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Team:Dundee/Project/Notebook
From 2013.igem.org
Notebook
Week 0
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 encoding protein phosphatase 1 (PP1). The problem was PP1-encoding gene has a PstI 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.
Week 1
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 amplify DNA encoding the three signal sequences we require (MalE, TorA and PrsA) and clone these DNA pieces into our plasmid (pUni-prom).
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 PP1 protein to allow us to perform Western Blots - looks like we have a lot of cloning ahead of us!
The Dry Team's first week included tutorials from our supervisors to get us up to speed with the biology, most of us had never done biology before so this was a steep learning curve!
Week 2
Yep, lots of cloning for the guys in the wet lab, most importantly ligating DNA encoding 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 engineering EnvZ and PrkC as possible PP1 detectors.
The team in the dry lab began work on “Operation Moptopus”, a modular computer acting as a lab in a box. We also began work on a model to compare the current detection method to our 1 hour detection method by analysing the production rate of algae.
Some time was also spent thinking about branding and our team logos and mascots, recruiting a graphic designer for some quality images, check out our Facebook profile picture!
Week 3
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 amplifying DNA encoding our possible PP1 detectors EnvZ and PrkC. Hopefully by next Monday we will have it cloned in proper!
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 bacterium.
Also this week we had a visit from a very special guest, our university rector and world famous movie star Brian Cox. Mr. Cox was very interested in our project, so much so that we might see some collaborative work from him in the future… Watch this space.
Week 4
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 5' regions of envZ and prkC.
The Dry team, this week, researched transcription and translation models and got together some rate constants from research papers. Our deterministic model began to take shape.
A few members of the team went to meet with Ranger George Potts in regards to the algal bloom problem at Clatto Country Park causing it to be shut over the summer. This proved to be very helpful, he also provided us with images of the park from the past and visitor numbers from over the years.
Brian Cox came back to visit us! We took him to the university radio station and he agreed to record some voiceovers for future use. Check out the youtube videos!
Week 5
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 lacking a functional Tat pathway that contain our TorA-PP1 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 MalE-PP1 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!
The detector team began cloning the PP1 gene downstream of envZ and prkC this week, hopefully it will go smoothly...
...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.
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. In addition, whilst developing the wiki website problems within the iGEM CMS were both noted and addressed. This information was to presented in the London Meet up to Randy Rettberg ( The President of iGEM) and all attending members of the YSB 1 in London.
We also featured in our local papers the Forfar Dispatch, Kirrie Herald and the Courier, getting the word out about synthetic biology and algal blooms to our local community!
