Transposase & C. perfringens gliding motility
Question 1 (4 points)
The experimenters used a plasmid-based mariner transposon mutagenesis system in this experiment with three unique features not present in other C. perfringens systems:
i.
The transposon is carried out on a replicating plasmid to allow every cell to have equally chances of random insertion ii. The transposon is under the control of an inducible promoter which is activated under certain conditions. In this case the promoter, Pbgal, a glalactose promoter, is induced under conditions by lactose, the sugar found in milk. iii. A negative selection center to remove the plasmid following transposition.
Question 2 (3 points)
Figure 2c shows the colony morphology of wild-type & mutant C. perfringens. The negative control, also having the same morphology as the wild-type, is HN13. HN13 shows the curvilinear flares characteristic of motile C. perfringens via gliding motility. The experimental strains are the HLL9 & HLL28 mutants, each containing the knockout of the flare gene. HLL9 mutants have more reduced miniflares compared to the wt whereas the HLL28 mutants have a more dramatic change in phenotype with even fewer flares so can’t perform gliding motility. The positive control is HLL50 with an in-frame deletion of the sagA gene required to form the filaments via end-to-end connections & thus have gliding motility. HLL50, as expected has no flare formation and is to control that the transposition wasn’t causing the flare-less phenotype.
The three strains containing plasmid pHLL42 are significant because they show flare formation can be partially rescued following complementation with the plasmid in all of the strains except the HLL50 strain containing the sagA deletion. The strains are also important because the rescued function proves the plasmid was incorporated.
Question 3 (2 points)
Transposons are mobile genes that can “hop” around in the genome and sometimes cause mutations in the genome. The transposase normally have a bias for what regions of the genome it