Question

The genome of the bacteriophage M13 was modified to include a CRISPR-Cas 9 operon with the

following features:
The operon includes a gene coding for protein Cas 9, and a gene coding a sgRNA.
The CRISPR-Ca9 operon is under the control of a bacterial constitutive promoter.
The sgRNA gene includes a spacer flanked by two repeats.
The sequence of the spacer matches a sequence of an antibiotic resistance gene present in an antibiotic resistant form of a bacteria.
Bacteria containing the antibiotic gene do not contain any sequence matching the phage M13 genome in their CRISPR array.

1) You need to predict the outcome of three different experiments. In each experiment the same M13 modified phage (M13_MDR) infect a different version of the antibiotic resistant bacteria.

a) In the first experiment, a single copy of an antibiotic resistance gene is contained in the E. coli genome.
Identify the correct statements in the following list and briefly explain why statements are correct or incorrect. (6 pts)

1. Upon infection, the operon contained in the M13_MDR genome is expressed and a programed CRISPR surveillance complex target the bacterial genome

2. Immediately after infection, the bacterial CRISPR-array leads to the production of a programmed CRISPR targeting the M13_MDR genome.

3. The sequence of the sequence in the antibiotic resistance gene targeted by M13-DMR encoded CRISPR-Cas9 complex starts with a PAM sequence

4. The synthesis of the programmed CRISPR surveillance complex is likely to lead to the death of the infected bacteria.

b) In the second experiment, bacteria contain a plasmid including an operon with 3 genes under the control of the same promoter.
The three genes are:
A gene coding for antibiotic resistance (targeted by M13_MDR) and located immediately downstream of the promoter

A gene coding for an unstable antitoxin
A gene coding for a stable toxin
A single transcript containing the 3 open reading frames is produced when the operon is activated. As long as the antitoxin is present at high concentration the bacteria will survive even when the toxin is produced.
Note the genome of bacteria does not contain any copy of the genes contained in the plasmid

Which one of the two following treatment is correct? Briefly explain. (5 pts)

1. M13-MDR infection restore bacterial antibiotic-sensitivity but leads to bacterial death

2. M13-MDR infection restore bacterial antibiotic-sensitivity and bacteria survive in the

   absence of antibiotic

c) In the third experiment, bacteria contain a plasmid that only contains the antibiotic gene under the control of its own constitutive promoter. Toxin and antitoxin genes are not present in the plasmid or the bacterial genome. There is no copy of the antibiotic resistance gene in the bacterial genome either.
Which one of the two following treatment is correct? Briefly explain. (5 pts)

1. M13-MDR infection restore bacterial antibiotic-sensitivity but leads to bacterial death

2. M13-MDR infection restore bacterial antibiotic-sensitivity and bacteria survive in the

   absence of antibiotic

Answer 1

1)
A) Although the microorganisms hold the antibiotic gene since the one copy, it's been observed that the spacer sequence that is contained in the CRISPR Cas9 system or maybe the operon within the M13 MDR stress is going to be activated as it's that are underneath the constitutive promoter as well as hence it'll be the chance that it'll impact guidebook via the sgRNA to the antibiotic gene belonging to the germs as well as because of to which the bacteria will likely be targeted by this particular operom after the an infection with M13 MDR stress

B) When the 3 genes have been included to the plasmid. Then it is going to be the situation that the M13 will bring the antibiotic gene for the bacteria and the bacteria will be old as it'll once again be targeted through the sgRNA that has a reduced performance of detection as a result of the spacers that has been included and hence the bacteria will encounter death.

C) Even after getting rid of the toxin as well as antitoxin gene the antibiotic gene remains contained in the bacteria and just one gene is able to result in the exact same activity as the above 2 experiments and hence result in the demise of the germs once again thanks the constitutive activation of this operon by M13 MDR.

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