pGLO Bacterial Transformation

DNA RNA Protein Trait

SAFETY FIRST!

• WEAR GLOVES• DO NOT OPEN PLATES• CLOROX LAB STATION WHEN DONE• BRING PLATES TO BISHOP TO BE DESTROYED.• WASH HANDS WITH SOAP – HAPPY BIRTHDAY

JUMP TO SLIDE 29

What was the point of this lab?

What was alive?

Escherichia coli, a bacterium

Glass E. coli by Luke Jerram

Escherichia coli (E. coli)

• Found in human large intestines• Model organism for molecular biology

• model organism – species that has been extensively studied to better understand biological phenomena and give insight to workings of other organisms

• “White rat” of molecular biology

light microscopescanning electron microscope

What was the source of “new genes”?

A plasmid, genetically engineered to carry specific genes

Symbol Bacterial structure Illustration of E. coli K-12

Plasmid containing a few genes

Circular bacterial chromosome

Plasmid• commonly found as small circular, double-stranded DNA molecules

in bacteria; a NORMAL component of prokaryotes (and a few eukaryotes!)

• physically separate from, and replicate independently of, chromosomal DNA within a cell

• carry genes that may benefit survival of the organism (e.g. antibiotic resistance)

• may be modified to express proteins of interest; these “man-made” plasmids are widely used as vectors in molecular cloning

Bacterial DNA

Plasmid DNA

Bacterial cell

Genomic DNA

DNA in nucleoid region – NO nucleus

Bacteria have a single circular chromosome

Bacteria have accessory DNA, small circular pieces called plasmids

pGLO, a recombinant plasmid & vector

• Recombinant plasmid – plasmid into which DNA fragments or genes have been inserted

• Vector – plasmid used experimentally as tool to clone, transfer, and manipulate genes

• Because bacteria divide rapidly, they can be used as “factories” to copy DNA fragments in large quantities

How did you get the plasmid intothe E. coli?

TRANSFORMATION - Process by which bacteria take up plasmids from the environment and express the plasmid genes

EXPRESS – make proteins

TRANSFORMATION IS A NATURAL PROCESS BUT SOME BACTERIA ARE “BETTER” AT IT THAN OTHERS. Our procedure was developed to improve the likelihood E. coli will transform.

Bacterial TransformationProcess by which bacteria take up plasmids from the environment and express the plasmid genes

Beta lactamase(ampicillin resistance)

pGLO plasmids

Bacterial chromosomal DNA

Cell wall

GFP

Transformation Procedure • Suspend bacterial colonies in Transformation

solution • Add pGLO plasmid DNA

• Place tubes on ice• Heat-shock at 42°C and place on ice • Incubate with nutrient broth • Streak plates: LB LB/amp/+ LB/amp/- LB/amp/ara/+

Reasons for Performing Each Transformation Step?

1. Transformation solution = CaCI2Positive charge of Ca++ ions shields negative charge of DNA phosphates

Ca++

Ca++

OCH2

O

P OO

O Base

CH2

O

PO

O

O

Base

OH

Sugar

Sugar

OCa++

Why Perform Each Transformation Step?

2. Incubate on iceslows fluid cell membrane

3. Heat-shockIncreases permeability of membranes

4. Nutrient broth incubationAllows beta-lactamase expression

Beta-lactamase(ampicillin resistance)

Cell wall

GFP

What is Nutrient Broth?

• Luria-Bertani (LB) broth• Medium that contains nutrients for bacterial

growth and gene expression– Carbohydrates– Amino acids– Nucleotides– Salts– Vitamins

How do you know which cells transformed (took in plasmid)?

pGLO has reporter genes• bla (ampr) • GFP• araC

3 Genes of interest in pGLO plasmid • Gene for Beta

Lactamase (bla)– Protein that

inactivates the antibiotic ampicillin

• Gene for Green Fluorescent Protein (GFP)– Aequorea victoria

jellyfish gene– Protein fluoresces

green after absorbing UV or blue light

• Gene for araC regulator protein– Protein that regulates

GFP transcription – “turns GFP gene on”

This is DNA!

ANY QUESTIONS?

STUDENT MANUAL

• Lesson 1 page 33• 1, 2, 4 – you should be able to answer• 3 safety ideas?• Does not produce toxic compounds which

make people sick• Grows well in lab but can’t survive outside

lab• Not able to infect animals and plants

STUDENT MANUAL

• Lesson 1 page 34• Phenotype – physical appearance• Starter Plate:

Let’s answer the questions together.

STUDENT MANUALNumber of colonies?

What is a colony?Descendants of a single cell!

STUDENT MANUALSize of of colonies?

STUDENT MANUALColor of colonies?

STUDENT MANUALDistribution of colonies?

STUDENT MANUALAppearance in UV light?

STUDENT MANUALAbility to reproduce in presence of antibiotic?

STUDENT MANUALYou should be able to answer questions 1 and 2

STUDENT MANUAL

• Lesson 2 page 42• 1? You Explain• 2? You Explain• 3? You Explain• 4? You have 2 Control plates – You Explain

STUDENT MANUAL

• Lesson 3 page 43• 1, 2, 3, 4 You do.• But first let’s predict!

PREDICTION RESULTS

Did bacteria get plasmid?Is bacteria ampicillin resistant?Will bacteria glow in the dark?

