Team:Dundee/Project/Notebook

         <p> <p>One intrepid team member ventured in to the lab before anyone else to give us a wee head start. Much of this time was spent fiddling around with the iGEM plasmid pSB1C3 and the gene central to our project, protein phosphatase 1 (PP1). The problem was PP1 has a SalI site within it, a site that we needed to use to get it in to pSB1C3. This meant doing a lot of quick changes to change the sequence.

         <p>This was the first week doing proper lab work for much of the wet team, and we were thrown in at the deep end! However thanks to help from our supervisors and other experienced team members we thankfully found our feet. We managed to isolate the three signal sequences we require (MalE, TorA and PrsA) and clone them into our plasmid (pUni). <br><br>

         In addition to this, a lot of time this week was also spent mapping out our project and our strategies – for example, attaching a HA tag to our construct to allow us to perform Western Blots - looks like we have a lot of cloning ahead of us! <br><br>

         <p>Yep, lots of cloning for the guys in the wet lab, most importantly ligating PP1 and HA tagged PP1 (PP1 HA) into pUniprom containing our signal sequences. This should give us the construct: pUniprom – Signal Sequence – PP1, which should act as our molecular mop. On the detector side of things we began thinking about EnvZ and PrkC as possible PP1 detectors. <br><br>

         <p> A mistake with primer sequences set us back a little this week. The primer for our PP1 gene with a HA tag attached added the incorrect restriction site. Nothing daunted however, we immediately ordered new (correct) ones in addition to primers for our possible PP1 detectors EnvZ and PrkC. Hopefully by next Monday we will have it cloned in proper!<br><br>

         In the dry lab we began to model the dimensions of our bacterial mops E. coli and B. subtilis and we set up a small Matlab programme, to show PP1 being packed in the periplasm or on the surface, and give us the maximum numbers for each bacteria. <br><br>

         <p> This week the Wet team had to be split into three, to cover more ground and get more work done! The team was split into Detector, E. coli mop and B. subtilis mop. We also got sequencing data back for our constructs saying they were all correct, meaning that we could go ahead with cell fractionation and run our first Western blot! In addition to this the detector team managed to clone the N terminus into EnvZ and PrkC.<br><br>

           <img id="image-6" src="http://placehold.it/600x300/EB3C8B/000000&text=Something Cool">

         <p> Our first Western Blot came through this week, suggesting that export of PP1 via the Tat pathway was working far better than export via the Sec pathway. In light of this, we started work on characterising cells without a Tat pathway that contain our Tat  construct and blotting them against normal wild type cells with our Tat construct to make sure that PP1 was being exported by the Tat pathway. Another blot of the Sec construct was performed, the results of which seemed to suggest that PP1 may be getting stuck in the membrane of E. coli during export. Not ideal! <br><br>

         The detector team began cloning PP1 into EnvZ and PrkC this week, hopefully it will go smoothly!<br><br>

         Unlike the B. subtilis mop. We are having to jump through some hoops to get PP1 in to a plasmid that B. subtilis will accept. This involves using an intermediate vector in order to get the right sticky ends on the ends of the construct.<br><br>

         The main aim of the dry team this week was to put together poster designs and presentation layouts for our trip to London next week. This also meant both teams started to write up all the information they had so far for the poster and wiki.<br><br>

           <img id="image-6" src="http://placehold.it/600x300/8066DB/000000&text=Something Cool">