Question

6. Transposon mutagenesis was performed to identify genes that are involved in the biosynthesis of theA amino acid alanine. A wildtype culture of E. coli was infected with a defective phage carrying Tn10 A and the culture was plated on LB-tet media to obtain tet-resistant colony forming units. (LB is a rich growth media for E. coli and contains all amino acids required for bacterial growth) Individual CFU were then transferred to a multiwell plate containing liquid LB-tet media and growth occurred in all wells (Panel A) 1 2 3 45 6 7 8 LB-Tet Next, a multiprong device was used to inoculate an agar plate containing M9 media (a minimal media containing glucose as the sole carbon source). As shown below, most of the Tn10 clones were able to grow on M9 but the cells from wells A5, A6, B2 and C2 were not (panel B below). Another plate was used to identify the Tn10 insertions specifically located in genes involved in alanine biosynthesis (panel C) 1 2 3 4 5 6 7 8 1 2 3 4 5 6 7 8 M9 A. What was the selective media used in panel C? B. Identify the well(s) in Panel A that contain cells you can use to further characterize the pathway of alanine biosynthesis (i.e. - which well contains cells that have a Tn10 insertion in a gene required for alanine biosynthesis)

Answer 1

What is Transposon mutagenesis?

The process in which that allow the genes to modify or interrupt the extant genes in the chromosome that causes mutation in the organism.

What is Tn10?

Tn10 is a kind of Transposon that is responsible for mutation, consist of flanking five genes. The function of Tn10 is to insert in five genes into the chromosome randomly that could be involved in both recognition and cleavage of target DNA during insertion.

A. What was the selective media used in panel C?

M9 media with β-alanine. When β-alanine is added to the media that results in the growth of the cells in the wells. The β-alanine helps in the growth of the cellwall of the E.coli bacteria. When the alanine biosynthesis gene is distrupted the Alanine production is reduced, hence addition of β-alanine helps in the growth of cellwalls.

B. Identify the wells in Panel A that contain cells you can use for further characterize the pathway of alanine biosynthesis.

1. This can be identified by comparing the panel A, panel B and Panel C, Where On comparing the panel B and C we found that C2 was capable of alanine Synthesis.
2. The  wells A5, A6 and B2 that contain Tn10 was not able to grown in M9 media in Panel B.
3. On comparing Panel B and Panel C we can conclude that C2 well is capable of alanine Biosynthesis.
4. When Comparing the Panel A and Panel C we conclude that the wells A5, A6 and B2 is not capable of Alanine Synthesis.
5. Hence, Except the Well such as A5, A6 and B2 in panel A, all the wells are responsible for the synthesis of Alanine.