Plasmids

Isolation of Plasmid DNA from Bacteria

Properties of a Cloning/Expression Vector (Plasmid)

Uses of Expression Vectors

PLASMID DNA ISOLATION FROM E.Coli

Lipopolysaccharide

Peptidoglycan

Plasma MembranePLASMID (3-20 kbp)

Chromosomal DNA (4 Mbp)

Proteins

Proteins

Proteins

Proteins

Proteins

How to isolate plasmid without a commercial kit.

Lipopolysaccharide

Peptidoglycan

Plasma MembranePLASMID (VECTOR) (3-20 kb)

Chromosomal DNA (4 Mbp)

Proteins

Proteins

Proteins

Proteins

Proteins

Sol 1 (EDTA, Glucose, Tris pH8) [Binds Mg2+ (interferes with cell wall integrity), breaks outer layer, inhibits DNAses, maintains osmotic pressure, buffers cells at pH 8]Lysozyme (Digests Peptidoglycan)Sol 2 (SDS, NaOH) (Detergent- lyses the cell, denatures proteins including DNAses)Sol 3 (KAc, Acetic Acid)Phenol/Chloroform extraction (Dissociate proteins from nucleic acids)Proteinase K (proteolytic enzyme to remove remaining proteins. Not denatured by SDS)RNAse (Digests RNA)Precipitation of DNA (Isopropanol rapidly ppts nucleic acids. Ethanol removes remaining salt from preparation)

The Qiagen Miniprep Kit you will be using

Buffer P1- Resuspension Buffer50mM TrisHCl pH 8.010 mM EDTA10 ug/ml RNAse A

Buffer P2- Lysis Buffer200mM NaOH1% SDS

Buffer N3- Neutralizing BufferPotassium Acetate/Acetic Acid

Chromosomal DNA +SDS-protein Complex

Plasmid in suspension

Use of Silica Gel Membrane Technology

Plasmid binds tightly to membrane under high [salt]

Elute Impurities

Plasmid is elutedunder low [salt]

using a commercial kit to isolate your plasmid

How to quantify of your DNA (Plasmid)

1. DNA can be quantified in an agarose gel by comparing the intensity of the fluorescence emitted by an ethidium bromide-stained DNA sample relative to a dilution series of a DNA standard of known concentration.

2. If the sample is pure (free of RNA, protein, phenol, agarose), aSpectrophotometric method based on measuring the amount of UV irradiationthat is absorbed by the bases is accurate and convenient.

-DNA concentration is determined from readings at 260nm.-Values are in optical density units (OD) where 1OD=50µg/ml

-Eg, if OD=0.2, [DNA]= 50µg/mlx0.2=10µg/ml-If DNA has been diluted, then dilution factor must be included

-Eg, (2µlDNA+98µlH2O) with 0.2OD will read:

0.2x50x(1/1000)x50= 500ng/µl

Explain?Can you think of a dilution factor(s) you could use that would allowyou to determine [DNA] without any mathematical calculations?

What is a plasmid ?

Expression VectorShuttle VectorCloning Vector

What are the 3 most important parts of an expression vector ?

1. Origin of replication

2. Selectable marker

3. Cloning sites

Ori

RepliconContains all info necessary to begin and end DNA replicationOrigin of replication (Ori) is a defined location within the replicon where DNA synthesis begins

The ColE1 Replicon

ColE1 naturally occuring plasmid of E.coli.replicates independently of the host genome.

DNA Pol

200-300 bp

RNA I : RNA II INTERACTION

ROP PROTEIN NEGATIVELY REGULATES RNA II BY STABILIZING RNA I: RNA II INTERACTION

Copy number of Vectors

Plasmid Replicon=Origin

Copy number

pBR322 ColE1 15-20

pT7T3.pac ColE1 500-700

Why does pT7T3.pac have a high copy number?

• There is a point mutation in the origin that alters the initiation of RNAI transcription, such that the RNAI:RNAII complex does not form as well as wild-type.

• The region of the origin that encodes Rop protein is deleted.

Why does pT7T3.pac have a high copy number?

2 REASONS

Selectable Marker (Ampicillin Resistance)

Ampicillin: -belongs to family of antibiotics called β-lactams -penicillin derivative

β-lactam ring

Interacts with and inactivates transpeptidase which is responsible for transpeptidation (cross-linking of sugars)

AmpR gene codes for β-lactamase which inactivates ampicillinby cleaving the β-lactam ring.

AmpR gene: to select for bacteria containing plasmid.

-Why does bacteria sustain plasmid replication?

Ori

EcoRINotI

HindIII

VP7

Uses of Expression Vectors in Genetic Research

Animal models of diseases (Various genetic techniques)Wide range: overexpressing genes to gene silencing

Genomic Studies

Gene therapy

There are many types of Expression Vectors that researcherscan choose from. Choice depends on the experiment.

