Transformation Of E. Coli With pGAL Plasmid

Question 1

Explain what is bacterial transformation

Answer 1

The uptake of exogenous DNA by cells, resulting in a newly acquired genetic trait that is stable and heritable .

• E.G. antibiotic resistance and presence of blue bacterial colonies indicating the expression of specific gene
• allows for the introduction of plasmids in bacterial cells
• makes possible the propagation, genetic expression and isolation of plasmid DNA

Question 2

What is plasmid?

Answer 2

A small, circular, double-stranded DNA molecule that exists outside the main bacterial chromosome

• can replicate independently
• Does not integrate into the bacterial chromosome
• The genes carried in plasmids often provide bacteria with genetic advantages, such as antibiotic resistance
• Used as tools to clone, transfer, and manipulate genes

Question 3

What is the Lac operon?

Answer 3

The Lac operon:

Three proteins encoded in a single expressionless unit of DNA
-an efficient mechanism for metabolizing glucose

-proteins produced only in the presence of lactose for its metabolism

The E. Coli strain used in this c,ass experiment is Lac Z-, but some can produce B-galactosidase

Question 4

Give the DNA map of pGAL

Answer 4

Amp^r gene- B-lactamaase is secreted inactivates ampicillin

LacZ gene- B-galactosidase cleaves X-gal and the pGAL transformed colonies will be blue

The pGAL plasmid- used in this experiment contains the complete lacZ gene, but no MCS

Repressor protein cannot bind to the lacZ gene on pGAL - no binding site, so lacZ gene on pGAL is always “turned on”

Question 5

What is competency ?

Answer 5

A physiological state in which bacterial cells are able to take up DNA and become genetically transformed
-naturally competent bacteria: Bacillus subtilis, Haemophilus influenzae, Neisseria gonorgaea, streptococcus pneumoniae

Question 6

How to prepare competent cells?

Answer 6

Treat with solutions containing chloride salts of metal cations such as calcium, rubidium or magnesium e.g., CaCl2

• Heat-shock treatment: rapid hot and cold temperatures
• Electroporation: place cells in electric field.

***These changes affect the structure and permeability of the cell wall and membrane such that DNA can pass through

Question 7

What are the expected results?

Answer 7

X-gal/control 1 = plate covered with white colonies(no ampicillin to kill bacteria)

AMP/X-gal/control 2= no growth (ampicillin killed bacteria)

AMP/X-Gal/pGAL= blue colonies that may have white satellite colonies(the only one that received transformed DNA)

Only bacteria containing recombinant DNA will grow and the antibiotic kills the others were killed by antibiotic/ampicillin

Question 8

Summarize the treatment of E. Coli cells with cold CaCl2

Answer 8

1. E. Coli from the source plate are resuspended in an ice-cold CaCl2 solution
2. Plasmid DNA is added to half of the cells before they are “heat shocked” in a 42 degrees Celsius water bath
3. The heat shock step facilities the entry of DNA into the cells
4. Recovery broth is added to the cell suspension, and the bacteria are allowed to recover for 30 minutes at 37 degrees Celsius
5. This recovery period allows the bacteria to repair their cell walls and to express the antibiotic resistance gene
6. Lastly, the transformed E. Coli are plated on LB plates
7. Spread the cells by titling the plate from side to side or by spreading with wire loop. Cover plates, leave upright for about an hour to allow the cell suspension to be absorbed by the agar.
8. Stack your group’s plate on top of one another and tape them together. Put your initials or group number on the taped set of plates
9. After cell suspension is absorbed by the agar for approximately one hour, your instructor will place the plates in the inverted position (agar side on top) in a 37 degree Celsius bacterial incubator overnight (18-24 hours)

Question 9

What are satellite colonies?

Answer 9

Are small colonies which may appear around a large blue colony

• are not resistant to ampicillin and have not been transformed with the pGAL plasmid.
• grow in a region of agar where B-lactamase has diffused and inactivated the antibiotic ampicillin
• the number of satellite colonies increases if the concentration of ampicillin is low or if the plates have been incubated for a long time