Bacterial Transformation Lab Quiz 3-15-22

Question 1

What does CaCI2 do to bacterial cells? Why is this important?

Answer 1

CaCI2 makes the bacteria membranes permeable to external DNA. This allows the DNA to take the plasmid.

Question 2

How does a Heat Shock work? What are the steps, and why do we do them?

Answer 2

Heat Shock is a process where the bacteria is cooled then suddenly heated up to a moderately high temperature for a bit then cooled back down again. The steps are leaving the bacteria in ice for more than 10 minutes, then warming them up to 42°C for 50 seconds, then using ice to cool them back down again for 5 minutes. We warm up the bacteria in the first place to make the membrane of them more permeable and open gates up to allow the plasmids to enter, then cool them back down again to trap them inside.

Question 3

What are the 4 important genes located on the plasmid pGLO and their functions?

Answer 3

ori = (origin of replication) tells the DNA polymerase where to start replicating
bla = beta-lactamase (ampicillin resistance gene)
araC = turns on GFP transcription if arabinose is present
GFP = Green Fluorescent Protein (from bioluminescent jellyfish)

Question 4

Explain the goal of our transformation experiment.

Answer 4

To understand and perform the process of bacterial transformation.

Question 5

Which tube is the negative control? Why do we use this?

Answer 5

The negative control is the LB only sample as none of the other elements involved aren’t in it, which are ampicillin, pGLO, and arabinose). We use this as to ensure no external factors are influencing the test as it’s expected to not respond/change.

Question 6

What will happen on the negative LB plate, negative LB/amp plate, positive LB/amp plate, and positive LB/amp/ara plate? Growth? Transformation? Glow?

Answer 6

Negative LB plate - growth as no amp is there to kill it, no transform and no glow as no pGLO
Negative LB/amp plate - no growth due to amp killing them, no transform and no glow as no pGLO
Positive LB/amp plate - growth as not all are killed by the amp due to pGLO, but no transform and glow since no arabinose
Positive LB/amp/ara plate - growth as not all are killed by the amp due to pGLO and there is glow and transform since there is arabinose

Question 7

What is the Promoter?

Answer 7

where the RNA Polymerase binds

Question 8

What makes the repressor?

Answer 8

araC

Question 9

How is the repressor removed?

Answer 9

The araC repressor is removed by the arabinose, which is an inducer. This clears the way for the RNA Polymerase to transcribe the gene.

Question 10

Transformed Bacteria are…

Answer 10

resistant to ampicillin and will glow brilliant green under UV light.