Question

You've discovered a gain-of-function mutant E. coli strain that makes red colonies on plates containing normal minimal medium; wild-type E. coli make white colonies. To identify the mutant gene causing this unuusal phenotype, you decide to use a plasmid library transformation approach. Which of the following do you need?

A. A DNA construct in which sequences from the 5' end and sequences from the 3' end of the mutant gene flank a drug resistance gene.
B. Sequencing reagents, such as dNTPs, ddNTPs, and DNA polymerase.
C. A computer to blast the base pair sequence of the bacterial genomic insertagainst the genome sequence of your E. coli strain.
D. Cloned transposons from an organism other bacteria.
E. An E. coli genomic library made from the wild-type strain.
F. An E. coli genomic library made from the mutant strain.
G. Minimal media plates with no drugs or other supplements.
H. Minimal media plates containing the drug for which your genomic library plasmids have a resistance gene.
I. A sequencing primer that hybridizes adjacent to the insertion site of the bacterial genomic fragment in the library plasmids, and pointing in the 5'-to-3' direction towards the bacterial genomic DNA insert.

Answer 1

The correct answers are options

B. "Sequencing reagents, such as dNTPs, ddNTPs, and DNA polymerase".

C. "A computer to blast the base pair sequence of the bacterial genomic insert against the genome sequence of your E. coli strain".

F. "An E. coli genomic library made from the mutant strain".

I. "A sequencing primer that hybridizes adjacent to the insertion site of the bacterial genomic fragment in the library plasmids, and pointing in the 5'-to-3' direction towards the bacterial genomic DNA insert".

Step-by-step explanation:

Plasmid library transformation approaches are very useful to characterize novel mutant bacterium strains. In order to perform a characterization study of this kind, several reagents and tools are needed, including:

- "Sequencing reagents, such as dNTPs, ddNTPs, and DNA polymerase". One important part of knowing what makes novel mutant bacterium different is to sequencing its DNA.

- "A computer to blast the base pair sequence of the bacterial genomic insert against the genome sequence of your E. coli strain". BLAST is a useful computer tool that compares and aligns DNA sequences, this allows to detect what are the nucleotides that are unique in the novel mutant bacterium.

- "An E. coli genomic library made from the mutant strain". Once DNA has been sequenced, an genomic library must be constructed to ensure that this novel mutant is preserved.

- "A sequencing primer that hybridizes adjacent to the insertion site of the bacterial genomic fragment in the library plasmids, and pointing in the 5'-to-3' direction towards the bacterial genomic DNA insert". Sequencing primers are needed when new genomic libraries are constructed. This short DNA sequences allow DNA polymerase to synthesize the DNA an replicate the genome of interest.