelRedinNaCl0.1M‐SizeMarkers:«SmartLadder»(Eurogentec)1000,800,700,600,500,400,300,200,100bp‐6XLoadingbuffer:Glycerol15%,BromophenolBlue0.12%‐10XTris‐Borate‐EDTA(TBE)Buffer:Tris‐Borate900mM,pH8.3,EDTA20mM

Protocol

1‐PCRproductelectrophoresis

Electrophoresisgelresolution%Gel LinearDNAsize

(kb)%Gel LinearDNAsize

(kb)

0.5 30to1.0 1.2 7to0.4

0.7 12to0.8 1.5 3to0.2

1.0 10to0.5 2.0 1to0.1

Agarosegelx%(w/v)in0,5XTBEbuffer.ChosetheagaroseconcentrationyouneedusingtheabovetableMake50mLofgel.Weightheagarose,transfertoan“erlen”(Erlenmeyerflask),add0,5XTBEandplaceinthemicrowave.Heat(usually30‐45secatmaxtemp,avoidboiling)todissolveagarose.Letcool(avoidbubblesandvigorousstirring)tobelow60°C,add1µLGelRed(dilution1/50000),gentlypoorinthetray,insertthecombandletthegelsolidify.

Runningbuffer:prepare450mLof0,5XTBEandfillthetank.

Samples: add x µl of 6X loading buffer to the 25 µL of each sample, and load in the expectedwells(respectchosenorder):samples,15µL,sizemarkerssolution,5µL.

Running:1h30at50volts.

2‐DNAfragmentsdetection,nativegelPlacethegelontheUVtable(WARNING:CLOSETHELIDTOAVOIDUVEXPOSURE),andturnontheUVlamptovisualizeDNA.Takeapicture(withyourteacher)..

MV401‐Purification and Analysis of a Recombinant ProteinSPECIFICATIONSHEET2:POLYACRYLAMIDEGEL

14

PolyacrylamideGelElectrophoresis

ProteindenaturinggelSDS‐PAGE(SodiumDodecylSulfate‐PolyacrylamideGelElectrophoresis)

Material

‐Bio‐RadApparatus,thickglassplates(SpacerPlates)withpermanentlybonded1,5‐mmspacers,thinshortplates,castingframe,15‐wellcomb,tank‐Acrylamide*(acrylamide/bis‐acrylamide29:1mix)40%‐SDS10%(w/v;weight/volume)‐StackinggelbufferTris‐HCl1M,pH6,8‐ResolvinggelbufferTris‐HCl1M,pH8,8‐5XRunningbuffer(Tris/Glycine/SDS):Tris125mM,Glycine960mM,pH8.3,SDS0.5%‐Polymerizationcatalysts:Ammoniumpersulfate(APS)10%(w/v)andTemed‐3XLaemmlisolution:Tris240mM,pH6.8,SDS6%,glycerol30%,DTT270mM(orβ-mercaptoethanol15%),BromophenolBlue0.03%‐Prestainedapparentmolecularweightmarkers«Biorad»:mixof10proteinsincluding2pink‐stained(bold):10,15,20,25,37,50,75,100,150,250kDa‐Stainingsolution:CoomassieBlueR2502.5%,ethanol50%,aceticacid10%(orCoomassieBlueinphosphoricacid,ethanol)‐Destainingsolution:ethanol20%,aceticacid7.5%(orH2O)

*WARNING!Liquidacrylamideistoxic:avoidskincontact,don’tswallow,USEGLOVEStopreparesolutionsand

pourgels.

Protocol

1‐GelpreparationSettheglassplates,allowinga1.5‐mmthickgel,intheGelCassetteSandwich.

Resolving gel: Acrylamide * 12%, Tris‐HCl 0.25 M, pH 8.8, SDS0.1%.

Prepare10mLpergelinasmallbeaker.Addthecatalystslast:APS0.1%andTemed0.05%.Homogenizegentlyandpour7.2mLofthesolutionsmoothlybetweentheplates.AVOIDBUBBLES.Immediatelyoverlaythegelsolutionwithdistilledwaterorethanol(fromthewashbottles).Allowthegeltopolymerize(30‐45min).Lettheremaininggelpolymerizeinthebeakerasapositivecontrolanddiscardinthebiologicaltrashafterwards.

Stackinggel:Acrylamide*5%,Tris‐HCl0.12M,pH6.8,SDS0.1%.Rinsethegelsurfacewithdistilledwater,andgetthe15‐wellcomb(1.5mmthick)beforepreparingthesolution.

Well

Short plate

Thick plate

Spacer

Resolvinggel

Stackinggel

MV401‐Purification and Analysis of a Recombinant ProteinSPECIFICATIONSHEET2:POLYACRYLAMIDEGEL

15

Prepare5mLpergel.Addthecatalystslast:APS0.1%andTemed0.1%.Homogenizegently;pour thesolutionsmoothlyontopof theresolvinggeluptotheglassplates’top.Insertthecombsmoothlyavoidingtrappingairbubbles.It’seasiertoinsertthecombstartingatanangle

andtoinserttheteethprogressively.Allowtopolymerizefor20‐30min.Runningbuffer:preparer1Lof1XTris‐Glycine‐SDSbufferandpourintoelectrophoresistank.Gentlyremovethecombandrinsethewellsthoroughlywithrunningbuffer.

SamplesandloadingDenaturesamplesat95°Cintheheatingblockfor5min.Load15µLofeachsample,and5µLofthe‘size’markerssolution.

2‐Electrophoresis

Running:applypowerandbeginelectrophoresiswithconstantcurrent(30mApergel)orconstantvoltage(110volts).Rununtilthebluemarkerreachesthebottomofthegel(usually1hrwithconstantamperage).Turnoff thepower supply and remove the tank lid.Gently remove thegel from theGel CassetteSandwich.

3‐Proteinstaining

Coomassiebluestaining.Rinsethegelfor5minindH2OtoremoveSDS(fromtherunningbuffer),thenincubateinstainingsolution for at least 1 hour under gentle stirring (rocking table) at room temperature. Save thestainingsolution.Destaining:incubateseveraltimesindestainingsolution(ontherockingtableatroomtemperature)untiltheproteinbandsareseenwithoutbackgroundstainingofthegel.

Recycledestainingsolutionthroughanactivatedcharcoalfilter.

DONOTLETTHEGELDRY.

MV401‐Purification and Analysis of a Recombinant Protein SPECIFICATIONSHEET3:BRADFORDDOSAGE

16

PROTEINQUANTIFICATION

BradfordcolorimetricassayMaterial

‐Microwellplates,platereader‐BovineSerumAlbumin(BSA)10mg/mL‐Bradfordreagent:G250CoomassieBlue0.1mg/mL;H3PO48.5%;Ethanol9.5%

Protocol

Inacidicsolution,G250CoomassieBluebindsside‐chaingroupsofbasicaminoacids(lysine,arginine,histidine)andfreeamino‐groupsofthepolypeptidechain.Thisgeneratesashiftoftheabsorbancemaximumfrom465to595nm.ThebindingoftheproteinstabilizestheblueformoftheCoomassiedye.

Theincreaseofabsorbanceat595nmisproportionaltotheamountofbounddye,andthustotheamount(concentration)ofproteinpresentinthesample.

1‐Standardcurve(oneperstudent!)

PreparetwoindependentseriesofBSAstandards.

Preparetubescontaining0to20µgdeBSAin100µLdistilledwater,finalvolume.Take20µLofeachdilution and distribute in the microwell plate in the order given. Add 200 µL Bradford reagent,incubate for a given duration (minimum 10min). Read absorbance at 595 nm and plot standardcurveA595nm=f(µgBSA).

Ifthecurveisnotlinear,resumetheexperiment…

2‐Samples

Assaythesamplesunderthesameconditions:

Takethenecessaryvolume(seeTable2)andadditto100µLdistilledwater(finalvolume).Take20µLofeachdilutionanddistributeinthemicrowellplatefollowingcoordinatesandreadingorder.

Don’tforgettoprepareablanktubewithoutprotein.Theabsorbanceofthissamplewillbesubtracted

fromtheabsorbancesobtainedfromthe“unknowns”.

Add200µLBradfordreagent,andproceedaswiththestandardassay.Ifabsorbancevaluesfalloutoftherangeofthestandardcurve,doanotherassaywithdilutedsamples.

MV401‐Purification and Analysis of a Recombinant ProteinSPECIFICATIONSHEET4:TRANSFERASEACTIVITY

17

Glutathione‐S‐TransferaseEnzymaticActivityAssay

Material

‐SpectrophotometerShimadzu,UV‐transparent1mLdisposablecuvettes‐1‐Chloro‐2,4‐DiNitroBenzene,CDNB,100mMinEtOH‐ReducedGlutathione,G‐SH,100mMinH2O‐Reactionbuffer10X:Na2HPO4430mM,KH2PO4147mM,NaCl1.37M,KCl27mM;pH7.3

Protocol

1‐ReactionG‐SH+CDNB+Glutathione‐S‐TransferaseG‐S‐DNB+HCl

ThesampleGSTcatalyzesthetransferofCDNB(artificialsubstrate)onreducedglutathione.ThisresultsinGS‐DNB(ε340nm=9.6103M‐1.cm‐1)formation.Productformationisfollowedbymeasurementofabsorbanceat340nmasafunctionoftime,forthevariousenzymeconcentrations.Activity is assayed during the initial‐rate period (initial velocity), with substrate saturation. Therefore, theinitialslopeoftheresultingplotofA340nm=f(t)givestheVmaxvalues.

Usingtheseoriginslopevalues,wecalculatetheactivity,ΔA340nm/min/µL,foreachfraction.◊Knowingthefractionvolume,wecalculatethetotalactivitywithineachfractionand,bycomparingfinal

andinitialfractions,thepurificationyield.◊ Knowing the protein quantity within each fraction, we calculate the ratio of activity over the protein

mass,ΔA340nm/min/mg,thatisthespecificactivityofeachfractionand,bycomparingfinalandinitialfractions,thepurificationfactor.

2‐Reactionmix:CDNB1mM,G‐SH1mMin1Xreactionbuffer

Becausethereactionmixisunstable,addtheG‐SHjustbeforedoingthespectrophotometermeasurements.Oncepreparedatroomtemperature,themixshouldbeusedwithin30min.Prepare enough volume for 12measurements (withoutG‐SH!), keeping inmind that eachmeasurement isdoneon1mLfinal.

3‐Sampleenzymaticactivityassay

Samples:assayactivityof5µLoffraction1(pureanddiluted1/10in1Xnativebuffer)and6.1,forbothGSTandGST‐GRDBD.

Kineticmeasurements: a) Insert cuvettewith990 µL reactionmixwithoutG‐SH in thespectrophotometer.Pressauto‐zeroat340nm.

b) Add 10 µL G‐SH in the cuvette, homogenize and measure activity over 30 sec(chemicalactivity1).

c)Thenadd5µLof thetestfractionto thecuvette,homogenizeandmeasureactivityover30sec(chemicalandenzymaticactivities2).

d)Theenzymaticactivityofthefractionisgivenbysubtracting1from2.

Beforeassayingeachfraction,pressauto‐zeroandre‐dothechemicalactivitymeasurement.The spectrophotometer calculates the slope value over 1 min. Write down the slope values (they are notsavedandthereisnoprinter).

Iftheenzymaticactivityofafraction is>1.5ΔA340nm/min,diluteafractionvolumeinnativebufferandresume

measurement(thiscanhappenfortheGSTfractions).Ontheotherhand,iftheactivityistoolow,assay10µLofthefraction.

MV401‐Purification and Analysis of a Recombinant Protein APPENDICES

18

APPENDIXI

ProteinexpressionsystemRecombinantproteininvivoexpressiontechniquesareoftenusedtoproduceproteinquantitieshighenough

toobtainantibodies,tostudytheirstructure,ortoexamineprotein‐proteinorprotein‐DNAinteractions.Prokaryoticsystemswerethefirstusedtoexpresslargequantitiesofproteins.Proteinsareexpressedathigh

yieldsand theirexpression is easily regulated.However,expression of eukaryotic proteins inprokaryotic systemshas a major drawback if this protein requires post‐translational modifications (phosphorylation, glycosylation,acetylation or proteolysis) in order to be fully active. Another frequent problem of protein overexpression inbacteriaproteininsolubility,whichgreatlyhindersthepurificationofafunctionalprotein.

To overcome these drawbacks, eukaryotic expression systems have been developed (in yeast, insect ormammaliancells).Insuchsystems,eukaryoticproteinsaremorelikelytobeproperlyfolded(conformation)andtobeproperlyprocessedtogetalltheirrequestedpost‐translationalmodifications.

Thechoiceoftheexpressionsystemisthereforelargelymotivatedbythefutureuseoftheprotein.

ExpressionasafusionproteininframewiththeGlutathione‐S‐Transferase(GST)

Glutathione:

Thistripeptideispresentinalllifeforms.ItisatypicalsinceitsN‐terminalglutamateisboundtocysteinebyanon α-peptidic bound. Glutathioneisasubstrateforvariousenzymes.

The arrow indicates the position of the covalent bond with agarose in theglutathione‐agaroseresin.

Theinducibleartificialpromoter«tac»:

The«tac»promoterusedisasyntheticconstruct:

The–35regioncontainstheTrppromotersequence(tryptophanoperon)anditsPribnowbox(the–10region)correspondstotheUV5‐lacpromotersequence(=lactoseoperonregionwhichbindsthelacIrepressor).The«tac»promoterusestherepression‐inductionsystemofthelactoseoperon.Itisextremelypowerfulandallowsonetocontroltheonsetoftranscription.Thisisofhighimportancebecausetheaccumulationofhighquantitiesofexogenousproteinscanperturborinhibitbacterialgrowth,and,consequently,reduceproteinexpression/yield.Similarly,iftheproteinproducedinbacteriaisunstable,itispossibletodelaytheonsetofproteinexpressionuntilahighdensityofbacterialcultureisreached,andtheninducethetranscriptionoftheclonedgeneatmaximalstrength.Suchstrategiesallowtheproductionofexogenousproteinswithquantitiesupto1to10%ofthetotalbacterialprotein.


19

APPENDIXIIAmplificationbyPolymerisationChainReaction(PCR)

1/PCR:generalitiesandprinciple

From a short double–stranded DNApriming region, Taq DNA polymeraseusessingle‐strandedDNAasatemplateto polymerize a new complementarychain. Single‐stranded DNA resultsfrom heating double‐stranded DNA attemperaturesclosetoitsmeltingpoint.The polymerization reaction startswhen a synthetic oligonucleotideprimer hybridizes to a given sequenceofthetemplate(seefigure).Thus,PCRmachines allow short cycles ofdenaturation (high temp.), primerannealing (low temp.) and DNAextension (intermediate temp.). EachcycleallowstheformationofacopyofthetargetDNA,whichinturnwillbecopiedduringthenextcycle.Therefore,thereisanexponentialamplificationofthetargetDNA sequencewitha theoretical valueof 2ndouble‐strandedDNAmolecules after n cycles (thatmeansmore thanamillioncopiesfor22cyclesandoverabillionafter32cycles).

2/Reactionconditions

• DNA: initial quality and quantity to amplify are two important factors. Degradation and impurities (chelating agents orinhibitors) lower the efficiencyof theamplification reaction. Toomuch templateDNA increasesprimermismatchesandnonspecificamplification.ToolittletemplateDNAlowersprimerannealingefficiency.

• Buffer:magnesiumconcentration is critical.WithoutMg++, theTaq polymerase is inactive,while excessMg++ reduces itsfidelity. Free Mg++ quantity depends on the concentrations of DNA, enzyme, dXTP and chelating agents. It is sometimesnecessarytotesttheoptimalMg++concentration,whichusuallyrangesbetween1mMto5mM.SomebufferscontainreagentmoleculesthatcanincreasePCRspecificityorefficiency(ex:BSA,glycerol,dimethylsulfoxide,TritonX‐100).

• ThermostableDNApolymerase:severaltypesareavailable,somewithhigherfidelity(e.g.,Pfu)thanothers(e.g,Taq),andothersmoreefficientlyamplifylargerfragments.We’reusingTaqpolymerase.

• Nucleotides:DNAsynthesisrequiresnucleotides(dATP,dTTP,dCTPanddGTP).Theirfinalconcentrationrangesfrom50to500µM,usually200µM.

• Primers:oligonucleotidesmust contain from 18 to 30nucleotides, have40 to60% GCbases,be specific and border thesequencetoamplify.Theymustnotbeself‐complementaryorcontainahairpinmotif.Primerconcentrationrangesfrom0.1to0.6µM.Toomuchprimerfavorsnon‐specificamplification;toolittleprimerlowerstheamplificationyield.

• Amplificationcycles:amplificationcyclenumberdependsontemplateconcentration.Annealingtemperaturedependsontheprimersequences.Extensiontemperaturedependsonthepolymeraseused,andelongationdurationdependsonthesizeofthefragmenttoamplify(ruleofthumb:1minute/kb).


20

APPENDIXIII

ElectrophoreticmobilityinthediscontinuousLaemmlisystemDiscontinuous systems permit loadinganelectrophoresisgelwith largevolumesofproteins solution.Proteinswill beprocessedthroughaporousstackinggelandalignedinathinbandontopofa lessporousresolvinggel,whichallowsindividualproteinstobeseparatedintowell‐resolvedbands.

Theelectrodebuffer (or runningbuffer), inwhich the cathodeand theanodeare submerged, contacts thegel. Thisbuffer containing Tris‐glycine is characterized by a high quantity of trailing ion, glycinate (low mobility), and abuffer/counter‐ion(Tris)atpH8.3.AtthispH,theglycinepopulationisnegativelycharged,sincethepHiis6.

Thesamplebuffercontainsthedissolvedproteins,amigrationmarker,adetergentandareducingagent,inthestackingbuffer.

The stackinggel is thegel inwhichall proteinsenter a largepore size acrylamide network. Thisgel contains abuffercharacterizedbyahighquantityoffrontion(Cl‐),thebuffer/counter‐ion(Tris)atpH6.8.

Theresolvinggelallowsseparationofthepreviouslystackedproteinsfromoneanother.ThisgelbuffercontainsCl‐ion,thebuffer/counter‐ion(Tris)atpH8.8.

Whenvoltageisapplied,Cl‐ionswillmoverapidlybecausetheyaremobileandstronglycharged.Theglycineionfront,entering the running buffer with the same pH as the stacking gel, is mostly zwitterionic and moves very slowly.Therefore,thiscreatesaregionoflowconductivity:sinceCl‐ions,verymobile,haveleft,buttheglycineions,notmobile,havenotarrived.Intheseconstantelectricconditions,thisvoltagedropregionwillalwayshavethesamesize(1‐2mm)andwillhaveatendencyto“aspirate”themoleculesendowedwithintermediatechargedensitiesandmobility.

Inthisdevice,proteinsaremoremobilethanglycinate

butlessthanCl‐.Thislowconductvityzonesweeps

theproteinsthroughthelargeporesofthestacking

gel. Because the acrylamide concentration in thestacking

gelislow,theproteinsarenotseparatedandare

stackedintoalowervolumethantheinitialvolume

loaded.

