Team:UC Davis/Notebook/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.

--Wednesday 9/07/11--

Today we continued to carry out the more runs with the four new levels of IPTG (0, 1, 2, and 5 mM). This included a a 0.33% arabinose run and two 0% arabinose runs (the first one didn't work well). After the 0.33% arabinose run, we were able to put together our data and construct our first 3D graph. The graph had an IPTG axis, an arabinose axis, and a fluorescence axis. Our graph as a whole supported the general trend that we expected to see. With more arabinose (increased repressor), we saw less fluorescence, and with more IPTG (derepressing the promoter), we saw more fluorescence. However, when looking at individual points, our data had a lot of noise. While all of our runs looked perfect individually, they looked bad when put together. Even after using the wild type to determine the relative fluorescence from each run, the data was very noisy and did not line up properly.

The inconsistency of our data tells us that we will need to reevaluate how we set up and carry out our Tecan runs. Firstly, we need to make sure that all runs are set up the same way. Instead of doing three runs a day with different incubation times, we are going to do two runs (10:00 AM and 10:00 PM), each with a 12 hours incubation time. The other thing we need to change is what we put on each plate. Right now, we think that we should do one mutant per plate instead of the usual six mutants per plate. This will allow us to test each mutant at 7 arabinose levels, and four levels of IPTG induction. While our data from the past week or two is mostly unusable, it has let us figure out some important information about which arabinose and IPTG levels to test at. For the LacI promoter/repressor pair, good levels of arabinose seem to be between 0% and 1%, and good IPTG levels seem to be between 0 mM and 5 mM.

--Thursday 9/08/11--

Because we won't be ready to characterize LacI mutants until the evening when our cultures grow up, we've decided to screen a set of R0040 mutants last night, and another set of R0051 mutants this morning. Tonight we will put in our first run with 7 arabinose levels and 4 IPTG levels. We will start with wild type.

--Friday 9/09/11--

This morning we put in another run with wild type. This run incubated for 12 hours at room temperature instead of for 6 hours at 37 °C. We hope to see whether or not these two methods of incubation cause any substantial difference in the data acquired from the Tecan reads. Next, we will put in a run for LacI mutant #1 (BBa_K611021)

--Saturday 9/10/11--

After finding that the last week of data collection was bad, we can't afford to take a weekend. The Tecan is still running 24/7. In order to make faster pace, instead of screening 1 mutant at 7 arabinose levels and 4 IPTG levels, we've decided to screen 4 mutants at 7 arabinose levels. This means that we'll be able to get a complete set of 2D data for all of our LacI mutants within two runs. Once we're done with this, we can fully characterize our pTet and cI lambda promoter mutants quickly. If time permits, we can go back to LacI and get the full amount of data for different arabinose levels.

--Sunday 9/11/11--

We've already finished our runs for characterizing the LacI promoter mutants at 0 mM IPTG and we have yet to gather successful set of our other two mutant types. So for today, we'll do another pTet screen and until we get new good mutants, we'll do the same sort of run that was mentioned on Friday (1 mutant, 7 arabinose levels, and 4 IPTG levels).