Team:Dundee/Project/Notebook2

         <p> Since the <em>B. subtilis</em> cloning was proving fruitless, we decided to try a change of tack. In order to skip out the intermediate plasmid step we ordered primers to do a PCR which would add in restriction sites, allowing us to ligate our gene construct straight in to the <em>B. subtilis</em> plasmid vector pDR110. <br><br>

         <p> We did some more Western blots on our Tat mutants (&Delta;Tat)  however the results are pretty rubbish, looks like we need a new sample!<br><br>

         On the <em>Bacillus front</em>, we performed the PCR to add in the desired restrciton sites to the genetic construct and began attempting to clone it in to the <em>B. subtilis</em> vector.<br><br>

         This week the dry team also decided to split the work. Craig started setting up Matlab programmes for our stochastic modelling. Rachel and Nasir started getting to grips with NetLogo, modelling software, so we can visualise our bacterial mops and how the Sec and Tat pathways work within the mops.

         <p> Blots of the E. coli mop this week suggest that PP1 is present in the &Delta;Tat in the Sphaeroplast and Membrane fractions, but not in the cytoplasm or periplasm fractions, which is interesting. Wild type fractions are not blotting properly, may need to make fresh competent cells. <br><br>

         The detector team also managed to clone PP1 into EnvZ this week so we almost have a completed detector!Efforts to get the mop cloned in to B. subtilis finally came to fruition. The construct was cloned in to the appropriate plasmid vector and transformed in to <em>B. subtilis</em> cells. On to characterise it!<br><br>

         <p> &Delta;Tat blots still have strong PP1 banding in the sphaeroplast and membrane fractions whereas the wild type refuses to blot well.<br><br>

         Western blots for PP1 in the <em>B. subtilis</em> mop have not proved to be effective, no results. Looks like a bad sample after Coomassie staining.

         <p> Much the same story with the <em>E. coli</em> mop blots, &Delta;Tat works but wild type doesn’t. We began testing ToxiMop this week!  Although unfortunately one of our control experiments was found to actually contain PP1, as we found by Western blot. Never mind! The repeat experiment will actually have an empty plasmid vector next time! <br><br>

         There is still no sign of PP1 in Bacillus so we performed some PCRs to ensure that the insert DNA is definitely there. It bloody well isn’t, so we decided to focus our efforts elsewhere with the E. coli model.<br><br>

         For our detector we managed to get our GFP reporter going for our EnvZ detector so hopefully we’ll have a working PP1 detector very soon, how exciting!<br><br>

         NetLogo model for the sec gate was completed and tested online. To our luck, it worked! We will now begin our Tat pathway model.