Thisconcentratesallproteinspeciesintothinbandsatthe surface of the resolving gel andwill increase theresolvingpoweroftheresolvinggel.

Intheresolvinggel,thepHis8.8,andtheglycineions

becomechargedandmigratefaster,almostasfast

asCl‐,andbecauseoftheirsmallsizearenotslowed

bythegelnetwork.Theproteinswillthenbe

separatedbasedontheirsize,whichisproportionaltotheirapparentmolecularweight.

MV401‐Purification and Analysis of a Recombinant Protein STRUCTURALSTUDY

21

Study of a 3D structure of a GRDBD/DNA complex

Introduction

Obtainingthe3Dstructureofaproteininvolvesknowingtherespectivepositionsoftheatomsandthusbetterunderstandingtheirinteractions.AsdescribedduringthisPracticalCourse(TP),therearethreetechniquesgivingaccesstostructuraldata:

1. X‐ray diffraction. This is a very powerful technique that gives structuralinformationaboutmaterialofallsizes:frommeresaltstoproteincomplexesoverseveral million Daltons (ribosomes, photosynthetic complexes). Proteins mustform crystals. This crystallization process is delicate and mostly empirical, thusoftenlimiting.

2. NMR (nuclear magnetic resonance). This technique is based on the capacity ofsomeatoms(1H, 13C, 15N,…)tointeractwithamagneticfield.Theparametersofthisinteractionareundertheinfluenceofthesurroundingatoms.Therefore,usingspecificandcomplexequations,itispossibletocalculatetherespectivepositionsoftheatoms.Thelimitingfactoristhusthesizeofthemoleculeintermsofatomnumber:themoreatoms,themoredifficultitistoextractstructuralinformation.Nowadays,thelimitforsolutionstructuredeterminationis40‐50kDa.

3. Cryo Electron Microscopy. This technique, which is under rapid development,allowsaccess totheenvelopeofmacro‐complexesthatarespread inaultrathinlayer(toavoidoverlapping).The2Dimagesobtainedaresummedanda3Dmodelcanbededuced.This techniquewasusedtosolvethestructuresofseveralviralparticles.

Allknownstructuresaredepositedinadatabase,"ProteinsDataBank"orPDB.Thisdatabankisfreeandcanbeaccessedfromdifferentwebsites:

‐ RCSB (Research Collaboratory for Structural Bioinformatics) www.pdb.org :historicalsiteforconsultationandsubmissionofstructuredata.

‐ PDBe http://www.ebi.ac.uk/pdbe/ : European version of the pdb.org site,administeredbyEBI(EuropeanBioinformaticInstitute)withadifferentinterface.

‐ PDBsumhttp://www.ebi.ac.uk/pdbsum/ : siteadministeredbyEBI.Less thoroughthanthepreviousonesbutmoreuser‐friendly,withlinkstoothersites likeGeneOntology,Pfam.Givesdiagramsofinteractionswithotherproteins.

Thereareseveralprogramstovisualizethesestructures,eitheronline,orafterprograminstallation.Thelatermethodallowsonetouseverypowerfulprograms,likePyMol,VMDorSwissPDBviewer.However,duringtheTPsession,wewilluseanonlineversion:AstexViewer.

MV401‐Purification and Analysis of a Recombinant Protein STRUCTURALSTUDY

22

Structurestudy

OpenSafariandconnectthroughyourstudentaccount.

Go to the PDBe site (http://www.ebi.ac.uk/pdbe/). To find the structures of the DNAbinding domains of the glucocorticoid receptor, select the “search” tab. If you enter“glucocorticoid receptor”, you will get over 400 answers. Refine your search byspecifyingthespeciesstudiedhere.

Ifyoudon’tfindanystructurecorrespondingtothedomainstudiedinthisclass,lookforthe3FYLstructure.

• Fromwhichspecieshasthecrystallizeddomainbeenobtained?• Whichtechniquewasusedtoobtainthestructureofthecomplex?• 3Dimagesappearautomaticallyontherightofthescreen.What isthenatureof

themoleculesthatcomposethecrystallizedcomplex?

Toworkdirectlyonthestructure,gothe“Viewin3D”tab,andselect‘AstexViewer’.Anewwindowopens after a few seconds. The structure appears ona black screen, theproteinin“Cartoon”formatandtheDNAin"Sphere".Youmaymodifytheformatusingthetabsontheleft.Imagespileup.Thus,tomodifytheDNA(tab“NucleicAcid")displayto"Line",for instance,youmustselect"line"andselect"Sphere".Tozoom,use"Shift+mouse click", tomove "Ctrl+mouse click ". To go back to former image, select "ResetView"orrightclickinthewindow(orCtrl+mouseclickonMac)and"View"and"Reset".

Colorcode:green=C,red=O,blue=N,yellow=S,violet=P,white=H

• Howmanymoleculesformthecomplex?• Whichresiduesformtheprotein/DNAinterface?Explain.• Usethe"Ligand"tab,select the"Sphere"display:youwill see4grayballsanda

moleculeformedof2greenand2redballs.Whichresidueisalwaysclosetothegrayballs?

• Whatisthenameofsuchstructure?

NIH Master de Sciences et Technologies / Master of Science and Tehnology Mention: Biologie Moléculaire et Cellulaire / Molecular and Cellular Biology

Fundamental module (UE) MV 401

Methods in Molecular and Cellular Biology

Practical Course « Methods of cellular analysis»

Practical room: “Biotechnology Workshops” Atrium, yellow area, third floor

Academic year 2012 - 2013

Faculty members in charge of the UE: Agnes Audibert, Sophie Louvet-Vallée. Faculty members in charge of the practical course « Methods of cellular analysis»: Anthi Karaiskou, Florence Bourgain-Guglielmetti. Faculty members in charge of the ”Biotechnology Workshops”: Adrien Six Secretary’s office: Carine Joseph, Tel: 01 44 27 35 35, e-mail: [email protected]

Paris 6 University Fundamental UE MV401

Methods in Molecular and Cellular Biology

CONTENTS

I- OBJECTIVES OF THIS PRACTICAL SESSION .........................................................................3

1. STUDY OF SOME ASPECTS OF THE MEIOTIC MATURATION OF THE XENOPUS OOCYTE. .....................3 2. SYNCHRONISATION OF CELLS IN CULTURE AND STUDY OF THE EXPRESSION PATTERN OF CELL CYCLE PROTEINS. ...................................................................................................................................3 3. OBSERVATION OF CYTOSKELETON DURING INTERPHASE AND MITOSIS IN CULTURED MAMMALIAN CELLS. ....................................................................................................................................................3

II- CELL CYCLE REMINDER............................................................................................................3

1. OVERVIEW..........................................................................................................................................3 2. CELL CYCLE STUDY USING MAMMALIAN CULTURED CELLS..............................................................5 3. CELL CYCLE STUDY USING XENOPUS OOCYTES.................................................................................6

III- PLANNING OF THE WEEK ........................................................................................................8

IV- EXPERIMENTAL PROCEDURES ..............................................................................................9

1. STUDY OF MEIOTIC MATURATION IN XENOPUS OOCYTE..................................................................9 A- DEFOLLICULATION............................................................................................................................9 B- SORTING OF OOCYTES ARRESTED IN PROPHASE................................................................................9 C- OBSERVATION OF OOCYTES UNDER A BINOCULAR MICROSCOPE ...................................................10 D- PREPARATION OF PROTEIN SAMPLES ..............................................................................................10

2. CELLS CULTURE AND SYNCHRONISATION.......................................................................................10 A- CELL CULTURE ............................................................................................................................11 B- CELL HARVEST AND PREPARATION OF THE PROTEIN LYSATE .....................................................13

3. PROTEIN ASSAY .................................................................................................................................14 4. POLYACRYLAMIDE GEL ELECTROPHORESIS OF PROTEINS ............................................................15

A- GEL PREPARATION ..........................................................................................................................15 B- PROTEIN SAMPLES PREPARATION....................................................................................................16

5. PROTEIN TRANSFER ON NITROCELLULOSE MEMBRANE AND IMMUNO-ENZYMATIC DETECTION................................................................................................................................................................16

A-REALISATION OF THE ELECTROPHORETIC TRANSFER......................................................................16 B- IMMUNO-ENZYMATIC REVELATION OF THE PROTEINS OF INTEREST: .............................................17

6. ANALYSE OF THE CELL CYCLE BY FLOW CYTOMETRY ..................................................................18 A- OBJECTIVE ......................................................................................................................................18 B- DNA STAINING PROTOCOL BY PROPIDIUM IODIDE .........................................................................18

7. IMMUNOFLUORESCENCE PROTOCOL ...............................................................................................20 A- OBJECTIVE ......................................................................................................................................20 B- PROTOCOL .......................................................................................................................................21

V- CONCLUSION................................................................................................................................22

VI- APPENDIX.....................................................................................................................................23

A- NUCLEOTIDES BIOSYNTHESIS PATHWAY ........................................................................................23 B- CELL CULTURE .................................................................................................................................24 C- CELL CULTURE MEDIA .....................................................................................................................26 D- LAMINAR FLOW HOOD .....................................................................................................................28 E- PRINCIPLE OF THE POLY-ACRYLAMIDE GEL ELECTROPHORESIS OF PROTEINS..........................29 F- OPERATING PRINCIPLE OF A FLOW CYTOMETER ...........................................................................31 G- OPERATING PRINCIPLE OF A FLUORESCENCE MICROSCOPE ........................................................33

I- OBJECTIVES OF THIS PRACTICAL SESSION The purpose of this week's practical course is to experiment several techniques of cell analysis. The biological questions and experiments that you will carry out to answer them will focus on the "cell cycle" theme. You will highlight some aspects of the cell cycle control and the cell signalling in two different models: Xenopus oocyte and a line of hamster cells in culture, called CHO.

1. Study of some aspects of the meiotic maturation of the Xenopus oocyte. A. Collection and sorting of oocytes. B. Induction of meiotic maturation by hormonal stimulation (progesterone) and effect of protein synthesis inhibition. C. Observation of maturing oocytes under a dissection microscope, and sampling at different stages. D. Analysis of two kinases: Cdk1 (Cdc2) and MAPK (quantity and activity), by immunoblot from the different samples.

2. Synchronisation of cells in culture and study of the expression pattern of cell cycle proteins. A. Culture and synchronisation of the cells by serum deprivation, addition of thymidine or nocodazole. Samples preparation. B. Study of the distribution of the cells in the cycle from the various samples by measuring DNA content by flow cytometry. C. Analysis of Cyclin A and Cdk1 (Cdc2) proteins by immunoblot from the different samples.

3. Observation of cytoskeleton during interphase and mitosis in cultured mammalian cells. A. Culturing the cells on glass coverslips. Samples preparation. B. Chromatin and microtubules visualisation by indirect immunofluorescence. II- CELL CYCLE REMINDER

1. Overview

Cell cycle consists of two major phases: interphase and mitosis (figure 1). Interphase

is a preparation phase for cell division or mitosis (M). It starts with the G1 phase (Gap 1), during which the cells receive signals required to induce their growth, and perform RNA and protein synthesis. The cells then enter the DNA synthesis phase (S), during which they double their DNA quantity. In the G2 phase (Gap 2), cells are getting ready for mitosis. The successive stages of mitosis (prophase, pro-metaphase, metaphase, anaphase and telophase) followed by cytokinesis, result in the formation of two daughter cells containing the same genetic material as the mother cell. The cell cycle duration and cycle number vary according to the cell type. Note that a cell can exit the cell cycle permanently or temporarily (e.g. in the absence of growth factors). It then enters a stage called G0 (Gap 0) or quiescent phase.

Many surveillance systems exist in order to interrupt the cell cycle progression when abnormalities are detected. These surveillance systems are called "checkpoints". Theoretically, if an element upstream of the checkpoint is defective, the cell does not go through to the next stage of the cell cycle. The checkpoint G1-S is named "restriction point" beyond which the cycle progression becomes independent of growth factors. The progress of the S phase is blocked when DNA is damaged and resumes after damages have been repaired.

The progression of the G2 to M phase occurs only when replication is complete and if no damage in genomic DNA is detected. Finally, exit from M phase needs as a prerequisite a proper mitotic spindle assembly. Figure 1: Cell cycle: checkpoints and their activation conditions

Progression in the cell cycle depends on different types of cyclin/CDK complexes (Figure 2). CDK protein is a cyclin-dependent (regulatory subunit) kinase (catalytic subunit). The kinase activity of the cyclin/CDK complexes is controlled by the synthesis/degradation of cyclins and also by phosphorylation/dephosphorylation of CDKs. In quiescent cells, cyclins and CDKs are absent or inactive. Figure 2: Sequential activation of cyclin-CDK complexes in mammals

2. Cell cycle study using mammalian cultured cells.

The CHO cell line will be used for this practical course. These cells, derived from the Chinese hamster ovarian epithelium, are immortalized: they can make an unlimited number of divisions. The use of cultured cells as a model for studying the cell cycle requires getting a population of synchronized cells. Synchronization will stop a cell population at a given phase of the cell cycle by using drugs, metabolites or by physical methods. If the treatment is reversible (non toxic), cells then simultaneously resume cycling after treatment arrest. The most commonly used treatments are listed below.

Some primary cells or immortalized (not transformed), have contact inhibition: they stop growing once they have formed a confluent cell layer and enter into quiescent G0. This is the case for the CHO.

a- Synchronisation in G0 phase

* Deficiency or deprivation in growth factors, for example by withdrawal of serum, can arrest cell proliferation, which then enter the G0 phase. * Some primary cells or immortalized (but not transformed) cells have contact inhibition: they stop growing once they have made a confluent cell layer and enter into the quiescent G0 phase. This is the case for the CHO.

b- Arrest in G1/S transition and in S phase * Excess of thymidine induces a feedback inhibition of the ribonucleotide reductase involved in the nucleotides biosynthesis pathway. The resulting deficiency in dCTP (see in appendix A the nucleotides biosynthesis pathway) leads to an inhibition of DNA replication and a cell cycle arrest at the G1/S transition or during S phase. * A mimosine treatment alters the metabolism of deoxyribonucleotides and thus DNA replication. * A treatment with hydroxyurea inhibits the ribonucleotide reductase and thereby blocks the reduction of ribonucleotides into deoxyribonucleotides. Thus DNA replication is impossible which leads to a blockage of cells in S phase. * A treatment with aphidicolin reversibly and specifically inhibits alpha DNA polymerase, and blocks cells in S phase

c- Arrest in G2 phase * Effective cell cycle arrest before mitosis can be experimentally induced by the addition of topoisomerase inhibitor molecules, such as genistein (inhibition of topoisomerase II), or camptothecin (inhibition of topoisomerase I). These drugs induce DNA breaks, then a G2 arrest to repair DNA damages (activation of the checkpoint of the DNA state).

d- Arrest during mitosis Cell progression in the cycle can be interrupted at different stages of mitosis by: * the use of drugs that inhibit microtubule polymerization such as nocodazole, vinblastine, vincristine, colchicine, colcemid. Depolymerization of microtubules induces cell arrest at an early stage of mitosis, in pro-metaphase, after activation of the "spindle checkpoint". * the use of taxol, which stabilizes microtubules and irreversibly stops cells in metaphase. A physical method (mitotic shake-off) allows to harvest mitotic cells. It consists in detaching the poorly adherent mitotic cells by gentle shaking and taping the culture dishes. The percentage of mitotic cells can be further increased by a prior synchronization of the cell population in mitosis.

3. Cell cycle study using Xenopus oocytes.

a- Introduction Unlike somatic cells in culture, the Xenopus oocyte has the advantage of being

physiologically arrested in two points of the meiotic division cycle: prophase (compared to a late G2 phase) of meiosis I and metaphase (M phase) of meiosis II (Figure 3). The resumption of meiosis occurs when the oocyte has accumulated enough reserves (ie the end of the growth period, primary oocyte), and in response to a hormonal signal, the progesterone secreted by the follicle cells. The signaling pathway activated in response to progesterone leads to activation of the MPF factor (M-Phase Promoting Factor or Cdk1-cyclin B complex) and thus to meiotic maturation (prophase I - metaphase II transition). Meiosis resumption is characterized by the migration of the germinal vesicle (GV) towards the membrane and nuclear envelope breakdown (GVBD for Germinal Vesicle Break Down) that cause delocalization of the black cortical pigments at the animal pole of the oocyte, resulting in the apparition on the surface of a white mark (maturation spot). Meiotic maturation allows the formation of fertilizable gametes arrested in metaphase of meiosis II (MII), laid in their external environment. Meiotic resumption, following metaphase II arrest, will be triggered by the oocyte fertilization.

Meiotic maturation offers the possibility to experimentally address the molecular mechanisms ensuring the G2/M transition of the cell cycle. The amphibian oocyte has the advantage of allowing cell biology approaches (microinjections, enucleations), because of its large size (> 1 mm in diameter) and biochemistry approaches, due to the large amount of available material (30-40 micrograms of protein per oocyte).

Figure 3: Oogenesis and meiotic maturation in Xenopus

b- MPF regulation mechanisms MPF is a heterodimer composed of a catalytic subunit, the Cdk1 kinase (also known

as Cdc2) and a regulatory subunit, cyclin B. Three levels of regulation control Cdc2 kinase activity: association with the regulatory subunit, i.e. with cyclin, dephosphorylation of two inhibitory residues (Thr 14 and Tyr 15) and, finally, phosphorylation of one activator residue, threonine 161 (Figure 4).

Cdc2 is inactive in prophase I oocytes and exists as two forms: a monomeric non-phosphorylated free form, and a cyclin B2-associated form, kept inactive by phosphorylation of Thr14 and Tyr15 of Cdc2, called pre-MPF.

Meiosis resumption is characterized by MPF activation. Several mechanisms may be responsible for this activation:

1) New complexes of active MPF can be generated by the association of free Cdc2 molecules with newly synthesized cyclin B1 molecules in response to progesterone (Table I).

2) The conversion of inactive pre-MPF into active MPF (Cdc2-cyclin B2): it is based on the dephosphorylation of both inhibitory sites of the Cdc2 kinase (Thr14, Tyr15) by a specific phosphatase, Cdc25 phosphatase (Figure 4). Cdc25 activation is correlated with its hyper-phosphorylation, which occurs just before the rupture of the nuclear envelope. The hyper-phosphorylation of Cdc25 depends, among other things, on Cdc2 activity, which creates a positive feedback loop between Cdc2 and Cdc25. Posttranslational activation of Cdc2 thus involves a self-amplification loop that allows the rapid formation of active MPF molecules from pre-MPF ones.

3) Accumulation of the Mos protein kinase in response to progesterone results in the activation, by successive phosphorylations, of MEK, MAPK and p90RSK kinases, occuring at the time of GVBD. Several recent studies argue for an involvement of this pathway during meiosis resumption.

