Team:Fatih Turkey/Experiments1


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Exposure Experiments

In this experiment, we aimed to stop gram negative growth in solid medium (LB) by exposing it to LALF via liquid culture of its host or supernatant. We used B.subtilis as the host of LALF. With the results; we wanted to confirm that both of our LALF and signal peptides work.
By adding E. Coli on Bacillus Subtilis;
Assay 1:  We put E. Coli on the B. Subtilis with LALF (K541915) biofilm. For this purpose, we first prepared a corn starch plate with liquid LB medium and added 100 ul B. Subtilis liquid medium as a point over it. For a qualified biofilm, we incubated it for 24 hours at 37˚C.
Later, we dropped 10 uL E. Coli with RFP as a single point and again incubated it.
In order to understand whether LALF protein or another factor killed E. Coli on the Bacillus biofilm, we prepared the same contrivance with a B. subtilis biofilm that does not produce LALF protein or does not include antibiotic.
Also, we prepared an alternative plate that does only include antibiotic in order to avoid a false result.
Assay 2: Without adding corn starch, production rate of biofilm by B.subtilis extremely decreases. This allows us to test the effectiveness of LALF in non-biofilm media. We put E. coli as a single point while B. subtilis spread is fresh. We aimed to see if B. Subtilis could kill E. coli without forming a biofilm.
We prepared a control group by preparing the same contrivance with only B Subtilis that does not produce LALF protein. Alternatively, we spread B. Subtilis that does not produce LALF protein but has antibiotic resistance and added E.Coli with RFP as a single drop over it.

 By adding E. Coli over B. Subtilis;
Assay 3: We added 100 ul liquid culture of B. Subtilis with LALF, over the normal LB medium applied E. coli with RFP as a single drop. The aim here is to test the effect of LALF contraversly comparing with Assay 2.
Over the fresh spread of E. coli, 100 ul of liquid culture of wild B. Subtilis strain as a single drop. As an alternative contrivance, we added B. Subtilis that has only antibiotic resistance over the fresh spread of E. coli with RFP likewise. We aimed to see the affect of antibiotic resistance over E. coli with RFP, by using B. subtilis with and without antibiotic resistance.
Assay 4: We repeated the Assay 3 by adding the supernatant of B.subtilis instead of its liquid culture. Of course, again we used fresh spread of E.coli with RFP and we added single drop of inhibitor solution.
Control groups were B.subtilis construct without LALF and antibiotic resistance. A second control group is designed to test antibiotic effect on inhibition by inserting antibiotic resistance of bacteria.