Hi, I would like to make sure if my answer is correct or makes any sense 1. Desi

Question

Hi, I would like to make sure if my answer is correct or makes any sense

1. Design and describe a strain/plasmid/gene combination that gives the following phenotypic result: You have created a plasmid that enables E. coli to have antibiotic resistance to kanamycin. In one strain of E. coli, the plasmid replicates and yields kanamycin resistant colonies. In a second strain of E. coli, the genotype of which only differs from the first strain by 1bp in total (there are no extra genes or missing genes and the chromosome sequences are nearly identical) the EXACT SAME PLASMID will replicate but does not provide antibiotic resistance. There are no modifications of the plasmid (methylation, etc...) that are different between both strains. (12.5 pts) The difference between the two strains could be in a CRISPR spacer region. In the first strain that does develop kanamycin resistance, the mRNA that would eventually go on to code for a protein enabling kanamycin resistance would not get degraded by the CRISPR system. However, a 1bp difference could allow for the CRISPR system in the cell to recognize the kanamycin resistance mRNA and degrade that mRNA. The plasmid would then still be replicated in the cell, but no proteins would be made to provide kanamycin resistance due to all of the transcripts getting degraded

Explanation / Answer

There are no modifications in the plasmid. Within the two strains, one strain would replicate and yield resistance to kanamycin whereas the other strain would not provide resistance to kanamycin. There is no deletion as no genes are missing. The only difference is in the size of the plasmid, which differs by 1 bp.

The explanation is correct. the difference is due to the CRISPR, but, the modification is in the crRNA, which is targeted. In this case, the CRISPR RNA is targeted so as to induce a mutation, where, the edited gene, kanamycin resistance, would be targeted. As this gene is targeted, there would be crRNA recognition, which would be done using a target locus. The Cas 9 would target this locus and there would be a marker free mutation with almost 100% recovery of the strain, such as in this case, where the difference is only of 1bp. This is also referred to as CRISPR mediated editing of the genome.

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