At the metaphase I/anaphase I transition, MPF activity drops due to the cyclins

degradation (Figure 4). As they are also neosynthesized, active MPF molecules are reformed allowing entry into metaphase of meiosis II. CSF (CytoStatic Factor) activation at that time allows blocking of the oocyte in MII until fertilization (Figure 4). It is clearly shown that the Mos/MEK/MAPK/p90Rsk pathway plays an essential role in the triggering of CSF activity.

Table I. Content in Cdc2 and B1 and B2 cyclins in Xenopus oocyte

MPF components Prophase (G2 phase) Metaphase (M phase)

Free Cdc2 Present inactive Present inactive Complex Cdc2-cyclin B1 Absent Present active = MPF

Dephospho-Cdc2 (T14 and Y15) Complex Cdc2-cyclin B2 Present inactive = pre-MPF

Phospho-Cdc2 (PT14 et PY15) Present active = MPF Dephospho-Cdc2 (T14 and Y15)

Figure 4:

MPF activation of oocyte

during meiotic maturation.

III- PLANNING OF THE WEEK

Xenopus oocytes CHO cells

Treatments Western Blot Cell culture Western Blot FACS Immuno fluorescence

Day 1 am Presentation of the practical course – Assignment of the talks

Day 1

Collection of ovaries Defolliculation Oocytes sorting: batches 1 to 4 Freezing batch 2 (PI)

Observation Plating: -‐ dishes L1 to L4 and F1 to F4 -‐ dish with coverslips

Day 2

Batches 3 and 4: Freezing MII and CHX Observation batches 1, 3, 4

Lysate PI, MII, CHX

L2, F2: -‐ Serum L3, F3: + Thy L4, F4: + Noco

Lysate L1 Fixation F1 Fixation of coverslips Ab I

Day 3

Lysate L2, L3, L4 Prot assay

Fixation F2, F3, F4

Ab II Mounting Observation

Day 4

Electrophoresis Transfer Ab I

Electrophoresis Transfer Ab I

Staining Acquisition

Observation

Day 5

Ab II Revelation

Ab II Revelation

Results analysis

Observation

Day 5 pm Debriefing and discussion of the results

IV- EXPERIMENTAL PROCEDURES

1. STUDY OF MEIOTIC MATURATION IN XENOPUS OOCYTE The aim is to study some aspects of meiotic phase (M) resumption using Xenopus

oocytes as a model. The biological question that will be addressed is the role of protein synthesis during meiosis resumption.

The study will be done firstly by observing oocytes under a binocular microscope and secondly by analyzing, using immunoblotting, proteins implicated in the cell cycle regulation at different stages of meiotic maturation.

In the ovaries, oocytes are surrounded by follicular cells, which under the effect of a hormonal stimulus (LH type), synthesize and release progesterone into the oocyte. The isolated oocytes are cultured in vitro after dissociation of follicle cells by enzymatic treatment (defolliculation); meiosis resumption is induced by addition of progesterone to the extracellular medium until the appearance of a spot of depigmentation (maturation spot) at the animal pole which characterizes meiosis resumption. A- Defolliculation - Abdominal incision of anaesthetized female, and removal of an ovary. - Ovary cut into fragments, then incubation in a physiological medium (Merriam medium1) containing successively the proteolytic enzyme (40 mg/100 mL of dispase) for 3 hours, followed by rinsing and incubation in collagenase (40 mg/100 mL) for 1 hour with gentle stirring.

- After a thorough rinsing in Merriam medium1, about 200 oocytes (all stages mixed) are placed in a small dish for each student’s pair to sort.

B- Sorting of oocytes arrested in prophase Only fully grown oocytes are competent for meiotic maturation induced by progesterone. Approximately 100 oocytes are selected under a binocular microscope: they are large (diameter > 1 mm), undamaged, with a homogeneous pigmentation. They are then divided into five batches.

# batch 1: fix 5 oocytes in 20% TCA for 15-20 min; keep them in Merriam1 in order to observe them the following day.

# batch 2: freezing at -20 °C of 5 oocytes: put the 5 oocytes in a 1,5 mL Eppendorf tube and suck all the medium using a syringe or a fine tip; they will be used to prepare the protein lysate "PI".

# batch 3: incubate 20 oocytes in a Petri dish overnight at 18 °C in 10 ml of Merriam in the presence of progesterone (10-6 M). The next day, you must determine the percentage of maturation (oocytes blocked in metaphase II). Then place 5 matured oocytes in a 1,5 mL Eppendorf tube and suck all the medium using a syringe or a fine tip, note "MII" on the tube and freeze at -20 °C; they will be used to prepare the protein lysate "MII". Fix 5 oocytes in 20% TCA for 15-20 min; keep them in Merriam1 for observation.

# batch 4: incubate 20 oocytes in 10 ml of Merriam with cycloheximide (protein synthesis inhibitor, 100 µg/mL) for 1 hour. Then add the progesterone (10-6 M) and incubate overnight at 18 ° C. The next day, you must determine the percentage of maturation, as for batch 3. Place 5 representative oocytes in a 1.5 mL Eppendorf tube and suck all the medium using a syringe or a fine tip, note "CHX" on the tube and freeze at -20 °C; they will be used to

1 Merriam buffer: 10 mM Hepes, 0,82 mMMgSO4, 88 mM NaCl, 0,33 mM Ca(NO3)2, 1 mM KCl, 0,41 mM CaCl2 ; pH 7,4.

prepare the protein lysate "CHX". Fix 5 oocytes in 20% TCA for 15-20 min; keep them in Merriam1 for observation. Caution ! the maturation spot that appears at the animal pole should be small with well defined edges. Some oocytes get damaged during the night and then show diffuse smudges. C- Observation of oocytes under a binocular microscope

On day 2, sorted and fixed oocytes can be observed under a binocular microscope, in an external view as well as in a meridian section (use a scalpel) to watch the germinal vesicle. Draw oocytes blocked in prophase I and metaphase II, in external view and cross-section. Specify the stage of the oocytes of batch 4. Question – Discussion:

See questionnaire sheet (distributed by the teachers) D- Preparation of protein samples You must first: * pre-cool the centrifuge

* set the heating blocks to 95°C * prepare an ice bucket

- The oocytes are lysed on ice with 10 µL of EB2 buffer per oocyte (50 µL for 5 oocytes), by pipetting using a yellow tip.

Caution ! Stay on ice as much as possible to avoid any proteolysis. Lyse all oocytes by

up and down pipetting. - The lysate is centrifuged during 5 min at 15 000 rpm at 4°C. - Supernatant is transferred to a clean tube. Determine the volume of 5X Laëmmli buffer3

to add, vortex, then denature the proteins by heating for 5 minutes at 95 ° C. - Rapidly cool in ice 5 min and centrifuge briefly before freezing these new prepared

samples. 10 µl of each sample will be analysed by electrophoresis, ie the equivalent of 1 oocyte. It is not necessary to assay protein concentration because we know the general amount of proteins per oocyte (see introduction).

2. CELLS CULTURE AND SYNCHRONISATION The goal is to study some aspects of the cell cycle regulation in cultured mammalian cells. The biological question to be discussed is the expression of a cell cycle regulator, cyclin A, in different phases of the cell cycle using synchronized CHO cells. The quality of synchronization will be determined by flow cytometry. In parallel, changes in microtubules and chromatin will be observed by immunofluorescence in cells in interphase and in different mitosis phases.

The usual precautions and principle of cell culture and the composition of media used

are presented in B and C appendices.

2 EB:80 mM β-glycerophosphate 80, 20mM EGTA, 15mM MgCl2, 1mM DTT, proteases inhibitors ; pH7.6. 3 5X Laëmmli: 312,5 mM Τris pH6.8, 50% glycerol, 0,1% bromophenol blue, 10% SDS, 250mM DTT.

A- Cell culture

a. Culture conditions and maintenance of the line: CHO cells are cultured at 37°C and 5% CO2 in DMEM medium (see appendix C) supplemented with: - 10 % of foetal calf serum - Penicillin (100 U/mL), Streptomycin (100 µg/mL) - L-proline (0.2 mM) For optimum growth and proper maintenance, cells must be transferred to new dishes after dilution. Thus, cells are passaged when they reach 80% confluence, to avoid selecting cells that have lost contact inhibition. The cells are detached from their support by a brief enzymatic treatment with trypsin in the presence of EDTA, and plated at a desired density. b. Cell numeration You will get two 10 cm diameter dishes, at the density of 4-8 million per dish of 10 mL of culture medium. Check under the microscope the cellular density in the dishes, and observe their morphology using phase contrast. Observe and describe the differences. These cells will be used to perform the various experiments, and must be dissociated with trypsin and counted. Caution! Use media, PBS (Phosphate Buffered Saline), and trypsin preheated to 37°C. Trypsin at 37°C is more efficient, and a cold culture medium can detach the cells.

- Suck up the culture medium with a Pasteur pipette and gently wash the cells twice with 5 mL of PBS.

Caution! PBS washes are important because trypsin is inhibited by serum. - Add 1 mL of trypsin previously heated to 37°C, thoroughly distribute the liquid

over the entire surface of the dish. Incubate at 37°C for 5 min. Check under the microscope that the cells are detached and therefore well rounded.

- Add 9 mL of complete medium to inactivate the trypsin. Caution! Inactivate the trypsin as soon as the cells are in suspension: a too long incubation alters the cell plasma membrane, which will not adhere anymore.

- Harvest the cells and gently detach those still adhering to the support by successive pipetting. Transfer them to a 50 mL sterile Falcon tube.

- Homogenize the cell suspension and add 10 µl of the cell suspension (using a pipetman) to a numeration slide (Kova-Slide). This slide is gridded with 9 large squares (volume of a large square: 0,1 µl), divided into 9 small squares (Figure 6).

- Count cells under the microscope in the 11 shaded squares in Figure 6, as explained in Figure 7. Remove the 2 extreme values. You therefore have the number of cells in 9 small squares (0,1 µl). You can deduce the concentration of cells (x.106 cells / mL).

Figure 6: Kova-Slide.

Figure 7: Cell counting

c. Cell seeding You have several dishes of sterile cultures. Note down the dishes and seed as indicated in the following table:

Dish code dish Total volume of medium Cells number L1 6 cm dish 4 mL 0,5.106 L2 10 cm dish 10 mL 2.106 L3 6 cm dish 4 mL 0,5.106 L4 10 cm dish 10 mL 1.5.106

coverslips 6 cm dish 4 mL 0,5.106 Well homogenize the dishes with back- and forward movements in order to distribute the cells evenly, and then incubate them at 37°C and 5% CO2. Indicate in your notebook the calculations that you carried out to determine the concentration of your cell suspension as well as the cell volumes you have taken to inoculate the different dishes (to be presented in a table). Cells in L1 to L4 dishes will be cultured under the conditions described below (Figure 8) and will then be lysed to prepare the samples analyzed by immunoblot. The “coverslips” dish will be treated the same way as the L1 dish and the coverslips will be collected to perform the immunofluorescence experiment (see Chapter 7). On day 1, a student or teacher will carry out a series of dishes marked F1 to F4 identical to what you did: they will be used for the FACS analysis. Finally, other dishes will be used to maintain the line for the next week practical course (Figure 8).

0,1 µl

Whatever the hemacytometer and the surface to enumerate, we count: all the elements located within the boundaries of this surface, and for the elements located on the lines, we count those which are on the left line (and not those which are on the right line, or vice versa) AND those which are on the top line (and not those which are on the bottom line, or vice versa). Cells counted in one square ( )

Cells excluded from the count of the square ( )

Figure 8: Culture and treatments of the cells

d. Synchronisation of the cells It will be achieved in different ways: - G0 arrest by growth factors deprivation (F2, L2): day 2 in the morning, remove the culture

medium, rinse twice the cells with 5 mL of PBS (37°C), then culture the cells for 24h in medium containing 0,1% serum. Harvest on the morning of day 3.

- G1/S arrest by an excess of thymidine for 24h (F3, L3): day 2 in the morning, add directly into each dish x µl of a 200 mM thymidine solution (2 mM final). On the morning of day 3, harvest the cells of the F3 and L3 dishes.

- Arrest in prometaphase in the presence of nocodazole (F4, L4): Day 2 in the evening, add directly into each dish y µl of a 1 mg/mL nocodazole solution (100 ng/mL final). In the morning of day 3, harvest the cells.

Cells in the F1, L1 and coverslips dishes are asynchronous proliferating cells that are not treated. They are harvested on day 2.

B- Cell harvest and preparation of the protein lysate a. Materials preparation:

Pre-cool the centrifuge at 4°C. Heat to 95°C the heat blocks. Install a bottle of PBS on ice near the sink of the room, with a 5 mL pipette, a pro-pipette, a P1000, a P200 and a vacuum pump. Prepare on ice the RIPA4 2X lysis buffer (ie 2 times concentrated as it is assumed that after washing the cells, 50 µl of PBS remain in the dish that will dilute the 50 µl of RIPA buffer). Prepare the 2X RIPA buffer containing the proteases and phosphatases inhibitors (complete 2X RIPA), from stock solutions (see with teachers).

4 1X RIPA buffer: 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 1% NP40, 0.1% SDS, 0.5 % sodium deoxycholate, 20 mM EGTA, 1 mM DTT; pH 7.3, supplemented with proteases inhibitors (Pepstatine A, Leupeptine, PMSF, Aprotinine) and phosphatases inhibitors (β-glycerophosphate, NaF).

b. Protocol for preparing protein lysates L1, L2, L3 (to be performed on ice): Values are given for a 6 cm-dish; the values for the 10-cm dishes are in parenthesis.

- Remove the culture medium with the vacuum pump and then add 5 mL (10 mL) cold PBS

- Remove PBS with the vacuum pump and then remove the excess with the P1000 by tilting the dish (IMPORTANT! Otherwise the sample will be too diluted)

- Add 50 µl (100µl) of complete 2X RIPA on ice. - Then scrape the cells always on ice. Transfer the lysate into a pre-chilled Eppendorf

tube and leave at least 10 min on ice. - Centrifuge 15 min at 15000 rpm at 4°C. - Transfer the supernatant into another Eppendorf tube clearly labelled. Transfer 10 µl

for the protein assay into another tube, also labelled, that will be stored at -20°C. - Measure the remaining volume and add the required volume of Laemmli3 5X buffer

(to be determined). - Denature proteins immediately by heating 5 min at 95°C, immediately cool on ice 5

min, centrifuge briefly (a few seconds) and store at -20°C. For L4 dish: You must recover adherent cells in G2 AND mitotic cells (round cells to be observed under the microscope) that are poorly adherent and can therefore easily come off.

- Do not wash the cells with PBS. Tap the dish on the bench to resuspend the mitotic cells (shake-off). Harvest the mitotic cell suspension using a pipette and transfer into a 15 mL tube. Centrifuge for 5 min at 1200 rpm. Pool all the mitotic cell pellets from the group. Wash the cells with ice-cold PBS. Resuspend the cell pellet with 1 volume of 2X RIPA, transfer in a pre-chilled Eppendorf tube and leave at least 10 min on ice. This is sample L5. The lysis protocol is the same as for L1, L2, L3 and L4. There will be one L5 sample for the whole room.

- After the shake-off, some G2 adherent cells remain on the L4 dish. To recover them, wash the dish with 10 mL of ice-cold PBS then follow the same protocol as for L1, L2, and L3. This is sample L4.

3. PROTEIN ASSAY

We use the BCA method which is compatible with the detergents present in the RIPA lysis buffer. The reagent5 consists of bicinchoninic acid (BCA) and cupric ions (Cu2 +) in an alkaline medium. Cuprous ions (Cu +) reduced by the presence of proteins form with BCA a stable and purple-colored product after incubation at 37°C (λmax = 562 nm).

Quantify proteins with 2 µl of each cell lysate, without forgetting to prepare a blank with 2 µl of 1X RIPA buffer. In parallel, perform a standard curve ranging from 0 to 32 µg (0; 2; 4; 8; 16 and 32µg) of proteins per tube using a bovine serum albumin solution (BSA) at 1 mg/mL.

BSA (standard range) or protein extract X µL H20 = qsp 50 µL (50-X) µL BCA reagent 1 mL

5 BCA: extemporaneous mix of 50 volumes of BCA pH 11,25 and 1 volume of 4 % CuSO4

Incubate at 37°C 30 minutes. Measure the absorbance for each sample at λmax = 562 nm. Write down in a table the measured absorbencies and draw a calibration curve (DO = f (µg of BSA) from the BSA values. Determine the extract volume to be pipetted to get 20 µg (or 10 µg) of proteins. 4. POLYACRYLAMIDE GEL ELECTROPHORESIS OF PROTEINS Polyacrylamide gel electrophoresis in denaturing conditions is used to separate proteins according to their apparent molecular weights (see Appendix E). A- Gel preparation

a. Gel casting. Install the plates and put a mark on the glass plate, at about 1 cm under the comb. Figure 9: Schematic diagram of the gel electrophoresis set up

Separating (or resolving) gel: prepare the following mix in a small beaker:

4 mL of acryl-bis/Tris/SDS6 solution 40 µl of 10% APS (ammonium persulfate) 4 µl of TEMED

Caution! Acrylamide is toxic; avoid any contact with the skin. Use gloves for preparing

solutions and pouring gels.

Mix the tube by inverting several times, do not vortex. Pour the gel immediately up to the mark on the plate. Overlay gently with distilled water and allow polymerization to occur.

At this stage, you can stop and leave the separating gel at 4°C. Make sure that water

does not evaporate and that the gel remains covered. 6 Separating gel solution: 0,375M Tris-HCl pH 8,7; 0,1% SDS; 12% acrylamide.

Stacking gel: prepare the following mix 2 mL of acryl-bis/Tris/SDS7 solution 20 µl of 10% APS (ammonium persulfate) 2 µl of TEMED

Mix the tube by inverting several times, do not vortex. Pour the gel immediately up to the top of the plates. Place the comb (10 well or 15 wells), avoiding bubbles. It is easier to insert the comb starting at one angle and to insert the teeth progressively. Allow polymerization to occur.

b. Installation of the plates inside the migration tank. Remove the plates from the casting racks. Remove the comb and rinse wells with distilled water. Mount the plates inside the migration tank and fill with electrophoresis buffer8 to the top of the plates. B- Protein samples preparation Load the samples following the order and indications given by the teachers. Caution: use the molecular weight marker only once, do not waste it!

- 10 µl for oocyte extracts - 5 µl of molecular weight marker (specification sheet available in the room) - X µl (i.e. 10 µg to 20 µg according to the size of the wells) of CHO extracts L1 to

L5.

Migrate at 100 Volts through the stacking gel and 120 Volts through the resolving gel. Let out the migration front in order to have the 20 kDa marker at the bottom of the gel.

