E8: Bacterial Transformation

The ability to introduce a specific gene into an organism and have it express that gene

Define genetic transformation.

constitutive genes lack regulation and are always on

inducible systems are turned on in the presence of a molecule that needs to be broken down

Compare constitutive vs. inducible gene expression.

E.Coli was used because it is a single celled organism that produces quickly and will not infect plants or animals and it cannot grow on plates with antibiotic (ampicillin)

pGLO can be used to control expression of fluorescent protein in transformed cells

What are some important characteristics of the strain and the plasmid in this experiment?

The bacteria with the pGLO DNA also codes for beta lactamase, which allows for the bacteria to be resistant to ampicillin

Why do we use LB/amp media to select for transformants?

it turns on the beta lactamase gene of the PGLO plasmid, which is an antibiotic resistant gene

What was the purpose of the arabinose?

LB/amp/arab produced glowing colonies because the arabinose activated the gene so it could be expressed which means glow.

Which plate, LB/amp or LB/amp/arabinose, produced glowing colonies? WHY?

the LB plate was the control without PGLO is the positive control (TMTC)

the LB/amp plate with no pglo is the negative control ( 0 colonies)

What are the control plates? What are their purposes?

CaCl2

Makes cells permeable to plasmid DNA

Ampicillin

Selects for the growth of transformed cells

pGLO plasmid

Provides the GFP gene

Induces GFP gene expression

Arabinose

heat shock

To increase the efficiency of transformation, cells will be placed in a 42°C water bath for 50 seconds during which step of the procedure?

1. Electroporation

2. Microprojectile

3. Protoplast formation

Which of the following is a method for making cells competent (permeable to plasmid DNA) ?

True

In our experiment, expression of the beta-lactamase gene (from the plasmid) is constitutive. (T/F)

False

HB101 is a strain of E. coli that is naturally antibiotic resistant.

False

During a transformation experiment with pGLO, the gene for GFP will remain turned off if arabinose is present in the media. (T/F)