New finding with some bugs

I have done 10 independent experiments to show that transformation frequency in ycaI mutant is about 5 folds higher than that of its wild type when Ampicillin concentration is 5 microgram per mL. Why my recent data shows that such effect disappeared, and transformation frequency of ycaI mutant is extremely low? Maybe the plate with which I picked up single colony to culutre is too old, I might have to restreak my plates with cells from the -70 refrigerator.

Nevertheless, after several repeats with large amount work (I am very tired this week because I have to go back home after 23:00 in these days), several points are clear. And I know some of the reasons why transformants can not be observed when protein inhibitor was used as the selection marker. Transformants appear much slower when protein inhibitor was used in transformation. The replica was done 10 h later than spreading and transformants can be observed when ampicillin was used as the selection marker 12 h later than replica. Few transformants can be observed when protein synthesis inhibitor was used as selection marker at this time. The latter type of transformants appear 12 h later than replica. That is, in all about 36 h is required for the formation of visible transformants. Transformants can be obviously observed until 48 h after plating or even later. This is quite a long time. In my previous experiments, I should have thrown plates away before the transformants visible. For transformants appeared on ampicillin plates, no such delay can be observed. All transformants appeared after 12 h later than replica. I also noticed that the 48 later appeared transformants has completely different layout comparing with other samples. Only the wild type behave like this. YcaI mutant has no such phenomenon. Both transformants appear 48 h later and the layout of transformants did not appear in YcaI mutant and samples pretreated with antibiotic (neither ampicillin pretreatment and spectinomycin pretreatment). With these information accumulated, it is quite possible that transformants appeared 48 h later should be the chromosomal transformants.

Strangely, MG1655 wild type did not produce transformants after 48 h. I need to redo this experiment and compare it with MG1655::dprA. Or I can do experiment in RR1, because recently I am very lucky in mutant construction. For more than one month, I failed to get dprA mutant in RR1. But luckily, I get it this week, and hofQ as well. Next week, I will have a lot of work to do. Firstly I need to isolate a large amount of DNA for my following experiments. But first of all, I need some rests......