5. PROTEIN TRANSFER ON NITROCELLULOSE MEMBRANE AND IMMUNO-ENZYMATIC DETECTION

A-Realisation of the electrophoretic transfer Use one nitrocellulose membrane and two sheets of Whatman paper per gel. Chill the transfer buffer to 4°C beforehand. Write your pair code using a pencil on the membrane. Then dip the membrane in the transfer buffer9. Remove the gel from the plates and discard the stacking gel. Install the gel and the nitrocellulose membrane inside the transfer cassette, as shown below, and according to the lab demonstration that will be done. Remove bubbles between the membrane and the gel. Place the transfer cassette into the tank previously filled with cold transfer buffer (be careful of its orientation). Transfer conditions: 1 hour at 100 V, at 4°C.

7 Stacking gel: 0,125M Tris-HCl pH 6,8; 0,1% SDS; 4% acrylamide 8 Electrophoresis buffer: 25 mM Tris, 0.193 M glycine, 0.1% SDS (pH 8,3) 9 Transfer buffer: 25 mM Tris, 0.193 M glycine, 20 % ethanol

Figure 10: Electrophoretic transfer mounting B- Immuno-enzymatic revelation of the proteins of interest:

a. Antibodies used Specificity Ab I Species Dilution used

Anti-Cdc2 Ab I mouse Will be specified during the course Anti-phosphoTyr15-Cdc2 Ab I rabbit Will be specified during the course Anti-MAPK Ab I rabbit Will be specified during the course Anti-phosphoMAPK Ab I mouse Will be specified during the course Anti-cyclin A human (C19) Ab I rabbit Will be specified during the course Anti-importin β Ab I goat Will be specified during the course Anti- Rabbit Ig - peroxydase Ab II 1/10 000 Anti- Goat Ig - peroxydase Ab II 1/30 000 Anti- Mouse Ig - peroxydase Ab II 1/10 000

b. Protocol

- Remove the transfer stack and incubate the membrane directly for one minute in a solution of Ponceau S10 in order to visualize the transferred proteins.

- Recycle the Ponceau S and rinse the membrane with distilled water (2 or 3 baths). - Make a photocopy or a scan to keep a record of the protein profile. - Cut the membrane according to the example given by the teachers knowing that the

molecular weights of the studied proteins are: Importin (95 kDa), Cyclin A (50-60 kDa), Cdc2 (34 kDa), MAPK (38 kDa). Write your pair code on each piece of the membrane.

- Transfer the membranes in blocking solution: 5% milk, TBS-Tween11 during 1 hour at room temperature.

- NB: After this saturation step, membranes should not dry!!! - Rinse once with TBS-Tween.

10 Ponceau S: 0.2% Ponceau (R250) in solution with 1% acetic acid 11 TBS-Tween buffer: 12.5 mM Tris-HCl pH 7.5, 136 mM NaCl, 1.5 mM KCl, 0.1 % Tween-20.

Add the first antibody diluted in TBS-Tween + 5% BSA. Incubate overnight at 4°C. - Wash 3x10 minutes in TBS-Tween. - Add the secondary antibody solution diluted in TBS-Tween, 5% milk. Incubate during

1 hour at room temperature. - Wash 3 times 10 minutes in TBS-Tween. - Incubate 5 min in the presence of 2 mL of the luminescent reagent and wrap in a saran

film. The reagent contains hydrogen peroxyde (substrate of peroxydase) and luminol (reagent).

- Place under a transparent plastic sheet and expose to an X-ray film (variable time that will be determined with teachers for optimal detection).

- In the dark room, develop, fix, and rinse the film.

Cut and paste the picture of the Ponceau S staining of your gel. Cut, paste and annotate the developed films obtained. Explain the Ponceau S and western blot results in parallel with the FACS results (see Chapter 6).

6. ANALYSE OF THE CELL CYCLE BY FLOW CYTOMETRY A- Objective Analyze the cell cycle by defining the cell DNA contents (2n, 4n or in between), which allows determining the percentage of cells in each phase of the cell cycle (respectively G0/G1, G2/M, S). The most commonly used fluorochrome on fixed cells is propidium iodide (PI). But other fluorochromes that quantitatively bind DNA may also be used: - In the UV: Hoechst 33342 or 33258 (respectively on live or fixed cells). - at 457 nm: Mithramycin. - at 488 nm: PI, 7-amino-actinomycin D (7-AAD), SYTOX Green, DRAQ5 - at 633 nm: TO-PRO-3 iodide. Staining should be specific for DNA: RNA must be removed by a prior hydrolysis in the case of PI. Staining should be quantitative: the fluorochrome must be present in saturating amount. A standard sample, whose DNA content is known, must be included in each experiment. B- DNA staining protocol by propidium iodide

a. Cell fixation. For the dye to be in saturating amount, about 1.106 cells will be used for the staining. Cells have to be dissociated with trypsin and counted (knowing how many were inoculated). Cells are centrifuged 5 min at 300 g (1200 rpm), aspirate the supernatant and leave approximately 200 µl on top of the pellet. Disrupt the cells by gentle pipetting. Add drop by drop with a P1000 and while vortexing, 2 ml of cold fix12, leave at least 1h at -20°C. Fixed cells can be stored several weeks at -20°C.

b. Cell staining. Centrifuge fixed cells 5 min at 300 g (1200 rpm), completely remove the supernatant and resuspend the pellet in 1mL PBS/RNAse (0,1 mg/mL final)/ propidium iodide (40 µg/mL 12 Fix: 70% absolute ethanol and 30% PBS, stored at –20°C

final). Cells are analysed straight away or in the next 3 days (if necessary cells are stored in the dark at 4°C). Caution! Propidium iodide needs to be handled with caution, using gloves. Tips and tubes that contained propidium iodide must be discarded in a special container.

c. DNA content analysis by flow cytometry.

Figure 11: Selection of cell populations to be analyzed.

The flow cytometer principle is shown in a diagram in Appendix F. To correctly analyse, you first need to eliminate the G1 cell doublets, easily identified by a shift of the PI fluorescence signal compared to the isolated cells and to select the singlet population (figure 11).

- Paste in your notebook the graphs obtained for the different experiments F0 to F4. - SUMMARIZE DATA IN A TABLE. - Conclude on the quality of the cell population synchronization. - Explain and interpret these data in parallel with those of the western blot. Question – Discussion: See questionnaire sheet (given by teachers)

Figure 12: Analysis of cell populations in the cell cycle phases. The percentage of cells in G1, S and G2/M is then determined using either manually positioned cursors, or a deconvolution software that is able to transform the graph into a sum of Gaussian curves (more accurate method).

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Propidium iodide Fluorescence

7. IMMUNOFLUORESCENCE PROTOCOL

A- Objective The glass slides that you seeded will allow to study, by immunofluorescence, the modifications of microtubules and chromatin during interphase and various stages of mitosis. First, cells need to be fixed in order to in situ immobilize the antigens, so that structures will not be damaged and that antigens will not be eluted during the different washing steps. Multiple fixation methods could be used, but the most common ones are:

- Methanol: it dehydrates the cells, causing precipitation of many molecules, while lipids and numerous small peptides are extracted. For antibodies to bind, it is necessary to rehydrate the cells: molecules may be eluted at different stages of washing. Thus, methanol is usually used for identification of poorly soluble antigens.

- Aldehydes (paraformaldehyde, formaldehyde, glutaraldehyde): they are cross-linking reagents that create intra- and intermolecular bonds, and thus immobilize molecules in their native form. They react mainly with proteins free amino groups, forming methylene bridges between proteins.

We will use the indirect immune-detection method to detect microtubules: the first antibody is revealed by a secondary antibody that carries the fluorescent marker, as shown below. Chromatin will be directly stained by propidium iodide intercalation. The principle of the fluorescence microscope is shown in Appendix G.

Figure 13: Indirect immunoreaction The choice of the fluorescent marker depends on the experiment made and the microscope filters that are available (see appendix G). Since we are using propidium iodide that emits at 617 nm, we chose a secondary antibody coupled to Alexa 488, a very stable fluorochrome, which emits at 519 nm.

Antibodies and dyes used are: Specificity Species Dilution Anti-tubulin α Mouse 1/1000 Anti- Mouse Ig – Alexa 488 1/1000 Propidium iodide 1µg/mL

B- Protocol Coverslips should always remain wet throughout the experiment; otherwise cells can be irreversibly damaged. Proceed with care because mitotic cells can easily detach. Prepare in advance: PBS at 37°in a tube and methanol at -20°C in another tube.

a. Fixation / permeabilization of the cells - Leave the coverslips at the bottom of the culture dish. Wash the cells with 4 mL PBS

at 37°C (T° which preserves the microtubule network). - Remove PBS (vacuum pump) then add ice-cold methanol (-20°C). Leave 5 min at -

20°C. Remove methanol (vacuum pump). - Grab coverslips with tweezers, drain on a paper towel, place them (cells upwards) on a

parafilm piece in a Petri dish. - Rinse 3x5 min with 300 µL of PBST13. b. First antibody incubation - Remove the liquid. - Gently deposit 35µl per coverslip of: PBST/3% BSA for the control coverslip and first

antibody DM1A diluted to 1/1000 in PBST/3% BSA for the second coverslip. - Incubate for 1h at room temperature or overnight at 4°C. In this case, you need to

add some wet paper in the dish to make a humid chamber with a lid. - Wash 3x5 min with PBST.

c. Second antibody incubation - Remove the liquid using a pipetman. - Add 35µl of the secondary antibody-Alexa 488 solution diluted to 1/1000 in

PBST/3% BSA; cover the dish with aluminium foil to keep in the dark for 30 min to 1h, at room temperature.

- Wash 3x5 min with PBST. d. Nuclei staining

Caution! Propidium iodide needs to be handled with caution, using gloves. Tips and tubes that contained propidium iodide must be discarded in a special container.

- Remove the liquid using a pipetman. - Add 35µl of the propidium iodide14 solution (1µg/mL) diluted in PBS; incubate in the

dark for 2-3 min - Wash 3x5 min with PBS. e. Coverslip mounting (lab demonstration will be done) - Add on a slide 5µl of mounting medium. - Grab the coverslip with tweezers and drain the remaining liquid by tilting it on a paper

towel. - Invert the coverslip on the slide, so that the mounting medium drop is well distributed

under the coverslip. - Seal the coverslip with 4 tiny spots of nail polish.

13 PBST: PBS + 0,1% Tween 14 Cells have been fixed with methanol, RNAse treatment is thus not necessary here.

f. Observation with the fluorescence microscope - Focus with the X40 objective, observe; - Observe with the X100 objective, after adding a drop of immersion oil on the

coverslip.

- Observe interphasic and mitotic cells and look for all stages of mitosis. Count the number of mitotic cells. What can you deduce? - Draw (paste pictures) the images that you observed at these different stages. Name the phases shown. - What results would you get if the cells had been pre-treated with nocodazole? V- CONCLUSION Write a conclusion of all the week experiments.

VI- APPENDIX

A- NUCLEOTIDES BIOSYNTHESIS PATHWAY Ribonucleotide diphosphoreductase (RNdPR) is an enzyme involved in the synthesis

of deoxyribonucleotides (dNDP) (figures 14 and 15) from the corresponding ribonucleotides (NDP). It catalyzes the reduction of ribose in deoxyribose. Its enzymatic activity is controlled by effectors, which are the products of the reaction (figure 16).

Figure 14 Figure 15

Figure 16

dTTP excess induces the retroinhibition of the ribonucleotides reductase and the reduction of UDP and CDP, thus the production of dTTP and dCTP.

B- CELL CULTURE

Cell culture is defined as a technique that aims to maintain functions of the cells in vitro. Under appropriate conditions, the cells (plant or animal) can survive, multiply and express properties of differentiated cells. There are several types of cell cultures:

● The organotypic culture: survival of an organ or tissue fragment while retaining its integrity and architecture (eg skin).

● Primary cell culture: cells are dissociated from each other, either mechanically or using enzymes, from a tissue, before being cultured. They can divide (fibroblasts), but in many cell types it is only a survival maintenance (hepatocytes).

● Cell lines: immortalized cells, capable of performing a limited or unlimited number of divisions, which are maintained in culture by successive subcultures (or passages).

1. Which environment for the cells? Some cells do not adhere to a substrate and are cultured in liquid medium

(hematopoietic cells, adipocytes, ...). Adherent cells require a solid pre-treated support. Support and culture medium are two parameters that are essential to survival, proliferation, migration and differentiation of the cells, at a temperature of 37°C and under CO2 controlled atmosphere (5% in general).

a. Support Adherent cells are grown on a plastic substrate (Petri dishes, flasks) having

undergone a cell culture specific surface treatment (electric field, magnetic field ...). This physical treatment increases the amount of charges of polystyrene and makes it more hydrophilic, allowing efficient interaction with the cell plasma membranes. Some supports are covered with proteins present in the extracellular matrix (collagens, laminins) that promote cell adhesion and differentiation.

b. Culture medium Synthetic media used are chosen depending on the cell type to grow. The most well

known are DMEM (Dulbecco's modified Eagle's medium), Ham F12 and RPMI-1640 media. The composition of the main culture media is detailed in a table in appendix C. These basic media are supplemented by addition of:

- 10 to 15 % of foetal calf or newborn calf serum.

Serum is a complex mixture of substances such as ions, vitamins, hormones, fibronectins and growth factors. We use animal sera from young individuals, the overall cytostimulant effect of serum being inversely proportional to the age of the donor. When cultured in presence of a suitable synthetic medium, most cells are only surviving. The presence of a number of mitogenic factors provided by the serum is needed to trigger cell division. The serum complement is inactivated by heat (56 ° C for 30 min) to prevent the cells to be lysed by the serum antibodies.

- Antibiotics and antifungal agents

They should be handled with caution, because they can have a toxic effect on cells. Antibiotics: penicillin (100 U/mL), streptomycin (100 µg/mL); antifungal agent: fungizone (0,25 U/mL).

2. Routine precautions in the culture room The culture room is a closed area that contains: - a laminar flow hood (see Appendix D) equipped with a liquid suction system

allowing changes of culture media. - a humidified thermostat CO2 incubator (5% CO2, 37°C), necessary for cell

maintenance. - an inverted microscope. - a low speed centrifuge. - a water bath at 37°C containing a non-foaming antiseptic.

a. Work preparation

This is an essential step, which avoids the comings and goings, source of contaminations. - Wash hands with soap before entering the culture room. - Put on a lab coat dedicated for cell culture. - Disinfect the surface of the hood with 70% ethanol.

Culture media and trypsin, maintained at 37°C in a water bath, must be disinfected with 70% ethanol before being placed under the hood.

b. Handling under the hood

Caution! Do not place anything within 10 cm from the edge of the hood. Do not overcrowd the hood working surface and always place the material as far as possible from the edges without disturbing the laminar flow.

- DO NOT MOUTH PIPETTE; use the pipetaid which are compatible with disposable pipettes.

- Begin with unscrewing bottle and tube caps. During use, caps will be held in your hand or placed face upwards as far as possible from the hood edges.

- Do not touch anything with the pipettes tip otherwise change pipette immediately. - Wipe right away a drop of liquid fallen on the work surface with a paper soaked in

water and then disinfect with 70° alcohol. - Never put your hand over open containers.

c. Tidying up the culture room

This procedure is absolutely necessary after each use, at the end of the day: - Put the culture media back in the fridge. - Wash with bleach the media suction pipe and empty the vacuum flask. - Turn off the water bath and the microscope. - Clean the work surface of the hood and turn on the UV ramp for 15 minutes.

C- CELL CULTURE MEDIA

Composition of some culture media (in mg/L) and importance of the components Media: DMEM HAM 12 RPMI Mineral salts CaCl2 anh (2H2O) 200 44 - Fe(NO3)3, 9H20 0,1 - - KCl 400 223,6 400 MgCl2, 6H20 - 122 - MgSO4, 7H20 (anh) 200 - 100 NaCl 6400 7599 6000 NaHCO3 3700 1176 2200 NaH2PO4, 2H2O 145 - - NaH2PO4, 7H2O - 268 1512 KNO3 - - - NaSeO3, 5H2O - - - CuSO4, 5H2O - 0,00249 - FeSO4, 7H20 - 0,834 - ZnSO4, 7H2O - 0,863 - CaNO3, 4H2O - - 100 Essential amino acids L arginine-HCl (base) 84 211 200 L cysteine base - - - L cystine (2HCl, HCl, H2O) 48 35,12 50 L glutamine 584 146 300 L histidine-HCl, H2O (base) 42 20,96 15 L isoleucine 105 3,94 50 L leucine 105 13,1 50 L lysine-HCl 146 36,5 40 L methionine 30 4,48 15 L phenylalanine 66 4,96 15 L threonine 96 11,9 20 L thryptophan 16 2,04 5 L tyrosine (Na2) 72 5,4 20 L valine 94 11,7 20 Non essential amino acids L alanine - 8,9 - L asparagine - 15,01 50 Acid L aspartic - 13,3 20 Acid L glutamic - 14,7 20 Glycine 30 7,5 10 L proline - 34,5 20 L hydroxy-proline - - 20 L serine 42 10,5 30 Vitamins and other cofactors Biotin - 0,0073 0,20 D pantothenate of Ca 4 0,48 0,25 Choline-HCl 4 13,96 3 Folic acid 4 1,3 1 Inositol 7,2 18 35 Nicotinamide 4 0,04 1 Pyridoxal HCl 4 0,062 - Riboflavin 0,4 0,038 0,2 Thiamine HCl 4 0,34 1

Vitamin B12 - 1,36 0,005 Pyridoxine HCl - 0,062 1 p-aminobenzoic acid - - 1 Other components D glucose 4500 1802 2000 Lipoic acid - 0,21 - Pyruvate of Na 110 110 - Hypoxanthine - 4,1 - Linoléic acid - 0,084 - Putrescine, HCl - 0,161 - Thymidine - 0,73 - Glutathion reduced - - 1 Bactopeptone - - - HEPES - - -

- Mineral salts

7 ions are essentials for the cells: sodium, potassium, calcium, magnesium, phosphate, carbonate and chloride. They help to maintain the osmotic pressure and are significantly involved in membrane transports and metabolisms. Na+, K+, Ca++ and Mg++

play a role in maintaining membrane potentials, enzymatic reactions as well as attachment and spreading of cells on their support. - Amino acids

Precursors for the protein synthesis, 12 AA are needed: 8 are essentials and 4 others (tyrosine, lysine, arginine and histidine) can’t be biosynthesized in vitro. Glutamine belongs to the essential amino acids. It is a precursor for the synthesis of nitrogen bases. It must be added to the culture medium immediately before use, as it is very instable in aqueous medium. - Vitamins

Vitamin requirements vary according to the cell types. The eight essential vitamins for cultured cells are: choline, folic acid, pyridoxal, riboflavin, thiamine, inositol, nicotonic acid and pantothenic acid. - Energetic substance

The core energetic substance is the D-glucose used mostly at 1g/L (5,5 mM).

- Buffer systems

They maintain a constant pH in the environment by neutralizing the acid and alkaline compounds which may appear due to the cell metabolism: - HCO3

-/CO2: depends on the concentration in HCO3- and in CO2 gas (5 to 10 %). - HEPES: in addition to the HCO3

-/CO2 system, it can target and keep a physiological pH (7,4). The pH of the medium is visualized thanks to a coloured indicator present in the medium: phenol red, which turns yellow in acidic condition (pH<6), and purple under basic condition (pH>8).

