Transformation of E.coli with pGal

Exchange of Genetic Information in Bacteria

1. Transformation

2. Transduction

3. Conjugation

Bacterial Transformation

Allows for the introduction of genetically engineered or naturally occurring plasmids

in bacterial cells

Natural and scientific uses!

Why do bacteria use plasmids?

They don’t usually contain genes vital to a bacterium’s day to day survival… But they do come with some other perks…

• Antibiotic resistance

• Provide a way for bacterial populations to quickly share useful genetic information

• Protect against stress related death

Transformation is not a super common occurrence! There is an evolutionary trade off… more on this later…

Why do scientists use plasmids?

• Scientists can force bacteria to keep them. Virtually all plasmids that are used to deliver DNA contain genes for antibiotic resistance. Once bacteria have been treated with a plasmid, scientists grow them in the presence of antibiotic. Only those cells that contain the plasmid will survive, grow and reproduce. The others will be killed by the antibiotic.

• They are copied independently. Plasmids can be copied numerous times, regardless of whether the bacterial host is replicating its own DNA, and every time a plasmid vector is replicated, so is the introduced DNA that it contains.

• They are circular. DNA that is circular is well suited to incorporate extra DNA sequences. That’s because it can be cut open without falling apart, then snap back together once new DNA has been incorporated.

• To make a foreign protein within bacteria. If the introduced DNA is a gene that encodes a protein, scientists can study the gene’s protein product by expressing it in bacteria.

pGal Plasmid•Multiple copies are present in E.coli•Replicates autonomously: does not integrate into the bacterial chromosome

Ori– Origin of ReplicationWhere DNA replication begins

B-Galactosidase•E.coli gene encoded by plasmid •In the presence of artificial galactosidases(X-Gal) the colonies will turn blue •Cells expressing B-Galactosidase will cleave X-Gal and produce a blue product

Ampr– Ampicillin Resistance • E.coli is NOT naturally resistant•Plasmid codes for B-lactamase = resistance gene!•B-lactamase is excreted into the media and inactivates ampicillin!

Restriction Sites

Molecule responsible for making colonies blue!

X-gal– present in the agar of some of the plates

B-Galactosidase– encoded by plasmid

Satellite Colonies– do not have pGal/B-lactamase but can grow bcamp has been deactivate by cells that have been transformed! Coooool!

Competency= Increasing Lab Efficiency!

Competence: the ability to take up foreign DNA and to become genetically transformed

How do we make the E.coli (lyphocells) more likely to take up our plasmid?

1. Calcium Chloride (CaCl2): weakens cell membranes

2. Heat Shock: facilitates entry of DNA into the cells

3. Recovery Period: allows bacteria to repair their cells walls and gives them time to express ampicillin genes

1.

2.

3.

Expected Results

????

Demonstrates that…

Colonies are white because the cells do not utilize X-gal. They do not contain pGal DNA which contains the gene for B- galactosidase.

Host cells are affected by ampicillin!•There is no bacterial growth•Without pGal cells do NOT have gene for B-lactamase and therefore are not resistant to the antibiotic

Cells have undergone successful transformation!•Are able to use X-Gal to generate the blue color.•Have acquired ampicillin resistance

Transformation Efficiency