Week 13

--Monday 9/05/11--

After a weekend of more runs, we got together and have decided to reevaluate our methods for collecting data. After looking at the results from the 2%, and seeing that it wasn't too different from the 1% run, we've decided to keep our arabinose testing range between 0% and 1%. As for IPTG levels, however, we're going to start trying something different. So far, we've been inducing at 0 mM, 0.25 mM, 0.5 mM, and 1 mM. We are instead going to try 0 mM, 1 mM, 2 mM, and 5 mM IPTG. This will allow us to see if the current levels of IPTG are sufficient for inducing the expression GFP through derepressing the repressor, or if we need to do more runs with these higher concentrations of IPTG.

We have also reevaluated how to set up each plate. We've noticed that the methods we had been using were yielding a large amount of variation within each triplicate. We decided that instead of inoculating each well from a plucked colony, we would instead inoculate from a liquid culture in exponential phase. This returned much better results and significantly reduced our standard error. In addition to this, we changed the way in which we add IPTG to our wells. The previous method had us adding the different volumes of the same IPTG stock to each well. We determined that this was diluting our LB and throwing off our measurements for wells with more IPTG. We now have been adding the same volume from different stocks of IPTG to reach the desired IPTG concentration. Since these changes, our data has looked near perfect!

--Tuesday 9/06/11--

The results from our new IPTG concentrations looked good, so we are going to try a few more runs with them! We plan to set up and run a 0.66% arabinose plate and a 0.33% arabinose plate for our R0010 mutants. Both of these will have the four new levels of IPTG induction: 0 mM, 1 mM, 2 mM, and 5 mM. Along with these two runs, we also plan on screening 84 R0051 mutants.