D- LAMINAR FLOW HOOD

A laminar flow hood (also called microbiological safety cabinet) is an enclosed, ventilated laboratory workspace for safely working with materials contaminated with (or potentially contaminated with) pathogens. The cabinet has a manipulation chamber partly open at the front. It is provided with an airflow device for protection of: - the laboratory worker, by driving the air flow away from the worker - and the environment, by evacuation of the air flow out of the cabinet through a very high efficiency filter. Type 2 PSM (conventionally used for cell culture) provides in addition the protection of the sample or experiment against contamination using a uniform and unidirectional airflow (laminar flow) directed downwards, through a very high efficiency filter. An airflow created at the front edge of the work surface forms a barrier between the worker and the experiment. An amount of air equal to the one that forms this barrier is exhausted from the chamber after filtration through a very high efficiency filter. The laminar flow is ensured by the geometry of the manipulation chamber and the air plenum upstream of the filter and also by a homogeneous distribution of the flow velocity. The flow intakes into the aspiration panels occurs at the front and at the rear of the work surface and then blown by the fan in the air plenum and directed to the exhaust filter or again in the manipulation chamber after filtration.

filters Front airflow intake grid

air

E- PRINCIPLE OF THE POLY-ACRYLAMIDE GEL ELECTROPHORESIS OF

PROTEINS

Electrophoresis is a technique of separation of proteins in a polyacrylamide gel submitted to an electric field. Acrylamide forms a crosslinked mesh that slows down the progression of the proteins in the electric field, proportionely to their size (smaller molecules are less retained by the mesh than larger ones). The polymerization of acrylamide is not spontaneous and therefore requires the addition of catalysts in the solution. Usually two types of catalyst must be combined: a reaction initiator: ammonium persulfate (APS) and an accelerator: TEMED (N,N,N',N'-tetramethylethylenediamine).

In practice, two electrophoresis gels are stacked (Figure 9): the first one is called stacking gel (or upper gel) and the second one separating gel (or lower gel). They differ in their percentage of acrylamide (4-5%, 7-15%, respectively) and in their pH (6,8 and 8,8 respectively). In the upper gel, wells are created for loading samples. The running buffer contains Tris-HCl and glycine. An important point is to obtain protein bands thin enough to allow a good resolution. One function of the stacking gel is precisely to concentrate the sample before it enters the separating gel. At pH 6.8, Cl- ions have the highest mobility and migrate faster. Proteins have a intermediate mobility and glycine ions (mostly zwitterionic), which have the lowest mobility, migrate more slowly. However, migration of Cl- ions causes a decrease in the gel conductivity behind them, which accelerates the migration of glycine ions. This results in a concentration ot the proteins between the Cl- ions ahead and glycine ions behind.

In native conditions, the only charges of the proteins are those of the lateral chains of amino acids (Lys, Arg, His, Glu, Asp), and those of the N-and C-terminal moieties. Under these conditions, protein migration depends on both their charge (different for each protein based on the amino acid composition) and their size, to pass through the acrylamide mesh pores.

In denaturing conditions, an anionic detergent such as sodium dodecyl sulfate (SDS: CH3-(CH2)10-CH2OSO3

— Na+) is highly bound to proteins (1.4 g of SDS per g of protein) and gives the complex a heavy negative charge, the sulphate ions one. The protein intrinsic charge becomes insignificant and the charge density (charge/mass ratio) becomes almost the same for all proteins. Under these conditions, the size of the protein is the only factor that determines the migration speed through the gel pores. This is why we can use this technique, not only to physically separate the various proteins in a sample, but also to determine their apparent molecular weight. This is done using standard proteins of known molecular weights.

It should also be noted that the protein sample is usually submitted to a reducing treatment (2-mercaptoethanol or DTT) that enables breaking intra- or inter-chain disulfide bonds. Reduction of intra-chain bridges has no significant effect on mobility. On the other hand, reduction of inter-chain bridges is essential to break the disulphide bonds between subunits, when they exist, and thereby allow the separation of subunits (Figure 17).

Figure 17: Migration of denatured proteins in a SDS-PAGE gel

Figure 18: Relationship between the molecular weight logarithm and the migration distance

Figure 18 shows the migration of a standard proteins mixture in a 12% acrylamide gel. The relationship between the logarithm of the molecular weight and the migration distance is equivalent to a straight line, at least in a given interval. Knowing the migration distance of a protein in a given gel, we can graphically determine its apparent molecular weight. We talk about apparent molecular weight because the density of the SDS binding is not exactly the same for all proteins and therefore can affect the speed of migration. In addition, some post-translational modifications such as phosphorylation may cause a significant change in the density of SDS binding and lead to important variations in migration speed. This is why, in some cases, phosphorylated and non-phosphorylated forms of the same protein can give apparent molecular weights differing by more than 5 kDa, whereas the mass of phosphate is only 80 Da. An interesting illustration of this will be shown in this practical course.

F- OPERATING PRINCIPLE OF A FLOW CYTOMETER

1. Equipment Figure 19:

2. Choice of parameters for the DNA content quantification

Figure 20

Doublets removal

Electric pulse analysis Signal generation

DNA content quantification

G- OPERATING PRINCIPLE OF A FLUORESCENCE MICROSCOPE It is a microscope equipped with one or more light sources (arc lamp, laser, etc.) and filters to select the excitation and emission wavelengths of the fluorescent markers used.

Figure 21 – Fluorescence microscope The light source is usually an arc lamp with high pressure of mercury or xenon vapor. The emitted light is polychromatic: the excitation wavelength is selected by means of the excitation filter. The light is then reflected by a dichroic mirror15 towards the objective lens which focuses the light beam on the sample. The fluorescence emitted by the sample goes back through the objective lens towards the dichroic mirror and passes through the latter. The blocking filter allows only the wavelengths for observation. Image is seen through the oculars or saved using a camera or a video camera. To each fluorescent marker corresponds a set of excitation filter + dichroic mirror + blocking filter: in all recent fluorescence microscopes these 3 elements are contained in a cube, that needs to be changed depending on the marker you want to observe. In the diagram above, the sample is illuminated using the objective lens to concentrate the light. This system, called "epi-illumination", provides high intensity light with objective lenses having a good numerical aperture (x20 and beyond). When using low magnification objectives (x10 or less) which generally corresponds to very low numerical apertures, the illumination of the sample is weak. In this case, we get a more intense light by "trans-illumination": rays (drawn in dotted lines) are focused on the sample by the condenser of the microscope, the excitation filter is placed under the sample, the blocking filter above and the dichroic mirror is no longer necessary.

15 A dichroic mirror is reflective to certain wavelengths and transparent to others.

Scholar year 2012/2013

MASTER IN LIFE SCIENCES AND TECHNOLOGIES MAJOR IN MOLECULAR AND CELLULAR BIOLOGY

OBLIGATORY COURSE BMC401 "METHODS IN MOLECULAR AND CELLULAR BIOLOGY"

CONSTRUCTION AND FUNCTIONAL SCREENING OF

A BACTERIOPHAGE LAMBDA GENOME LIBRARY

Pedagogic responsibility: Laure BIDOU ([email protected]) and Mathilde GARCIA ([email protected])

Teaching location: Genetics teaching department -‐ Building B, 3rd floor Administrative support: Carine JOSEPH; tel: 01 44 27 35 35; e-‐mail: [email protected]

Proposed courses for the second semester of Genetics (M1S2): BMC 437: Genetic approaches of normal development and pathologies BMC 439: Molecular and medical bacteriology BMC 440: Cellular genetics BMC 441: Evolutionary and human genomics BMC 442: Introduction to epigenetics BMC 414: Molecular cancerology BMC 420: Populational genetics BMC 421: Molecular genetics: the E. coli model BMC 422: Multifactorial genetics and human diseases BMC 434: Genome structure and dynamics BMC 452: Bioinformatics and exploratory genomics BMC 457: Cancerology: experimental approaches M2 in Genetics, orientation toward Research M2 GCC: Genetics of complex systems (from yeast to humans) BMC 521: Genome analysis BMC 513: Mice genetics BMC 521: Molecular and cellular genetics M2 in Genetics, orientation toward Professional degree M2 GGB: Genetics and management of biodiversity

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PROGRAM OF THE PRACTICAL COURSE MONDAY (9h – 18h):

- Introduction - Enzymatic digestion of bacteriophage λ DNA and pUC19 vector - Ligation of bacteriophage λ and pUC19 DNA fragments - Preparation of agarose gel

TUESDAY (9h – 19h): - Verification of DNA digestion and ligation by gel electrophoresis - Preparation of competent bacteria DH5α and DH5β - Transformation of bacteria with ligation products and functional screening

WEDNESDAY (9h – 18h): - Analysis of pUC19 and bacteriophage λ DNA maps - Interpretation of gel electrophoresis results - Analysis and interpretation of transformation results - Liquid culture of clones obtained by transformation

THURSDAY (9h – 19h): - Plasmid extraction using Alkaline Lysis Mini-Prep Protocol - Restriction analysis of recombinant plasmids (mapping) (part I) - Resistance test of selected clones to bacteriophage λ infection - Interpretation of results

FRIDAY (9h – 18h):

- Restriction analysis of recombinant plasmids (part II) - Interpretation of results - Summary of the DNA library and of the functional screen - In silico analysis of cloning - General discussion

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CONSTRUCTION OF THE GENOMIC LIBRARY -‐ EXPERIMENTAL APPROACH

The aim of this practical course is to obtain a representative library of the bacteriophage λ DNA (see Annex, page 24 for definition) and to identify the phage genomic locus that once integrated into host bacteria confers resistance to multiple bacteriophage λ infections (immunity to super-‐infection).

During the course you will get familiar with some molecular biology/microbiology techniques: DNA restriction and cloning (in vitro and in silico); transformation of bacteria with plasmid DNA, bacterial culture, plasmid DNA extraction from bacteria, infection of bacteria with bacteriophage λ.

The experimental work is organized as follows:

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VECTOR pUC19

A plasmid pUC19 is a bacterial vector, 2686 base pairs in length (see the detailed plasmid map in Annex, page 25), used for the construction of DNA libraries. It contains a fragment of the pBR322 plasmid, which carries the β-‐lactamase gene conferring resistance to ampicillin (gene AmpR) and an origin of replication (ORI). Various unique restriction sites allowing cloning of DNA fragments are assembled in a 59 base pairs long sequence called Multiple Cloning Site (MCS).

This artificial sequence was introduced at the beginning of a coding region of the gene lacZ from Escherichia coli: this region, lacZα, is responsible for the synthesis of the N-‐teminal part of β-‐galactosidase (α-‐peptide), under the control of the promoter/operator of the lactose operon (P/O lacZ). The MCS does not contain a STOP codon in the open reading frame defined by the translational START codon ATG of the lacZ gene. Under these conditions, the presence of pUC19 in a bacterial cell leads to the production of the α-‐peptide, lengthened by few additional amino acids encoded by the nucleotide sequence of the MCS. This peptide on its own does not posses the enzymatic activity of β-‐galactosidase, however, can confer this activity in genetically engineered bacteria containing the C-‐terminal part of β-‐galactosidase (ω-‐peptide). Restoration of the β-‐galactosidase activity by interaction of α+ω peptides is called α-‐complementation. After transformation of E.coli with pUC19, β-‐galactosidase activity can be detected in each individual bacterial clone by letting cells grow on a medium containing X-‐gal (5-‐bromo-‐4-‐chloro-‐3-‐indolyl β-‐D-‐galactopyranoside), a substrate of β-‐galactosidase which when metabolized renders bacterial colonies on agar plates blue.

When a DNA fragment (insert) is integrated in the MCS, the synthesis of α-‐peptide is no longer possible. By the consequence, there is no α-‐complementation and the bacterial colonies remain white in the presence of X-‐gal. Blue/white screening of bacterial colonies permits only the selection of clones corresponding to a recombinant plasmid (pUC19 + another DNA fragment) and elimination of clones containing an empty vector. A. β-‐Galactosidase catalyzes hydrolysis of β-‐galactosides (such as lactose) into monosaccharides

(galactose and glucose): B. β-‐galactosidase also catalyzes hydrolysis of an artificial β-‐galactoside called X-‐gal: C. α-‐Complementation scheme:

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BACTERIAL STRAINS Two bacterial strains of Escherichia coli are used during this practical course: DH5α : F -‐, gyrA96, hsdR17, recA1, endA1, supE44, thi-‐1, ∆(lacZYA-‐argFV169), (ph80dlacZ∆M15) DH5β : derivative of the strain DH5α

It has the same genotype as DH5α but in addition contains a plasmid pTP, a derivative of pBR322, carrying a tetracycline resistance gene and a mutation within the lacI gene promoter, named lacIq. This mutation results in increased gene transcription and, hence, in super-‐expression of the lactose repressor in bacteria.

BACTERIOPHAGE λ

The bacteriphage λ is a temperate parasite virus of Escherichia coli. Actually, upon infection, it can choose between two propagation strategies:

-‐ it can multiply independently of its host genome and generate new infectious particles, finally leading to the lysis of infected bacteria – this is called a lytic cycle.

-‐ under certain conditions the phage DNA can integrate itself into the bacterial chromosome in the form of a prophage – this is called a lysogenic cycle. In this case the replication of the phage genome is conditioned by the replication of the bacterial genome.

The choice between two propagation strategies is a result of a complex regulation conducted by at least 4 viral proteins, products of genes cI, cII, cIII et cro respectively, as well as interactions between some of them with bacterial proteins, products of genes hflA and hflB (hfl stands for high frequency of lysogeny). As a result of these interactions the virus can best adapt its life cycle to the physiological state of the infected bacteria. Details of these regulation mechanisms were presented elsewhere (see notes of the L2 lectures “Cell regulation”, LV205).

The genome of bacteriophage λ represents a double-‐stranded DNA 48502 base pairs (bps) long (see the detailed mapin Annex, page 36). It can exist in two forms:

-‐ in a linear form in the viral particle, -‐ in a circular form in the infected bacteria.

This circularization of the genome upon infection is facilitated by the presence of single-‐stranded 12 nucleotides long cos sequences present at each extremity of the λ genome.

The λ genome has been entirely sequenced. This sequencing revealed the existence of a certain number of genes defined by the open reading frames (ORFs) for which it has not been possible to isolate mutants. By consequence, the function of these genes is unknown. During this practical course we are going to explore the interest of different cloning techniques to study such genes.

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DAY 1

ENZYMATIC DIGESTION of PHAGE λ DNA and pUC19

Each pair of student receives: - One Eppendorf tube containing 4 µl of λ DNA at 500 ng/µl = tube λ - One Eppendorf tube containing 4 µl of pUC19 DNA at 500 ng/µl = tube pUC19 Reaction mixes are done directly in these tubes. Verify that each tube indeed contains 4 µl of the desired reagent!

λ DNA digestion by the restriction enzyme BamHI, tube "λ BamHI" - DNA: 4 µl - 10x Restriction buffer B: 2 µl - H2O, sterile: 12 µl - BamHI (10 U/µl): 2 µl (added by the supervisor) Final Volume 20 µ l

Incubate for 2 hours at 37°C in a water bath.

pUC19 DNA digestion by the restriction enzyme BamHI and dephosphorylation, tube "pUC19 BamHI+P"

- DNA: 4 µl - 10x Restriction buffer B: 10 µl - H2O, sterile: 82 µl - BamHI (10 U/µl): 2 µl (added by a supervisor) - Phosphatase (1 U/µl): 2 µl (added by a supervisor) Final Volume 100 µ l

Incubate for 2 hours at 37°C in a water bath.

PRECIPITATION of DIGESTED DNA

At the end of the reaction each digested DNA is precipitated, on one hand to remove salts and on the other hand to reduce the volume:

- Add: 180 µ l of sterile ΔH2O to the tube "λ BamHI" 100 µ l of sterile ΔH2O to the tube "pUC19 BamHI+P" - Adjust salt content to 0.25 M NaCl by adding 1/20 of the volume of 5M NaCl mix the content of each tube. - Add 2 volumes of 100% ethanol and mix by inverting tubes several times. - Keep tubes for at least 30 minutes (during the lunch break) at -20°C (precipitation). - Centrifuge 15 minutes at 12 000 rpm: to know where the pellet is after centrifugation it is important to place tubes in the rotor with the cap attachment site toward the exterior. - Verify the presence of DNA pellets and carefully eliminate the supernatant by the help of a Pipetman P200: the pellet is only loosely attached to the tube wall. - Add 1 ml of 70% ethanol to the DNA pellet (wash). - Centrifuge again 10 minutes at 12 000 rpm. - Eliminate the supernatant and let it dry in 50°C incubator. VERY IMPORTANT: the pellet should be completely dry without any traces of alcohol!

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- Resuspend: - λ DNA in 20 µ l of sterile H2O concentration = < 100 ng/µl - pUC19 DNA in 100 µ l of sterile H2O concentration = < 20 ng/µl

LIGATION

Important note: The objective of a ligation is to obtain a maximum number of recombinant plasmids containing a single fragment (insert) of the bacteriophage λ genome. Factors determining the efficiency of ligation: 1) compatible 5’/3’ ends. For this purpose λ DNA and pUC19 should be digested by the same or compatible restriction enzyme(s); 2) dephosphorylated 5’ ends of pUC19 to limit vector re-circularization; 3) optimal λ DNA and pUC19 ratio in the ligation mixture to increase the probability that one molecule of the plasmid encounters only one molecule of the λDNA to be cloned. This ration is usually determined experimentally. In our case the best compromise is obtained by providing a 20-fold access of λ DNA fragment’s extremities over plasmid’s extremities; 4) optimal total DNA concentration that determines a speed of the reaction and a quantity of the enzyme necessary to accomplish the ligation.

- Prepare 3 Eppendorf tubes: L (ligation), C1 and C2 (controls) as follows:

- Incubate at 16°C over night.

QUESTION: What for the two controls, C1 and C2, are needed? - Save your tubes "λ BamHI" and "pUC19 BamHI+P" at 4°C for tomorrow to use in a control gel electrophoresis.

AGAROSE GEL ELECTROPHORESIS: Gel preparation

- Prepare 0.5X TBE from 5X TBE stock provided by a supervisor (2L for the entire class). - Pour 40 ml of 0.5XTBE in an erlen of 500 mL. - Weight the quantity of agarose necessary to make a 0.8% agarose gel and add this agarose to

the erlen containing the buffer. Note the total weight of the erlen with the content! - Cover the elren by inserting a smaller erlen (25 ml) upside down. - Heat the mix in the micro-oven until the agarose is completely dissolved.

Supervise the heating to prevent the overflow and evaporation of the solution. - Weight the erlen and correct for the evaporated liquid by adding water.

