Question 1

Why do bacterial colonies appear blue on both the IX and IX/ARA plates in the CRISPR experiment described?

Accepted Answer

Because the lacZ gene is functional, allowing the bacteria to metabolize X-gal and produce a blue pigment.

Question 2

If the bacterial colonies did NOT have a functional version of the lacZ gene, what color would you expect the colonies to be on plates containing IPTG and X-gal?

Accepted Answer

White

Question 3

What is the role of arabinose in the IX/ARA plate during the CRISPR experiment?

Accepted Answer

It induces the expression of the Cas9 protein, enabling genome editing.

Question 4

In the context of the experiment, which component is necessary for Cas9 to target and cut the lacZ gene?

Accepted Answer

sgRNA

Question 5

Sample D contains bacteria from the IX/ARA plate transformed with pLZDonorGuide. Which of the following is TRUE about this sample?

Accepted Answer

It contains both sgRNA and donor template DNA, and Cas9 is ON.

Question 6

Which of the following best describes the expected outcome for Sample B (IX plate, pLZDonorGuide, Cas9 ON/OFF)?

Accepted Answer

Cas9 is OFF, so no genome editing occurs; colonies are blue if growth occurs.

Question 7

If a sample shows white colonies on IPTG/X-gal plates, what does this indicate about the lacZ gene?

Accepted Answer

The lacZ gene has been disrupted or cut, preventing functional β-galactosidase production.

Question 8

Which sample(s) would you expect to show evidence that the lacZ gene has been cut by Cas9?

Accepted Answer

Samples C and D, because Cas9 is ON and sgRNA is present in D.

Question 9

Of the samples that show evidence of lacZ gene cutting, which would also show evidence of repair via homologous recombination?

Accepted Answer

Sample D, because it contains both sgRNA and donor template DNA with Cas9 ON.

Question 10

A student observes blue colonies on a plate containing IPTG, X-gal, and spectinomycin after transformation with pLZDonorGuide and arabinose induction. What is the most likely explanation?

Accepted Answer

The lacZ gene was either not cut or was successfully repaired, restoring function.

Question 11

Which of the following best explains why spectinomycin is included in the plates for the CRISPR experiment?

Accepted Answer

To select for bacteria that have taken up the plasmid containing the spectinomycin resistance gene.

Question 12

Suppose you observe no growth on a plate containing IPTG, X-gal, and spectinomycin after transformation. What is the most likely reason?

Accepted Answer

The bacteria did not acquire the plasmid and are sensitive to spectinomycin.

Question 13

Which of the following equations correctly represents the relationship between the number of blue colonies ($$N_{blue}$$), total colonies ($$N_{total}$$), and the frequency of successful lacZ gene repair ($$f_{repair}) if $$only repaired colonies are blue?

Accepted Answer

$$f_{repair} = \frac{N_{blue}}{N_{total}}$$

Question 14

A researcher wants to maximize the number of colonies with a repaired lacZ gene. Which combination of components should be present in the transformation?

Accepted Answer

Cas9 ON, sgRNA present, donor template DNA present, and arabinose present.

Question 15

If a plate contains IPTG, X-gal, and arabinose, but no spectinomycin, what is a likely consequence for the experiment?

Accepted Answer

Non-transformed bacteria may grow, making it difficult to distinguish successful transformants.

Question 16

Which of the following best describes the function of IPTG in the CRISPR experiment?

Accepted Answer

It induces the expression of the lacZ gene by acting as a gratuitous inducer of the lac operon.

Question 17

A student hypothesizes that the presence of both sgRNA and donor template DNA is necessary for successful genome editing and repair. Which sample from the experiment best tests this hypothesis?

Accepted Answer

Sample D (IX/ARA, pLZDonorGuide, Cas9 ON)

Question 18

If the efficiency of homologous recombination repair is 20% in Sample D, and you plate 1000 bacteria, how many blue colonies would you expect to see, assuming all other factors are ideal?

Accepted Answer

$$200$$

Question 19

Which of the following statements best explains why the color of the colonies provides evidence regarding the state of the lacZ gene?

Accepted Answer

Only functional lacZ gene produces β-galactosidase, which cleaves X-gal to yield a blue product; non-functional lacZ results in white colonies.

CRISPR Lab: Interpreting Colony Color, Gene Editing, and Experimental Results

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