Several properties of vectors are essential for function.

Features to have: 1. high level of expression2. tight control3. subtle modulation

Site-Directed Mutagenesis (Use of Vectors and PCR)

TCTATGGACCAGTACGATACCAGTA.....CGACCTACGTAGACTAGACGGATAGAG AGATACCTGGTCATGCTATGGTCAT.....GCTGGATGCATCTGATCTGCCTATCTC

GAATTCTCTATGGACCAGTACGATACCAGTA.....CGACCTACGTAGACTAGACGGATAGAGGGATCCCTTAAGAGATACCTGGTCATGCTATGGTCAT.....GCTGGATGCATCTGATCTGCCTATCTCCCTAGG

Addition of EcoRI(GAATTC) and BamHI (GGATCC) sitesand cloning into vector of choice

TCGATGGACCAGTACGATACC (mutant forward primer extended) 5’T C G ATGGACCAGTACGATACC----->extension AGATACCTGGTCATGCTATGGTCAT.....GCTGGATGCATCTGATCTGCCTATCTC

(First annealing is imperfect because 5’ end of primer is single stranded for 3 nt. After cycle 1, annealing is perfect through all 21 nt and the mutation is copied into product by extension from reverse primer).

TCGATGGACCAGTACGATACCAGTA.....CGACCTACGTAGACTAGACGGATAGAG AGCTACCTGGTCATGCTATGGTCAT.....GCTGGATGCATCTGATCTGCCTATCTC

Using PCR to Add Additional Restriction Sites

Enzymes do not have a high efficiency if they are perched at the end of the DNA molecule.

Enzyme Oligo Length Chain Length %CleavageBamHI CGGATCCG CGGGATCCCG

CGCGGATCCGCG

81012

109090

USES OF VECTORS TO GENERATE TRANSGENICS

MMTV-LTR Gene XVector Backbone

CGCGTTCTTGAAAAGACAGGTAAAATGCGAGTTCCAGAATGGGTAGAATTGTAAAGT

CTGCACGAT CCATATGATCCAGATTGGTATTATATTAGATGTGC

TGCTTTAGTTCGTCATATTTATATTCGAAG AGTAACAAAAA

TTTTTGGAGGACGCAAACGTAATGGTACTCATCCTAGCCATTTCTGTCGATCGGCAG

GTGGTGTTGCTCGCAAAGCTCTTCAGAGCTTGGAACAACTTAAACTCATTGAAAAATC

TCCAGTTGGTGGACGTA CGTAGAGATTTAGATCGCATTGC

CAAAAAACAACTTAAGTTACAAGAAACTCTTGTTCTC

WILD TYPE:

MUTANT 1:

MUTANT 2:

MUTANT 3:

MUTANT 4:

MUTANT 5:

MUTANT 6:

*

Cloning sitesOriAmpr

Vector Backbone

Excision/Purification

Implant embryos into oviductof surrogate mouse

Genotype progeny (potential founders)using Southern Blot or PCR

Identify founders (express transgene) to generate transgenic lines

Analyze phenotype, biochemical analyses of tissues,Cross transgenics with other transgenics (bigenics)

Integrate transgene into pronucleus of 1 cell embryo (RANDOM INTEGRATION)

Transgenic

Surrogate

Generating Knockout Mice using Expression Vectors in Embryonic Stem Cells

Generate Targeting Vector & transfect ES cells derived from blastocyst

Homologous recombination of genomic and targeting alleles

Random Recombination

Random recombination & negative selection

Injection of Genetically Engineered ES cells into Blastocysts

Neomycin + Ganciclovir

Knockout mouse

                                Epidermal growth factor receptor (Egfr)

Phenotype depends on genetic background:   CF-1 background:

  Degeneration of the inner cell mass   Peri-implantation death

  129/Sv background:   Placental defects   Death at mid-gestation

  CD-1 background:

  Abnormalities in skin, kidney, brain, liver, and gastrointestinal tract (Threadgill et al., 1995)   Death by 3 weeks

  129/Sv X C57BL/6 background:   Death at birth

  129/Sv X C57BL/6 X MF1 background:   Death by postnatal day 20

  Phenotypic abnormalities in newborn mice (Sibilia and Wagner, 1995)

  Open eyes   Rudimentary whiskers   Immature lungs   Defects in the epidermis

Database of Gene Knockouts

             

                                               

                                 GENE KNOCKOUTS LISTED ALPHABETICALLY ACCORDING TO THE NAME OF THE GENE

The list can be scrolled through sequentially, or accessed by clicking any of the following letters:A B C D E F G H I J K L M N O P Q R S T U V W X Y Z

Example:

Embryonic Lethality How do we overcome

this limitation?

                                                                                                                                                                

The End