7

L C1 C2

Ligation buffer 10X 2.5 µl 1 µl 1 µl

λ BamHI (< 100 ng/µl) (your own tube) 8 µl - -

pUC19 BamHI+P (< 20 ng/µl) (your own tube) 2 µl 4 µl 4 µl

Sterile water 10.5 µl 7 µl 6 µl

T4 DNA ligase (1 U/µl) (added by the supervisor) 2 µl - 1 µl

Final volume 25 µl 12 µl 12 µl

Optionally: if you do not want to bother with the weight correction, prepare 0.7% agarose. Due to evaporation the final concentration will be about 0.8%

- Let the mix cool on the bench for 5 to 10 minutes. - Prepare the electrophoresis chamber by scotching up the two ends of the plexi tray and place

a comb (one or two combs per tray, see with a supervisor) paying attention to its position (low for our experiment).

- Put the plexi chamber on a paper towel in case it leaks. - Pour the gel (thickness = ± 4 mm) while avoiding bubbles. It is highly recommended to supervise

gel solidification at the beginning to detect any eventual leaks and pour another gel if necessary.

- Let the agarose solidify for about 30 min. - Cover the gel with 10 ml of 0.5X TBE buffer to prevent gel drying and store over night at 4°C

(see with your supervisor).

PREPARATION of COMPETENT BACTERIA: Bacterial pre-culture

Each pair of students prepares only one single strain of competent bacteria: DH5α or DH5β.

Prepare culture media (one person for the entire group) - Take an erlen containing 50 ml medium LB and under sterile conditions complement the

medium with 10 mM MgSO4 and 0.4% maltose (final concentrations, see Annex, page 26 for concentrations of stock solutions) this LB-Mg-maltose medium is used for DH5α culture.

- Take out 25 ml of the complemented LB-Mg-maltose medium into a Falcon-50 ml tube and add tetracycline to a final concentration of 15 µg/ml for DH5β culture.

Bacterial pre-culture (each pair of students) - In a Falcon-15 ml tube put 4 ml of the culture medium required for the strain you are using,

either DH5α or DH5β - Inoculate this culture medium with one isolated bacterial colony of DH5α or DH5β, taken from

a Petri LB plate using a metallic loop heat sterilized and cooled (or a sterile yellow tip). - Incubate this pre-culture at 37°C over night with agitation in an incubator. - Put one erlen containing 50 ml of LB medium over night at 37°C (for tomorrow’s culture).

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DAY 2

AGAROSE GEL ELECTROPHORESIS: Gel migration

Sample preparation The quality of DNA digestion by BamHI and ligation are verified by agarose gel electrophoresis. Untreated samples, pUC19 (50 ng/µl) and λ (100 ng/µl) DNA should be loaded on the same gel. These samples are supplied by your supervisor and already contain 2 µl of native pUC19 or undigested λDNA, respectively. Directly add sterile water and sample-loading buffer.

o Prepare 7 Eppendorf tubes according to this table:

Aliquot Sterile water 5X samples loading buffer

Load

pUC19* 2 µl 6 µl 2 µl 10 µl pUC19 BamHI+P 7 µl 1 µl 2 µl 10 µl C1 6 µl 2 µl 2 µl 10 µl C2 6 µl 2 µl 2 µl 10 µl L 7 µl 1 µl 2 µl 10 µl λ BamHI 6 µl 2 µl 2 µl 10 µl λ* 2 µl 6 µl 2 µl 10 µl

* Provided by your supervisor - For calibration, load 8 µl of the molecular weight marker called “1kb ladder” (ready to use solution, nothing to add, see Annex - page 26 for composition). Keep the rest of the marker aliquot at -20°C for future use.

Sample loading and migration - Take your agarose gel from the fridge, remove scotch and place the gel including the support

in the electrophoresis running tank. Pay attention to the orientation of the combe and electrodes!

- Fill the tank with the running buffer (0.5X TBE) to slightly cover the agarose gel. - Carefully remove the comb: the wells shall never remain dry. - Deposit samples by the aid of a Pipetman P20 and change a tip between every sample. - Connect the tank to a power supply. Pay attention to the polarity! - Migrate at a constant voltage: 80 V (about 30 mA/tank) for approximately 1h.

Gel staining and image acquisition

ETHIDIUM BROMIDE IS EXTREMELY GENOTOXIC. WEARING GLOVES WHILE MANIPULATING GELS IS MANDATORY!

- Delicately slide your gel in a container with a staining solution (ethidium bromide at 0.5 mg/ml in water or 0.5X TBE prepared by supervisors). Pay attention not to brake your gel!

- Let gels in the staining solution for at least 30 min (during a lunch pause) in obscurity. - Take your gel from the staining solution and rinse it briefly with running water. - Slide the gel on the UV transilluminator.

9

The UV waves (330 nm) cause skin and eye damage! When observing gels always close the plexi cover of the transilluminator or wear special glasses or a full facial shield.

- Observe your results directly on the transilluminator and take a photo through the

videocamera. Verify a quality of the image before trashing the gel to a yellow trash container for toxic waste.

PREPARATION of COMPETENT BACTERIA

Bacterial culture - Sterilely complement 50 ml of LB medium (left yesterday night at 37°C) with the supplements:

10 mM MgSO4, 0.4% maltose and 15 µg/ml tetracycline when necessary as the 1st day (see Prepare culture media). Each pair of students needs to prepare its own medium. Add supplements directly to the LB medium in an erlen of 500 ml. Pay attention whether you need to add the antibiotic or not!

- Inoculate this medium with an aliquot of the over-night pre-culture, which has grown to saturation (stationary phase):

- 0.75 ml of DH5α - 1.5 ml of DH5β - Incubate at 37°C with agitation in the incubator until the optical density at 600 nm (OD600)

equals 0,5 (reached in about 2h30min).

- Regularly monitor OD after 2h of culture incubation: prepare a blank for the spectrophotometer by placing 1 ml of LB medium in a disposable plastic cuvette (one for the entire class). In a second cuvette, pipet 1 ml of your culture. Attention: Immediately return your cell culture in the incubator! Also note that you are supposed to work in sterile conditions!

From this step on, ALL operations are done on ice since the temperature of competent bacteria shall never be above 4°C.

All solutions, tubes used for centrifugation, and centrifuges need to be imperatively pre-cooled in advance.

Preparation of chemically competent bacteria by the method of calcium chloride

- Prepare T1 solution (one person for the entire group) by completing given T1 volume with MnCl2 to final concentration of 50 mM (see Annex, page 26 for composition and concentration of stock solutions). You will need 15 ml of T1-MnCl2 for each pair of students/bacterial culture. Keep on ice.

- Cool down the bacterial culture by placing your erlen in an ice-water bath for 10 min (ice + some water for better conductivity).

- Pour the cooled down culture in a pre-chilled centrifugation tube with a round bottom (tubes hold about 35 ml and you will not be able to transfer the entire culture, do not overfill the tubes since the content may spill during centrifugation).

- Centrifuge 10 min at 4°C and 2 500 rpm, then discard the supernatant into a liquid waste bin. - Delicately tap the tube to disaggregate cells (do not keep cells outside of ice for more than

few seconds at the time) Never vortex: bacteria are extremely fragile!

- Delicately resuspend the bacterial pellet in 1 ml of ice-cold T1-MnCl2 solution by delicately rotating the tube on the surface of ice (see with a supervisor).

10

- When bacteria are resuspended, add the rest 14 ml of the ice-cold T1-MnCl2 solution to bacteria.

- Incubate bacteria on ice for at least 1 hour (during a lunch pause). - Centrifuge 10 minutes at 4°C and 2 500 rpm, then carefully eliminate the supernatant.

Attention: do not discard the T1-MnCl2 solution in a liquid waste bin containing bleach (toxic fumes form if you do)! Discard the supernatant in a Falcon-50 ml and

than trash in the sink.

- Delicately tap the tube to disaggregate cells (do not keep cells outside of ice for more than few seconds at the time).

- Add 1 ml of ice-cold T2 solution (see Annex for composition, page 26) and gently rotate the tube on ice to resuspend cells.

- Transfer cells into a pre-chilled Eppendorf tube and snap freeze in dry ice/ethanol solution (-78°C) prepared by supervisors. Leave in dry ice until you are ready for bacteria transformation.

TRANSFORMATION

Set one water bath to 42°C. Each pair of students is transforming only the bacteria strain they have prepared with the ligation (tube L) and controls (tube C1, tube C2). We will also set up a negative control (tube N) and a positive control (tube P) for transformation. For experimental procedure see the scheme below (page 14).

QUESTION: Why these two controls are needed: what information will they give?

How can they validate your experimental results? The bacterial transformation is done in the following successive steps: Step 1: Absorption and penetration of transforming DNA into bacteria

- Defreeze competent bacterial cells on ice.

- Prepare 7 pre-chilled Eppendorf tubes and deposit each transforming DNA at the bottom of the tube as indicated in the table below:

Tube name Transforming DNA

Volume Cells: DH5α or DH5β Volume

Nα or Nβ - 100 µl C1α or C1β 4 µl of C1 100 µl C2α or C2β 4 µl of C2 100 µl L1α or L1β 4 µl of L 100 µl L2α or L2β 4 µl of L 100 µl L3α or L3β 4 µl of L 100 µl Pα or Pβ 3 µl of native pUC19 (100 pg/µl) 100 µl

VERY IMPORTANT: Change a tip between every tube while distributing the DNA.

- Carefully mix defrosted competent bacteria and distribute in different tubes, 100 µl in each.

VERY IMPORTANT: Change a tip between every tube while distributing the competent bacteria to prevent the mix up between different tests.

11

- Let incubate transformation tubes 25 min on ice (absorption phase). - During this incubation time, pre-heat 4 ml of LB medium in a water bath set to 37°C. - Perform a heat shock of bacteria by putting transformation tubes at 42°C for 90 seconds. - Put tubes immediately back on ice.

Step 2: Expression phase

- Add to each tube 400 µl of LB medium pre-heated to 37°C. - Carefully resuspend bacteria by inverting each tube few times. - Incubate tubes at 37°C for 30 minutes.

At this point, each transformation tube contains a final volume of 500 µ l. - For four tubes, P, L1, L2 and L3, half part (250 µl) is going to be used for the functional screen (see Functional screen) and the rest half is going to be spread on LB plates (see Spreading). - For tubes N, C1 and C2 you can immediately proceed to Spreading instructions (see below).

FUNCTIONAL SCREEN

Only tubes P, L1, L2 et L3 are going to be used for an infection of bacteria by the mutant bacteriphage λcI ts857.

- Prepare 4 Eppendorf tubes containing 50-70 µl of mutant phage λcI suspension (titer = ± 1010 pfu/ml): under these conditions, the multiplicity of infection (number of phages per bacterium) is between 2 and 5.

- For the strain DH5α, add to each tube containing the phage well-resuspended bacterial cultures: • + 250 µl of cells from the tube Pα = Tube Pαλ

• + 250 µl of cells from the tube L1α = tube L1αλ • + 250 µl of cells from the tube L2α = tube L2αλ • + 250 µl of cells from the tube L3α = tube L3αλ

- For the strain DH5β, perform the same operation (tubes: Pβλ, L1βλ, L2βλ, L3βλ, respectively). - Incubate the four tubes of infected transformed bacteria for 15 minutes at 37°C without agitation (the absorption phase of bacteriphage on bacteria). Then proceed to the Spreading on LB agar plates.

SPREADING of BACTERIA on LB AGAR PLATES

- Start by identifying LB agar plates: LB agar plates color code (on the side): LBA = 1 line, LBAX = 2 lines, LBAT = 3 lines, LBATX = 4 lines. - Take appropriate plates. A pair of students will need 12 plates in total: 1 plate of LBA and 11 plates of LBAX (for DH5α) or 1 plate of LBAT and 11 plates of LBATX (for DH5β)

Important: check the absence of contaminations (bacteria or fungi) on the agar surface of plates! - Label all plates in advance (see the table below). Write on the bottom of the agar side in one corner (not all over the plate since you will count colonies later). - In advance, sterilely deposit about ten glass beads on the surface of each LB agar plate. - Add the bacterial suspension as indicated in the table.

Pay attention to the correspondence: tube - plate! Change a tip between every tube!

12

tube Quantity to deposit

LB plates for DH5α

LB plates for DH5β

Incubation T°C

N all* LBA LBAT C1 all* C2 all* L1 all* L2 all* L3 all* P50 50 µl of the tube P P200 200 µl of tube P

LBAX LBATX

37°C

L1λ all* L2λ all* L3λ all* Pλ all*

LBAX LBATX 42°C

* To deposit all cells from 250-500 µl of culture centrifuge bacteria 2 min at maximum speed, verify the presence of a cell pellet and eliminate all but 100µl of the supernatant. Gently resuspend all the bacteria in this remaining volume and spread the totality on plates.

- Close the plate and horizontally shake it back and forth allowing beads to cross the agar

surface. It is recommended to proceed with spreading by a set of 6 plates. - Let plates dry, then remove beads in the metalic container bin. - Put plates in the incubator at the appropriate incubation temperature, lead side down to avoid

condensation of liquid on the agar surface.

13

Scheme. Experimental procedure of bacteria transformation, functional screen and spreading of transformed bacteria on LB agar plates.

14

DAY 3

ANALYSIS of GEL ELECTROPHORESIS RESULTS

Results analysis constitutes an essential and very important part of the experimental work: it should clearly appear in your lab book and to be well structured and explicit to demonstrate your scientific approach and analytical skills! To help you in this work, please follow our recommendations:

Gels annotation: The photography of the gel should be stuck in the lab book and appropriately annotated to give the following information:

o Identity of each sample loaded in each lane; o DNA quantity (ng) loaded in each lane.

Critical analysis of the obtained results: The analysis of migration profiles of each sample should allow you to answer the following questions:

o The efficacy of enzymatic digestion of pUC19 and λ DNA; o The efficacy of ligation; o The efficacy of dephosphorylation of digested pUC19.

To answer each question you should proceed in three steps: (1) Observation: description of all bands that you observe (number; position comparing to the

1kb ladder and other samples, if needed; intensity) (2) Interpretation: analysis of migration profiles given the experimental procedure and

conditions you have used (DNA configuration: linear or circular, fragment’s size etc) (3) Conclusion: this part should summarize and bring answers to all questions you addressed in

this study. Note: don’t forget to be critic in the analysis and interpretation of the results and take into account limits of the used experimental approach (sensibility, resolution, etc).

In silico analysis of the bacteriophage λ genome: Interpretation of the experimental results will require some additional information that you can obtain thanks to in silico (informatics) analysis of the bacteriophage λ genome. For this purpose, you will use a special software “ApE” to extract further information:

o Number and size of fragments generated by the enzymatic digestion of λDNA by BamHI; o Number of λDNA-BamHI fragments that could be cloned into pUC19-BamHI; o Number of all possible recombinant plasmids obtained after ligation of λDNA-BamHI and pUC19-BamHI.

To perform the in silico analysis you can use a computer in the computer classroom: - Switch on the computer and log-in in the “etudiant” session. - Select a folder “TP master” and copy it into a desktop under your own initials. Go in the menu

"Fichier", than "Dupliquer" or press “cmd+D” or “⌘+D”. A new folder "copie de TP master" will appear on the desktop. Rename the folder by adding your initials.

- By double click to the icon, open your folder and check if the folder contains three files: lambda, pUC19-fragmentIVA and pUC19-fragmentIVB.

- Open the ApE by double clicking on the icon ApE on the desktop. - Open the file “lambda” within the ApE through: File > Open > ”Your folder” > lambda.

15

The sequence of the λDNA appears in a window in a conventional form: one strand in orientation 5’->3’. You can find some useful information in the upper part of the window: size, DNA configuration: linear or circular.

Note: You can modify DNA configuration, if needed, by a click on the corresponding key. - ApE allows you to find restriction sites within the DNA thanks to menu “Enzyme” and the

following functions:

menu ENZYME FUNCTION UTILITY ENZYME SELECTOR Allows the choice of a restriction enzyme

GRAPHIC MAP Displays the scheme of the DNA (linear or circular) with the position of restriction sites of a given enzyme

DIGESTION Gives DNA fragments size after a total digest of the DNA by the given enzyme. Here, the result depends on DNA configuration!

Use these functions to find BamHI restriction sites within the λDNA and size of fragments following the BamHI digestion. - Use the in silico analysis to interpret your gel elecrophoresis results and to conclude about the

configuration of the λDNA (linear or circular) and a number of λDNA-BamHI fragments that could be cloned into pUC19-BamHI.

Analysis of genetic maps of pUC19, without and with λDNA fragments:

The ApE software allows you to perform in silico cloning of the plasmid pUC19 and λDNA after digestion with BamHI. The results of this experiment, that is, all possible recombinant plasmids are present in Annex, pages 28-32. Analysis of the recombinant plasmids will allow you to:

o Establish a protocol for plasmids mapping: digestion of recombinant plasmids by restriction ezymes to identify each cloned λDNA fragment.

o Interpret the results of the plasmid mapping and of the functional screen later on. To facilitate the analysis you should annotate all recombinant plasmids with the following information:

(1) sites of BamHI; (2) positions of the pUC19 backbone and the λDNA fragment; (3) size of the λDNA fragment; (4) position of all genetic elements of pUC19: ORI, AmpR gene, P/O lacZ, MCS, lacZα; (5) orientation of the lacZα transcription and the first start codon, 5’ATG3’.

SELECTION of CLONES and BACTERIAL CULTURE

Each pair of students will pick 10 colonies of DH5α and 10 colonies of DH5β .

Media Preparation - Sterilely prepare 50 ml of medium LB-Mg-maltose directly in an erlen of 250 ml. Add ampicillin

to the final concentration of 100 µg/ml (see Annex, page 26 for the stock solution). - Split this medium into two Falcon-5O ml tubes, 25 ml each. -To one tube add tetracycline: to the final concentration of 15 µg/ml as previously. This tube

now contains LBAT medium. The other tube contains LBA medium. Save the rest of the medium, LBA and LBAT, in Falcon-50 ml for tomorrow at room temperature.

- Distribute the medium in 20 sterile Falcon-15 ml tubes: 10 tubes with LBA and 10 tubes with LBAT, 2 ml of the medium per tube.

16

Clones Selection - Select 10 clones for each of the studied bacterial strains (20 clones in total):

• 6 clones from LBAX plates without infection (37°C): L1α or L2α or L3α • 6 clones from LBATX 37°C plates without infection (37°C): L1β or L2β or L3β • 4 clones from LBAX 42°C plates with infection (42°C): L1αλ or L2αλ or L3αλ • 4 clones from LBATX 42°C plates with infection(42°C): L1βλ or L2βλ or L3βλ

QUESTION: What colonies you should select for analysis of clones and functional screen: blue

or white? Why? Pay attention to tubes labeling, to medium used (LBA for the strain DH5α and LBAT for the strain DH5β) and distinguish well between the clones originating from non-infected plates from clones originating from infected plates. Here is the proposed labeling: - 10 clones DH5α: α1, α2, α3, α4, α5, α6, α7λ, α8λ, α9λ, α10λ. - 10 clones DH5β: β1, β2, β3, β4, β5, β6, β7λ, β8λ, β9λ, β10λ. - For each selected clone, proceed as indicated on the scheme:

1. With a toothpick (or a steril yellow tip) pick from the plate the selected clone. 2. Drop the toothpick in the corresponding Falcon-15 ml tube and close the tube. 3. Put the tubes over night in the platform-shaker incubator set to 250 rpm and 37°C.

