Week 12

--Monday 8/29/11--

--Wednesday 8/31/11--

Today marked the beginning of our characterization of R0010 mutants. We plan on characterizing six mutants at a time by running many 96-well plates (8 rows, 12 columns) through the Tecan. We will have six rows for mutants, one row for wild type, and a final row for our controls. Each plate will have a constant arabinose level in all wells (except the controls) and will have 4 levels of IPTG induction across each row. Not only will we be able to get a lot of data about how each mutant responds to various repressor concentrations, but we will determine a good range of arabinose and IPTG levels to test over in the upcoming weeks.

The first characterizing run will be for our "good mutants" #1-6 at 0% arabinose. The four levels of IPTG that we have decided to start with are 0 mM, 0.25 mM, 0.5 mM, and 1 mM.

--Thursday 9/01/11--

We are starting to gather data at a very fast pace. The current schedule is to let each plate incubate for either 6 hours at 37 °C or 18 hours overnight at room temperature, and then to run each plate for 6 hours in the Tecan at 37 °C. This allows us to get in three runs a day: one at 8:00 am, one at 2:00 pm, and one at 8:00 pm. Thankfully we have a hardworking team that is dedicated to keeping the Tecan running 24/7!

So far, we have been able to add runs with 0.1% arabinose, 0.01% arabinose, and another 0% arabinose (all at 0 mM, 0.25 mM, 0.5 mM, and 1 mM IPTG levels) to our list of completed runs.

--Friday 9/02/11--

Today we reevaluated the arabinose levels that we plan to use in our run. The original plan was to try different values on a logarithmic scale (0%, 0.001%, 0.01%, etc.), but the strain we are currently using (bw22826) shows a more linear reaction to various arabinose levels. We are instead going to try value between 0% and 1%, where we believe 0% to be minimal repression and 1% to be near maximal repression.

We have put in a 0.5% arabinose run (still with varying IPTG levels of 0 mM, 0.25 mM, 0.5 mM, and 1 mM) and are setting up a 0.75% and a screen for C0012 repressor mutants.