Exercise 16: Bacterial Transformation Using the pGLO Plasmid

What is the process for artificial or induced transformation?

An electrical field can be applied (electroporation) or a chemical can be added to the bacterial cells to cause them to become competent and absorb the DNA.

Explain why bacteria on plate (LB/Amp/Arabinose) fluoresce and bacteria on plate (LB/Amp) don't.

Arabinose activates araC and GFP genes which produce the fluorescence. [the arabinose promoter is activated (GFP gene) and then transcription is activated once the arc protein binds to the arabinose promoter.

When the GFP gene is expressed, how do we know?

Bacterial cells transformed with the pGLO plasmid will fluoresce under UV light when the GFP gene is expressed.

What chemical allows for larger circular pieces of DNA (plasmids) to be absorbed in artificial transformation or induced transformation?

CaCl2

What is the purpose of calcium chloride (CaCl2) in the transformation procedure? What would happen if you forgot to add the CaCl2 tot he transformation mixture?

CaCl2 increases the permeability of the cell membrane; the larger pieces of DNA (plasmids) can't be absorbed

What process involves bacterial cells transferring plasmids from one cell to another in nature?

Conjugation

If competent E. coli cells are transformed with the pGLO plasmid what is expressed?

E. coli cells will produce GFP protein and fluoresce under UV light.

What are the characteristics of engineered recombinant plasmids?

Recombinant plasmids contain unique genes. Competent bacterial cells can be transformed with these engineered plasmids. Expression of the plasmid genes will cause the bacteria to produce new proteins and express new traits.

What does the araC protein bind to and what happens next?

The araC protein will then bind at the arabinose promoter to activate transcription of the GFP gene.

What happens at the "ori" site?

This is the site where DNA replication will begin on the plasmid.

What does it mean if the GFP gene is under the control of the arabinose promoter?

This means expression of the GFP gene is determined by activation of the arabinose promoter.

Explain why some bacteria were able to grow in the presence of ampicillin and other bacteria were unable to grow.

only +pGLO bacteria can grow because bla gene (ampicillin resistant gene) is present

What are agar plates containing ampicillin considered?

selective media; selects for E. coli cells that have been transformed with the pGLO plasmid

If cells are transformed with a plasmid containing an antibiotic resistance gene, they will be able to grow in the presence of ___.

that antibiotic

What is the araC gene on the pGLO plasmid do?

the araC gene produces the araC protein

The GFP (Green Fluorescent Protein) gene on the pGLO plasmid is under the control of what?

the arabinose promoter

What does the bla gene code for?

the protein beta-lactamase

If arabinose is absent what color colonies grow?

white colonies

What does araC acts as what?

an activator to turn on the arabinose promoter when arabinose is present

What is beta-lactamase?

an enzyme that breaks down the antibiotic ampicillin

What is an example of a newly acquired trait?

antibiotic resistance

Fluorescence is produced how?

arabinose is in the environment which activates the arabinose promoter on the GFP gene, but transcription doesn't occur until the araC gene binds to the arabinose promoter and transcription of the GFP gene occurs

For the arabinose promoter to be activated, what must be present?

arabinose must be present, as well as a protein called araC

What must be present for the araC gene to be activated?

arabinose must be present; When arabinose is present, transformed cells will express the GFP gene to produce GFP proteins. This will cause them to fluoresce under UV light.

What gene does the pGLO plasmid contain?

codes for the Green Fluorescent Protein (GFP)

Where is the GFP gene found?

found naturally in the bioluminescent jellyfish Aequorea Victoria, and allows the jellyfish to fluoresce and glow in the dark

Bacteria that produce beta-lactamase will be able to survive what?

in the presence of ampicillin (they become ampicillin resistant)

If the plasmid does not contain an origin of replication ("ori" site) what does this mean?

it means the plasmid won't be copied by the cell and it will not be passed down to daughter cells in the next generation

Which plate contains growth of untransformed bacteria? Why are the bacteria able to grow?

- LB plate because it's like a general TSA plate

What is the purpose of the heat shock step of the transformation? What would happen if you forgot to heat shock the bacteria before plating?

-denatures DNA -won't allow plasmids to be incorporated into DNA

If the media contains only LB what grows?

-pGLO / +pGLO

What are the important components of the pGLO plasmid in our particular lab?

"ori" site, the bla gene, and the arc gene

If the media contains LB/Amp what grows?

+pGLO

Why did growth occur on both (LB/Amp) and (LB/Amp/Arabinose) plates?

+pGLO has bla gene which allows for ampicillin resistance

If the media contains LB/Amp/ARA what grows?

+pGLO with green (fluorescence)

What does the "ori" site stand for?

"Origin of Replication"

What is Genetic Transformation?

Genetic transformation is a naturally occurring process in which small fragments of naked DNA are absorbed by a bacterial cell.

Where are plasmids found and what are their characteristics?

Plasmids are found naturally in bacterial cells and usually contain one or more genes that provide the bacteria with genetic advantages, but are not necessary for routine cellular processes..

What are plasmids?

Plasmids are small double stranded circular pieces of DNA commonly used for transformation in the lab.