DAY 4

PLASMID EXTRACTION by the ALKALINE LYSIS METHOD (MINI-PREP)

Extraction of plasmid DNA

- At room temperature prepare solution II containing 0.2 N NaOH and 1% SDS (see Annex, page 26 for stock solutions). Attention: 10N NaOH provokes skin burns!

1) Bacterial resuspension - Verify that all bacterial cultures have grown. If not, take as many as you can for futher

experiments.

17

- Pour by inverting the falcon tube about 1.8 ml of each over night culture in one Eppendorf-2ml tube. If a toothpick or a tip is on your way pick it out from the tube with the help of Pipetman P200 containing a yellow tip – change the yellow tip between every culture to avoid cross-contamination!

- In each Falcon tube it remains about 200 µ l of culture, which you should save on the bench for the resistance test to bacteriophage λ infection at the end of this afternoon.

- Centrifuge the Eppendorf tubes 1 minute at the maximum speed in the table-top centrifuge. - Carefully decant the supernatant in the liquid waste bin and tap the Eppendorf against the

paper towel spread on the bench to completely remove the liquid from the tube. The cells pellet should be as dry as possible!

- Resuspend the pellet in 100 µ l solution I (see Annex, page 26) by gently pipetting using a Pipetman P200.

2) Cell lysis and DNA denaturation - To each tube add 200 µ l of solution II. - Homogenize the mix by gently inverting the tubes three times. Do not use the vortex to

prevent shearing of genomic DNA. - Incubate tubes 1 minute at room temperature.

3) Neutralization and precipitation of DNA - To each tube add 150 µl of an ice-cold solution III (see Annex, page 26, tube placed on ice). - Mix by gently inverting the tubes three times. - Incubate tubes for 5 minutes on ice. - Centrifuge for 5 minutes at 12 000 rpm at room temperature. - Immediately transfer the supernatant (about 400 µl of the clear lysate) in an Eppendorf-1.5ml

tube. Absolutely avoid to take the white precipitate! Pay attention to properly label all tubes! - Add to each tube 800 µ l of 100% ethanol (2 volumes) and mix well. - Incubate for 10 minutes at room temperature. - Centrifuge for 10 minutes at max speed at room temperature: pay attention to the position of

tubes in the rotor (cap bars toward the exterior). - Verify the presence of a pellet at the bottom of Eppendorfs (on the side of the cap bar). - Carefully remove the supernatant. - Add 500 µl of 70% ethanol. - Centrifuge 5 minutes under the same conditions as previously. - Verify the presence of a pellet and determine the volume of TE-RNase (see Annex, page 26

for composition) which you will add to resuspend the pellet in the final step: between 50 to 150 µ l (≤2 mm pellet in 50 µl of TE-RNase, ≥4 mm pellet in 150 µl, see with a supervisor).

- Delicately remove the supernatant by the help of a Pipetman P200, then a Pipetman P20. VERY IMPORTANT: the DNA pellet adheres very loosely to the tube. Pay attention not to aspirate it by the Pipetman. Remove a maximum of the supernatant with the P20 and let the rest evaporate by placing open tubes in the 50°C incubator until the pellets are perfectly dry (the RNase does not tolerate alcohol!) - Resuspend dry pellets in the previously determined volume of TE-RNase solution. - Vortex briefly and let tubes incubate at room temperature for about 30 minutes (resuspension

of DNA and action of RNase). Each tube contains about 10 µg of a plasmid DNA and will be used for the restriction analysis (plasmid mapping).

18

PLASMID RESTRICTION ANALYSIS (MAPPING)

Enzymatic digestion of plasmids

The aim of this experiment is to identify in each clone the fragment of bacteriophage λ inserted into pUC19. For this purpose the recombinant vector should be digested by two different restriction enzymes and restriction products should be analyzed by agarose gel electrophoresis. The genomic DNA library is considered to be of high quality if all λDNA fragments are present in the entity of bacterial clones. QUESTION: Why two different restrictions of recombinant plasmids are needed to

determine the identity of the λDNA fragment? Taking into account the employed cloning strategy and plasmid maps (see pages 28-32), propose the most appropriate restriction enzymes for plasmid mapping.

The protocol for plasmid mapping is the following: 5 µl of the purified plasmid DNA (about 1 µg) is digested in the final volume of 20 µl with 10 units of a restriction enzyme in the appropriate restriction buffer (buffers are supplied as 10 times concentrated, 10X). Each plasmid (20 tubes in total: 10 with DNA extracted from DH5α bacteria and 10 with DNA extracted from DH5β bacteria) is digested by two different enzymes. In total, 40 tubes are subjected to enzymatic digestion.

To obtain better experimental reproducibility and for the practicality, it is preferred to proceed as follows:

- First, prepare all tubes with 5 µl of plasmid DNA to be digested: pay attention to tube numbering (n° of clones, bacterial origin). In total, 20 tubes with DNA extracted from DH5α and 20 tubes with DNA extracted from DH5β .

- Next, on ice prepare two tubes of the reaction mix (mix 1 and mix 2). In practice, the reaction mix is prepared for more samples than required (it allows to avoid pipetting errors during mix distribution). That is, for 20 digestions the mix is prepared for 22 digestions, as follows:

- 10X restriction buffer 44 µl - sterile ∆ H2O 264 µl - restriction enzyme (10 U/µl) 22 µl (added by a supervisor)

- Without significant delay distribute 15 µl of each mix to every Eppendorf tube containing the plasmid DNA, in total, 20 tubes with mix 1 and 20 tubes with mix 2. Attention: change a tip each time not to mix different digestions.

Note: You can verify that digestion conditions used here are as follows: 10 units of enzyme and 1X restriction buffer in the final volume of 20 µl per tube.

- Put digestion tubes for 2 hours at 37°C either in the water bath or in the incubator.

Analytical agarose gel Results of enzymatic digestions are controlled by gel electrophoresis on a 1% agarose gels. Gels are prepared and used as described above on page 7, with two combs in the “low” position per gel. Two gels are necessary to analyze all your digestion reactions.

- At the end of the digestion, add directly 5 µl of 5x loading buffer to all digestion tubes while changing the tip each time (final volume is 25 µl)

Note: Digestion tubes that you are not loading on the first gel are kept at -20°C. - Load 8 µl of each digestion on the gel: the rest of samples (17 µl) is stored at -20°C in order

to re-run another gel, if necessary. 19

- Load also 8 µl of the molecular weight marker “1kb ladder” on both sides of each loading line, that is 4 lines with 1kb ladder per gel. To distinguish your gel form others, move the loading position of the first standard by one with respect to your neighbor: the pair of students N°1 loads the standard in a well N°1, the pair of students N°2 loads the standard in a well N°2, …

- At the end of migration, color the gel by EtBr and observe your results under UV. After the migration of the first gel is done, take the plexi and immediately prepare a second agarose gel, which is going to be stored in TBE 0,5X at 4°C and used tomorrow morning under the same conditions as the first one.

Note: Don’t forget to properly annotate your gels in the lab book and discuss your experimental results!

RESISTANCE TEST to BACTERIOPHAGE λ

Each of 20 clones used for plasmid mapping are also tested for the sensitivity or resistance to bacteriophage λ infection. For this purpose you will use the remaining bacterial cultures (about 200 μl) from Falcon-15ml tubes stored at your bench. - On the bottom of two LBA plates for DH5α clones and two LBAT plates for DH5β clones

reproduce the drawing presented in the figure below: arrows indicate the starting position and the direction of each “spread”.

- On each plate, spread the bacteriophage λcI (titer = ± 1010 pfu/ml) by spotting 50 µl of the phage suspension provided by a supervisor and letting this drop slide over the agar in a straight line by vertical inclination. Let dry.

- By the help of a metallic loop, flame sterilized and cooled, spread on the agar in the direction perpendicular to the “spread” of the bacteriophage λcI your bacterial suspensions to be tested, 5 per plate (in one single movement). Let dry.

OR you can also proceed as for the spreading of the phage, by letting a drop slide over the agar, always perpendicular to the “spread” of the bacteriophage λcI, 10 µl of well-mixed bacterial culture.

- “Spread” in the same way the two control bacterial strains, one sensitive λS, and the other resistant λR to bacteriophage λcI respecting the order of the scheme. Let dry.

- Turn the 4 plates upside down and put them in the 42°C incubator.

20

DAY 5

PLASMID MAPPING Continuation of the digestion analysis by analytical gels (see DAY 4).

RESULTS INTERPRETATION with a help of IN SILICO ANALYSIS

Your experimental results should be completed by computer analysis thanks to the ApE

software and will concern only two recombinant plasmids containing fragment IV of the λDNA in two orientations A and B.

Following the analysis of your own experimental results and those of other students you will emit several hypothesis. To validate them you will require some additional information concerning bacteriophage λ that you will be able to find using BLAST (Basic Local Alignment Search Tool). This work will be done via Internet and the database of the National Center for Biotechnology Information (NCBI) at the following link:

http://www.ncbi.nlm.nih.gov/BLAST/ Annotation of recombinant plasmids

- Switch on the computer and log-in in the “etudiant” session. - Select the folder “TP master” and copy it into the desktop under your own initials. Go in the

menu "Fichier", than "Dupliquer" or press “cmd+D” or “⌘+D”. A new folder "copie de TP master" will appear on the desktop. Rename the folder by adding your initials.

- By double click to the icon, open your folder and check if the folder contains three files: lambda, pUC19-fragmentIV sens A and pUC19-fragmentIV sens B.

- Open the ApE by double clicking on the icon ApE in the desktop. - Open files with recombinant plasmids within ApE through: File > Open > ”Your folder” > pUC19-

fragmentIV sens A or pUC19-fragmentIV sens B. Each sequence appears in a new window in a conventional form: one strand in orientation 5’->3’. You can find some useful information in the upper part of the window: size, DNA configuration: linear or circular. The pUC19 sequence is marked in small characters as well as the λDNA is in large characters. Note: You can modify DNA configuration, if needed, by a click on the corresponding key. ApE allows you to annotate the known genetic elements within the sequence (resistance gene, replication origin, well-known promoters, etc). To do this, go to the menu FEATURES and use the following functions:

menu FEATURES FUNCTION UTILITY ANNOTATE FEATURES USING LIBRARY

Use a database to find and put in colour DNA sequences of interest. Putting your cursor on the underlined region the annotation of the sequence appears

LIST FEATURES Displays in another window the list and discription of the known annotations

- Use these functions to annotate your recombinant plasmids and display them via the function

ENZYME>GRAPHIC MAP. Compare the results with your own finding that you have done in Annex, page 31.

21

ApE allows to analyse open reading frames (ORFs) using the menu ORF. This menu contains several functions that may serve you to find an ORF of interest:

menu ORF FUNCTION UTILITY

ORF MAP

Display the map of all potential ORFs in 6 frames of the sequence of interest. By a click on the key “right” you can modify the scale of the map. The analysis of the potential ORFs has been done for two recombinant plasmids and the result is present in Annex, page 33.

FIND NEXT

Starting form the cursor position within the sequence, this function allow you to underline the coding sequence closest to 3’end. ATTENTION: the search for ORFs is done uniquely for the strand displayed on the monitor. If the ORF of interest is positioned on the complementary strand, transform your sequence into the complement via the function EDIT>Reverse-complement

- Mark all annotations of genetic elements on plasmid maps in Annex, page 31. - Compare ORF maps of two recombinant plasmids pUC19-fragmentIV-A and pUC19-fragmentIV-

B. This analysis will allow you to reveal certain features of one ORF that could explain a bias in presence of fragments in the bacteriophage λDNA library. Propose a hypothesis concerning the ORF of interest and consequences of its presence within the plasmid and transformed bacteria.

Characterization of the ORF of interest

- ApE allows aligning of a sequence of the suspicious ORF with a bank of protein sequences deposited in NCBI. For this purpose use the function BLAST in the menu TOOLS.

menu TOOLS

FUNCTION UTILITY

BLAST SEQUENCE AT NCBI

Aligns the sequence of interest with a bank of protein sequences deposited in NCBI. Options: -Selection only : tick off -BLAST program: blastp (alignement of protein sequences) -Expect: 100 -Low complexity region: tick off

Use this function to characterize a protein encoded by the underlined ORF sequence. The results will appear in a window of the Internet navigator. At the end, this work will allow you to approve or not your hypothesis to explain your experimental results.

22

ANNEXES

1/ PRINCIPLES OF GENOMIC DNA LIBRARY CONSTRUCTION …………………………….. 24

2/ INTRODUCTION OF A PLASMID pUC19 …………………………………………………………………… 25

3/ SOLUTIONS …………………………………………………………………………………………………………………….. 26

4/ AGAROSE GEL …………………………………………………………………………………………………………………. 27

5/ MAPS of RECOMBINANT PLASMIDS ……………………………………………………………………….. 28

6 / Map of ORFs in 6 frames …..………………………………………………………………………………………….. 33

7/ DNA and protein sequences of an ORF of the recombinant plasmid pUC19-

fragmentIV sens B …………………………………………………………………………………………………………….. 34

8/ Gene and protein sequences of the bacteriophage λ git gene ………………………………. 35

9/ Genome map of bacteriophage λ ……………………………………………………………………………………. 36

23

PRINCIPLES OF GENOMIC DNA LIBRARY CONSTRUCTION

24

INTRODUCTION of a PLASMID pUC19

The pUC19 plasmid is a cloning vector using Escherichia coli as a host. It contains 2686 bps. By agreement, the numbering of nucleotides starts at the first base T of the sequence 5'T1CGCGCGTTT…3' (arrow on the map below). In position 1624-2482 it contains the gene of resistance to ampicillin (AmpR) coding for the β-lactamase (protein of 286 amino acids), and in position 876 a bacterial origin of replication ORI. The part of the lacZ gene coding for the β-galactosidase, lacZa, is placed under the control of the promoter/operator of lacZ (P/O). The MCS or multiple cloning sites (MCS) (position 396-454) is introduced at the beginning of the coding sequence of the lacZa gene (see the detailed sequence of this region at the bottom of this page): the ORF of the fusion MCS-lacZa gene is situated at the position 238-469.

Detailed structure of the MCS

Only the 5'-3' strand is shown using a conventional numbering. The MCS bases are in bold. The cutting sites of different restriction enzymes in this MCS are shown: note that the cutting site of EcoR I is a mixed site 5'gaattc3'. The beginning of the sequence of the peptide α supplemented with a few amino acids encoded by the MCS (including the methionin of the start codon 3'gta5') is represented here below.

25

SOLUTIONS

Stock Solutions:

Maltose 20% final concentration: 0,4%

MgSO4 v1 M final concentration: 10 mM

Tetracyclin 4 mg/ml final concentration: 15 µg/ml

Ampicillin 25 mg/ml final concentration: 100 µg/ml

NaOH 10N final concentration: 0,2N

SDS 10% final concentration: 1%

MnCl2 1M final concentration: 50 mM

Solutions for competent bacteria:

Solution T1: K acetate 30 mM Solution T2: MOPS pH 7 10 mM KCl 100 mM KCl 10 mM CaCl2 10 mM CaCl2 75 mM Glycerol 15% Glycerol 15% MnCl2 50 mM (to add in a last minute) Solutions for plasmid DNA extraction (mini-prep):

Solution I: Glucose 50 mM Solution II: NaOH 0.2 N EDTA 10 mM SDS 1% Tris-HCl pH 8 25 mM

Solution III: K acetate 3 M pH 4.8 with glacial acidic acid

TE-RNase: Tris-HCl pH 8 10 mM EDTA 1 mM RNase 50 µg/ml Digestion and ligation buffers:

Buffer B, 1X content: Tris-HCl pH 8 at 37°C 10 mM MgCl2 5 mM KCl 100 mM Triton X-100 0.02% BSA 100 µg/ml

Buffer E, 1X content: Tris-HCl pH 7.5 à 37°C 50 mM MgCl2 10 mM NaCl 100 mM Triton X-100 0.02% BSA 100 µg/ml

Ligation buffer, 1X content: Tris-HCl pH 7.8 à 25°C 40 mM MgCl2 10 mM DTT 10 mM ATP 0,5 mM

26

AGAROSE GEL 1kb DNA ladder (0.1 µg/µL)

27

Solutions for agarose gel:

TBE, 5X content:

Tris base 0.5 M Boric acid 0.61 M pH 8,3 EDTA 10 mM Sample loading buffer, 5X content:

EDTA 50 mM SDS 0.5 % Glycerol 25% Bromophenol blue 0.12%

RECOMBINANT PLASMID

pUC19-FRAGMENT I

27

28

183 Nde I 1235 Bbe I 1235 Kas I 1235 Nar I 1235 Sfo I 1

396 Eco RI 2402 Eco ICRI 1402 Sac I 1412 Sma I 2412 Uth SI 2412 Xma I 2417 Bam HI 2498 Sex AI 1

936 Fsp AI 2957 Fsp AI 2

1466 Eco NI 21537 Eco RI 21552 Sty I 2

1768 Afe I 11867 Psr I 1

2723 Nae I 12723 NgoMIV 12819 Eag I 1

3366 Sma I 23366 Uth SI 23366 Xma I 23434 Nco I 13434 Sty I 23440 BsiWI 13574 Pas I 2

4715 Bst BI 1

6113 Asc I 16468 Pme I 2

6643 Cla I 26908 BsrGI 2

7180 Cla I 27439 Pas I 2

9249 Eco NI 2

2 Afl II 101451 Stu I 10329

1 Sna BI 105752 Sph I 10761

2 Bsa I 113391 Tth 111I 11558

1 Bfr BI 124381 Nsi I 12438

1 Ppu 10I 124381 Apa I 12677

1 PspOMI 12677

2 Ssp I 142942 Pme I 14302

2 Afl II 162232 BsrGI 16621

2 Bam HI 172581 Xba I 172641 Sal I 172702 Sph I 17282

1 Hin dIII 172881 Pci I 17647

2 Bsa I 186072 Ssp I 19342

1 EcoO109I 195151 Pss I 19515

puc19-fragment 1 sens B

19527 bpSites <= 2

183 Nde I 1235 Bbe I 1235 Kas I 1235 Nar I 1235 Sfo I 1

396 Eco RI 2402 Eco ICRI 1402 Sac I 1412 Sma I 2412 Uth SI 2412 Xma I 2417 Bam HI 2

1054 BsrGI 21452 Afl II 2

3371 Pme I 23381 Ssp I 2

4998 Apa I 14998 PspOMI 15237 Bfr BI 15237 Nsi I 15237 Ppu 10I 1

6114 Tth 111I 16336 Bsa I 2

6914 Sph I 27100 Sna BI 1

7346 Stu I 17530 Afl II 2

8421 Eco NI 2

2 Pas I 102352 Cla I 10495

2 BsrGI 107672 Cla I 11032

2 Pme I 112051 Asc I 11560

1 Bst BI 129602 Pas I 14100

1 BsiWI 142351 Nco I 142412 Sty I 14241

2 Sma I 143092 Uth SI 143092 Xma I 14309

1 Eag I 148561 Nae I 14952

1 NgoMIV 14952

1 Psr I 158011 Afe I 159072 Sty I 16123

2 Eco RI 161382 Eco NI 16204

2 Fsp AI 167162 Fsp AI 16737

1 Sex AI 171762 Bam HI 17258

1 Xba I 172641 Sal I 172702 Sph I 17282

1 Hin dIII 172881 Pci I 17647

2 Bsa I 186072 Ssp I 19342

1 EcoO109I 195151 Pss I 19515

puc19-fragment 1 sens A

19527 bpSites <= 2

51 Bsm BI 291 Drd I 2

179 Apa BI 2179 Bst API 2183 Nde I 2215 CstMI 2235 Bbe I 1235 Kas I 1235 Nar I 1235 Sfo I 1245 Bgl I 2

387 Bce AI 2387 Bce fI 2396 Eco RI 2408 Acc 65I 1408 Kpn I 1412 Ava I 2412 Nli 3877I 2412 Sma I 1412 Uth SI 1412 Xma I 1417 Bam HI 2