PREDICTION RESULTS

Did bacteria get plasmid?Is bacteria ampicillin resistant?Will bacteria glow in the dark?

STUDENT MANUAL

• Lesson 3 page 44• 1, 2 You do• 3 Badly worded. Infer cells are expressing the

bla gene – making a protein that inactivates ampicillin

• 4 Hint what plates should you compare and why?

STUDENT MANUAL

• Lesson 3 page 45• 1 The sample did not flouresce• 2, 3 You do• 4 You must answer based on you resultsIf successful, state evidence from platesIf unsuccessful, state evidence from plates

STUDENT MANUAL

• Lesson 3 page 46• Yes, E. coli grow on the LB plate• 1, 2 you answer• 3 a. What are the 2 factors?• 3 b. What does arabinose do? What does UV

light do• 3 c. You answer

STUDENT MANUAL

• Lesson 4 follow the instructions and do the math.

• Page 48 #1. Some of you have no colonies on the LB/amp/ara/+pGLO plate

USE THIS AS NUMBER OF COLONIES: 50PAGE 51: REPORT TO BISHOP AS SOON AS YOU HAVE NUMBERS. WE WILL SHARE NUMBERS LATER.

Grow? Glow?

• Follow protocol

• On which plates will colonies grow?

• Which colonies will glow?

From Wikipedia, the free encyclopediaJump to: navigation, search Left: pGLO under ambient light

Right: pGLO visualized under ultraviolet lightThe pGLO plasmid is an engineered plasmid used in biotechnology as a vector for creating genetically modified organisms. The plasmid contains several reporter genes, most notably for the green fluorescent protein (GFP) and the ampicillin resistance gene. GFP was isolated from the jelly fish Aequorea victoria. Because it shares a bidirectional promoter with a gene for metabolizing arabinose, the GFP gene is expressed in the presence of arabinose, which makes the transgenic organism fluoresce under UV light. GFP can be induced in bacteria containing the pGLO plasmid by growing them on +arabinose plates.

pGLO is made up of three genes that are joined together using recombinant DNA technology. They are as follows:-Bla, which codes for the enzyme beta-lactamase giving the transformed bacteria resistance to the beta-lactam family of antibiotics (such as of the penicillin family)-araC, a promoter region that regulates the expression of GFP (specifically, the GFP gene will be expressed only in the presence of arabinose)-GFP, the green fluorescent protein

Like most other circular plasmids, the pGLO plasmid contains an origin (ori), which is a region of the plasmid where replication will originate. The pGLO plasmid was made famous by researchers in France who used it to produce a green fluorescent rabbit named Alba.

Other features on pGLO, like most other plasmids, include: a selectable marker, Ori (origin of replication), and an MCS (multiple cloning site) located at the end of the GFP gene. The plasmid is 5371 base pairs long. In supercoiled form, it runs on an agarose gel in the 4200-4500 range.[1][2][3]

                                             

The green fluorescent protein GFP consists of 238 amino acids, linked together in a long chain. This chain folds up into the shape of a beer can. Inside the beer can structure the amino acids 65, 66 and 67 form the chemical group that absorbs UV and blue light, and fluoresces green. (Credit: Image courtesy of Nobel Foundation)

Links to Real-world • GFP is a visual marker

• Study of biological processes (example: synthesis of proteins)

• Localization and regulation of gene expression• Cell movement• Cell fate during development• Formation of different organs• Screenable marker to identify transgenic

organisms

Using GFP as a biological tracer

http://www.conncoll.edu/ccacad/zimmer/GFP-ww/prasher.htmlWith permission from Marc Zimmer

Transformation Procedure Overview

Day 1

Day 2

What is a plasmid?

• A circular piece of autonomously replicating DNA

• Originally evolved by bacteria

• May express antibiotic resistance gene

or be modified to express proteins of interest

What is Transformation? • Uptake of foreign

DNA, often a circular plasmid

GFP

Beta-lactamase

Ampicillin Resistance

Transcriptional Regulation

• Lactose operon

• Arabinose operon• pGLO plasmid

Transcriptional Regulation

B A DaraC

B A DaraC

RNA Polymerase

Effector (Arabinose)

araC B A D

ara Operon

RNA PolymeraseZ Y A

Z Y ALacI

Effector (Lactose)

Z Y ALacI

lac Operon

Gene Regulation

RNA Polymerase

araC

ara GFP OperonGFP Gene

araC GFP Gene

araC GFP Gene

Effector (Arabinose)

B A DaraC

B A DaraC

RNA Polymerase

Effector (Arabinose)

araC B A D

ara Operon

The Many Faces of Plasmids

Scanning electron micrograph of supercoiled plasmid

Graphic representation

How do you know which cells transformed (took in plasmid)?

pGLO has reporter genes• bla (ampr) – transformed cells will grow on agar

containing ampicillin, an antibiotic– Codes for enzyme called beta lactamase

• GFP – transformed cells will glow in UV light– Codes for green fluorescent protein

• araC– regulates expression of GFP; GFP expressed only in

presences of arabinose, a sugar

Methods of Transformation

• Electroporation– Electrical shock makes cell membranes

permeable to DNA

• Calcium Chloride/Heat-Shock– Chemically-competent cells uptake DNA after

heat shock