502 Ava I 2502 Nli 3877I 2521 Nco I 2

630 Fal I 2759 Nde I 2

897 Alf I 21005 Rle AI 11017 Bfr BI 21017 Nsi I 21017 Ppu 10I 21073 Hpa I 1

1183 Bfr BI 21183 Nsi I 21183 Ppu 10I 2

1568 Eco RV 21671 Bsu 36I 11716 Btg ZI 21766 Msc I 11773 Cla I 11860 Bsa AI 11860 Pml I 12007 Tst I 1

2285 Eco RI 2

2505 Bst BI 1

2678 Apa BI 22678 Bst API 2

2776 Bsp EI 22960 Btg ZI 2

3205 Bst EII 13319 PflMI 2

3547 Bsp EI 23679 Ale I 1

3702 Fal I 23728 Alo I 1

3818 PflMI 23881 Xba I 2

3993 Avr II 24067 Avr II 2

2 Alf I 42041 Bmg BI 4220

2 Bsa BI 43582 Nco I 4488

1 Xcm I 46952 Ahd I 4924

2 Hae IV 49241 Bpu 10I 4969

2 Eco RV 54412 Bsa BI 57371 Bsg I 58751 Bgl II 5964

2 Bam HI 60432 Xba I 60491 Acc I 60551 Sal I 60551 Sbf I 6060

1 Pci I 64322 Drd I 6534

1 Alw NI 68432 Bce AI 69182 Bce fI 6918

2 Ppi I 71232 Ahd I 7320

2 Hae IV 73201 Bsa I 73921 Bsr FI 7405

2 Bgl I 74392 Ava II 7463

2 Psp 03I 74632 Vpa K11AI 7463

2 CstMI 76782 Ava II 7685

2 Psp 03I 76852 Vpa K11AI 7685

2 Ppi I 79661 Aat II 82431 Zra I 8243

1 EcoO109I 83001 Pss I 8300

2 Bsm BI 8309


8312 bpSites <= 2

RECOMBINANT PLASMID

pUC19-FRAGMENT II

29

51 Bsm BI 291 Drd I 2

179 Apa BI 2179 Bst API 2183 Nde I 2215 CstMI 2235 Bbe I 1235 Kas I 1235 Nar I 1235 Sfo I 1245 Bgl I 2

387 Bce AI 2387 Bce fI 2396 Eco RI 2408 Acc 65I 1408 Kpn I 1412 Ava I 2412 Nli 3877I 2412 Sma I 1412 Uth SI 1412 Xma I 1417 Bam HI 2

496 Bgl II 1585 Bsg I 1

719 Bsa BI 2

1019 Eco RV 2

1490 Bpu 10I 11531 Ahd I 21531 Hae IV 2

1756 Xcm I 1

1972 Nco I 22098 Bsa BI 22240 Bmg BI 12250 Alf I 2

2393 Avr II 22467 Avr II 2

2579 Xba I 22637 PflMI 2

2725 Alo I 12753 Fal I 22777 Ale I 1

2913 Bsp EI 23136 PflMI 2

3254 Bst EII 13500 Btg ZI 2

3684 Bsp EI 23777 Apa BI 23777 Bst API 2

3955 Bst BI 1

2 Eco RI 41751 Tst I 4447

1 Bsa AI 46001 Pml I 4600

1 Cla I 46871 Msc I 4694

2 Btg ZI 47441 Bsu 36I 4788

2 Eco RV 48922 Bfr BI 52772 Nsi I 5277

2 Ppu 10I 52771 Hpa I 5387

2 Bfr BI 54432 Nsi I 5443

2 Ppu 10I 54431 Rle AI 54552 Alf I 5557

2 Nde I 57012 Fal I 5825

2 Nco I 59392 Ava I 5958

2 Nli 3877I 59582 Bam HI 6043

2 Xba I 60491 Acc I 60551 Sal I 60551 Sbf I 6060

1 Pci I 64322 Drd I 6534

1 Alw NI 68432 Bce AI 69182 Bce fI 6918

2 Ppi I 71232 Ahd I 7320

2 Hae IV 73201 Bsa I 73921 Bsr FI 7405

2 Bgl I 74392 Ava II 7463

2 Psp 03I 74632 Vpa K11AI 7463

2 CstMI 76782 Ava II 7685

2 Psp 03I 76852 Vpa K11AI 7685

2 Ppi I 79661 Aat II 82431 Zra I 8243

1 EcoO109I 83001 Pss I 8300

2 Bsm BI 8309


8312 bpSites <= 2

46 Pfo I 151 Bsm BI 291 Drd I 2

235 Bbe I 1235 Kas I 1235 Nar I 1235 Sfo I 1276 Pvu I 2306 Pvu II 2

396 Eco RI 2402 Ban II 1402 Eco ICRI 1402 Sac I 1408 Acc 65I 1408 Kpn I 1412 Sma I 2412 Uth SI 2412 Xma I 2417 Bam HI 2

588 Sap I 2597 Bsu 36I 1649 Bsa BI 2

974 Bpm I 21417 Psp XI 11418 Sci I 11418 Xho I 1

1534 Bsa AI 11634 Csp CI 1

1811 Psr I 11919 Stu I 22077 Sex AI 22114 Sca I 22152 Afl III 22176 Rle AI 22187 Bcl I 1

2377 Xcm I 12420 BsrGI 2

2699 Hpa I 2

2952 Tst I 23004 Dra III 2

3090 Ale I 23090 Sgr AI 13091 Age I 13091 Bsr FI 23109 Hpa I 2

3169 Eco RI 23299 Sma I 23299 Uth SI 23299 Xma I 2

3377 Rle AI 23438 Stu I 2

3696 Bse RI 13906 Sex AI 2

4444 Ahd I 24548 Dra III 2

2 Tst I 47222 Bsa XI 4803

1 Bst EII 49101 Aar I 5018

2 BspMI 50182 Ale I 5353

2 BsrGI 55242 Aat II 58802 Zra I 5880

2 Fal I 59872 EcoO109I 6118

1 PpuMI 61182 Pss I 6118

1 San DI 61181 Sty I 6123

1 Msc I 6298

2 Bsa BI 67141 Alf I 67762 Fal I 6826

1 Bss HII 69082 Bam HI 6944

1 Xba I 69502 BspMI 6960

1 Sbf I 69611 Sph I 6968

1 Hin dIII 69742 Pvu II 7155

2 Bsa XI 71862 Sap I 72102 Afl III 73331 Pci I 73332 Drd I 7435

2 Ahd I 82211 Bsa I 82932 Bsr FI 83062 Bpm I 8311

2 Acl I 84512 Pvu I 8593

2 Sca I 87042 Acl I 8824

2 Aat II 91442 Zra I 9144

2 EcoO109I 92012 Pss I 9201

2 Bsm BI 9210


9213 bpSites <= 2

RECOMBINANT PLASMID

pUC19-FRAGMENT III

30

46 Pfo I 151 Bsm BI 291 Drd I 2

235 Bbe I 1235 Kas I 1235 Nar I 1235 Sfo I 1276 Pvu I 2306 Pvu II 2

396 Eco RI 2402 Ban II 1402 Eco ICRI 1402 Sac I 1408 Acc 65I 1408 Kpn I 1412 Sma I 2412 Uth SI 2412 Xma I 2417 Bam HI 2453 Bss HII 1

530 Fal I 2579 Alf I 1

643 Bsa BI 21063 Msc I 1

1238 Sty I 11242 EcoO109I 21242 PpuMI 11242 Pss I 21242 San DI 1

1369 Fal I 21481 Aat II 21481 Zra I 2

1837 BsrGI 22004 Ale I 2

2342 Aar I 12343 BspMI 22450 Bst EII 12553 Bsa XI 22633 Tst I 2

2810 Dra III 22912 Ahd I 2

3454 Sex AI 23665 Bse RI 1

3923 Stu I 23984 Rle AI 2

4062 Sma I 24062 Uth SI 24062 Xma I 2

4192 Eco RI 24252 Hpa I 24267 Ale I 24269 Sgr AI 14270 Age I 14270 Bsr FI 2

4354 Dra III 24403 Tst I 2

2 Hpa I 46622 BsrGI 49411 Xcm I 4975

1 Bcl I 51742 Rle AI 51852 Afl III 5209

2 Sca I 52472 Sex AI 5283

2 Stu I 54421 Psr I 5543

1 Csp CI 57211 Bsa AI 5827

1 Psp XI 59421 Sci I 5943

1 Xho I 5943

2 Bpm I 6387

2 Bsa BI 67081 Bsu 36I 6763

2 Sap I 6772

2 Bam HI 69441 Xba I 6950

2 BspMI 69601 Sbf I 69611 Sph I 6968

1 Hin dIII 69742 Pvu II 7155

2 Bsa XI 71862 Sap I 72102 Afl III 73331 Pci I 73332 Drd I 7435

2 Ahd I 82211 Bsa I 82932 Bsr FI 83062 Bpm I 8311

2 Acl I 84512 Pvu I 8593

2 Sca I 87042 Acl I 8824

2 Aat II 91442 Zra I 9144

2 EcoO109I 92012 Pss I 9201

2 Bsm BI 9210


9213 bpSites <= 2

46 Pfo I 191 Drd I 2

235 Bbe I 1235 Kas I 1235 Nar I 1235 Sfo I 1245 Bgl I 2306 Pvu II 2

364 Bmr I 2396 Eco RI 2402 Ban II 2402 Eco ICRI 1402 Sac I 1408 Acc 65I 1408 Kpn I 1412 Sma I 2412 Uth SI 2412 Xma I 2417 Bam HI 2456 Bse RI 1

619 PflMI 2662 Csp CI 1667 Bsa AI 1667 Dra III 1667 Pml I 1

739 Bsg I 1984 Bmg BI 2

1108 Bpl I 11620 Btg ZI 21651 Sex AI 11697 Age I 11697 Bsr FI 21763 Sac II 1

1948 Acc I 22099 Bst EII 22148 Bsp EI 22261 Sma I 22261 Uth SI 22261 Xma I 22428 PflMI 22520 Bcg I 22559 Btg ZI 22696 Ban II 22698 Blp I 12731 Sph I 22754 Afl III 22754 Pci I 2

2981 Eco RI 2

3851 Bst XI 23876 Eco NI 2

4256 Psh AI 24260 Bmg BI 2

4393 Psi I 14797 Bcl I 1

1 Afe I 50922 Sbf I 51472 Pst I 5148

2 Psh AI 52201 Alo I 5424

1 Eag I 54952 Sty I 5644

2 Bst EII 57741 Xcm I 5975

1 Tth 111I 60261 Msc I 6109

1 Bbv CI 6335

2 Eco NI 66232 Bsp EI 6700

2 Sty I 7133

2 Sap I 73481 Bmt I 74701 Nhe I 7470

2 Bst XI 75472 Bam HI 7650

1 Xba I 76562 Acc I 76621 Sal I 76622 Sbf I 76672 Pst I 76682 Sph I 76742 Pvu II 78612 Sap I 79162 Afl III 80392 Pci I 80392 Drd I 8141

1 Ahd I 89272 Bmr I 89771 Bsa I 89992 Bsr FI 9012

2 Bgl I 90462 Acl I 9157

1 Sca I 94102 Bcg I 94481 Xmn I 95272 Acl I 9530

1 EcoO109I 99071 Pss I 9907


9919 bpSites <= 2

RECOMBINANT PLASMID

pUC19-FRAGMENT IV

31

46 Pfo I 191 Drd I 2

235 Bbe I 1235 Kas I 1235 Nar I 1235 Sfo I 1245 Bgl I 2306 Pvu II 2

364 Bmr I 2396 Eco RI 2402 Ban II 2402 Eco ICRI 1402 Sac I 1408 Acc 65I 1408 Kpn I 1412 Sma I 2412 Uth SI 2412 Xma I 2417 Bam HI 2

514 Bst XI 2597 Bmt I 1597 Nhe I 1

718 Sap I 2934 Sty I 2

1367 Bsp EI 21439 Eco NI 2

1731 Bbv CI 11958 Msc I 12038 Tth 111I 12083 Xcm I 1

2292 Bst EII 22423 Sty I 22572 Eag I 12636 Alo I 1

2843 Psh AI 22918 Sbf I 22919 Pst I 22975 Afe I 1

3270 Bcl I 1

3674 Psi I 13807 Bmg BI 23807 Psh AI 2

4186 Eco NI 24210 Bst XI 2

2 Eco RI 50862 Afl III 53132 Pci I 5313

2 Sph I 53361 Blp I 5368

2 Ban II 53712 Btg ZI 55082 Bcg I 5541

2 PflMI 56342 Sma I 58062 Uth SI 58062 Xma I 5806

2 Bsp EI 59192 Bst EII 5967

2 Acc I 61191 Sac II 63041 Age I 6370

2 Bsr FI 63701 Sex AI 64152 Btg ZI 6447

1 Bpl I 69542 Bmg BI 70831 Bsg I 7328

1 Dra III 73971 Csp CI 73991 Bsa AI 7400

1 Pml I 74002 PflMI 7443

1 Bse RI 76112 Bam HI 7650

1 Xba I 76562 Acc I 76621 Sal I 76622 Sbf I 76672 Pst I 76682 Sph I 76742 Pvu II 78612 Sap I 79162 Afl III 80392 Pci I 80392 Drd I 8141

1 Ahd I 89272 Bmr I 89771 Bsa I 89992 Bsr FI 9012

2 Bgl I 90462 Acl I 9157

1 Sca I 94102 Bcg I 94481 Xmn I 95272 Acl I 9530

1 EcoO109I 99071 Pss I 9907


9919 bpSites <= 2

183 Nde I 1235 Bbe I 2235 Kas I 2235 Nar I 2235 Sfo I 2276 Pvu I 2

364 Bmr I 2396 Eco RI 2402 Ban II 2402 Eco ICRI 1402 Sac I 1408 Acc 65I 1408 Kpn I 1412 Ava I 2412 Nli 3877I 2412 Sma I 1412 Uth SI 1412 Xma I 1417 Bam HI 2

655 Hpa I 2702 BsrGI 1

852 PflMI 21043 Age I 2

1192 Age I 21202 Ava I 21202 Nli 3877I 2

1332 Nru I 21796 Bss HII 2

2056 Bpu 10I 22121 Rsr II 1

2399 Asc I 12400 Bss HII 2

3061 Bst XI 23106 PpuMI 2

3637 Psi I 13710 Sph I 2

4218 Psr I 1

5190 Hpa I 25294 Pci I 25341 Ban II 25367 Aar I 1

5464 Mlu I 15507 Bgl II 1

5649 Alo I 25927 Bst EII 15950 PpuMI 25989 Csp CI 2

6268 Psh AI 16544 Mfe I 1

6980 Bpu 10I 27206 Eco NI 2

2 Csp CI 78412 Bst XI 7984

2 Bbe I 87452 Kas I 87452 Nar I 87452 Sfo I 8745

2 Eco RI 94522 Ahd I 9750

1 Sex AI 100151 Nco I 101761 Sty I 10176

2 Hin dIII 10283

2 Alo I 115502 Bsa I 11709

1 Bst BI 117871 Afl II 11794

2 PflMI 119001 Bbr 7I 119761 Bbs I 119761 Pml I 12062

2 Eco NI 125772 Nru I 12616

2 Bam HI 126921 Xba I 126981 Sal I 12704

2 Sph I 127162 Hin dIII 12722

2 Pci I 130812 Ppi I 13772

2 Ahd I 139692 Bmr I 140192 Bsa I 14041

2 Pvu I 143411 Sca I 14452

2 Ppi I 14615


14961 bpSites <= 2

RECOMBINANT PLASMID

pUC19- FRAGMENT V

32

183 Nde I 1235 Bbe I 2235 Kas I 2235 Nar I 2235 Sfo I 2276 Pvu I 2

364 Bmr I 2396 Eco RI 2402 Ban II 2402 Eco ICRI 1402 Sac I 1408 Acc 65I 1408 Kpn I 1412 Ava I 2412 Nli 3877I 2412 Sma I 1412 Uth SI 1412 Xma I 1417 Bam HI 2493 Nru I 2527 Eco NI 2

1047 Pml I 11133 Bbr 7I 11133 Bbs I 11204 PflMI 2

1315 Afl II 11322 Bst BI 11400 Bsa I 2

1552 Alo I 2

2826 Hin dIII 22933 Nco I 12933 Sty I 13093 Sex AI 13354 Ahd I 2

3657 Eco RI 2

4364 Bbe I 24364 Kas I 24364 Nar I 24364 Sfo I 2

5119 Bst XI 25262 Csp CI 2

5898 Eco NI 26128 Bpu 10I 2

6565 Mfe I 16837 Psh AI 1

7114 Csp CI 27158 PpuMI 27181 Bst EII 1

7453 Alo I 21 Bgl II 76021 Mlu I 7645

1 Aar I 77412 Ban II 77682 Pci I 7815

2 Hpa I 7919

1 Psr I 88842 Sph I 93991 Psi I 9472

2 PpuMI 100022 Bst XI 10042

1 Asc I 107082 Bss HII 107091 Rsr II 10987

2 Bpu 10I 110522 Bss HII 11313

2 Nru I 117772 Ava I 11907

2 Nli 3877I 119072 Age I 119172 Age I 120662 PflMI 122521 BsrGI 12407

2 Hpa I 124542 Bam HI 12692

1 Xba I 126981 Sal I 12704

2 Sph I 127162 Hin dIII 12722

2 Pci I 130812 Ppi I 13772

2 Ahd I 139692 Bmr I 140192 Bsa I 14041

2 Pvu I 143411 Sca I 14452

2 Ppi I 14615


14961 bpSites <= 2

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