Team:Cambridge/Diary/Week 4

Monday

Scandal in the lab! It appears that someone at Caltech has edited our wiki, initially inserting the word 'chicken' into our project page. Then they broke our Java script - potentially accidentally. Easily rectified, but still irritating, especially since the project description deadline was yesterday. Have we been the victim of a not-so-droll ivy league-ish prank? Or is some oddly minded person co-opting this student's account? We've emailed their project supervisor, so hopefully it shouldn't happen again.

One night seems to have been too little time to accurately determine if the transformation worked, as we now have lots of e.coli colonies on the plates. The bacillus plates have not been so successful, but the plasmid was not optimized to bacillus, potentially explaining their failure. We will need to make sure that we include more controls in future to work out where our experiments fail, if they should fail.

In any case, we were able to induce the pBAD promoter with arabinose in the e.coli cells. Results can be seen in the lab book page. We also streaked some further plates to grow up some more transformed e.coli, and inoculated some cells in liquid medium which will hopefully be ready to be tested tomorrow.

The construct we are hoping to submit to DNA 2.0 has also been partially designed. The fusion of the lux α subunit and the mOrange protein has been completed, as described in the project page (link in when a little more complete). The v.haveyi operon that we are hoping to have codon optimized has also been tided up, with more appropriate spacings between the genes.

Additionally, Andreas managed to finish his Arduino device. It now gives a response that is proportional to the ratio of the light intensities on the two light dependent resistors. By using a couple of coloured filters over the top of these resistors, we can measure the necessary spectral properties of our engineered luciferases. All the engineers need to do now is come up with a user interface.

Tuesday

Apologies from the editing parties at Caltech, apparently the remaining changes were not meant to be there. Unfortunately they also edited Brown's wiki. A storm may be brewing, depending how seriously they take the changes...

Anyway, apart from that little drama, lots happened in the lab today. Jolyon recieved his magnesium ion riboswitch primers - see what we did with them in the lab book. Paul and Andreas also made a short film link about their instrumentation.

Tom worked on making the construct we need to get ordered from DNA 2.0 using their program Gene Designer. He ran into a few issues, but Jim A. was able to give a few hints, and put him into contact with some friendly people in Newcastle who specialize in working with bacillus. If they don't know the answers, no one will.

Paul and Emmy are running into slight trouble with getting the right arabinose concentration for induction of luciferase production for the E. coli. It seems that with the optimum concentration suggested by previous years' teams there was very little light- so we added more arabinose. Cells are still not giving out any visible amount of light after 5 hours, and we now suspect that we might have killed the cells with too much sugar.

We're also having some problems deciding what approach to take with the input side of things. Our system should be very flexible, so it would be good to use many different biobricks from the registry. However, very few have been characterized in bacillus. Ideally we would want to find some way of transferring existing biobricks from e.coli so that they were reliable in bacillus, but we may find getting the right sensory proteins into bacillus complex. The idea was floated that we might want to approach the problem as a business problem, and do some market research into what the hypothetical end consumers of our product might want to sense. This would mean pitching to a specific group, potentially obscuring the main point of the project (the breadth and flexibility of the lux based system and its quantitative measurement of concentrations). For example, if we decided to create an agricultural kit as our prototype kit, we would want to create nitrate sensors, iron sensors, osmolarity sensors, fluride sensors etc. However, this would mean leaving out some very interesting additional sensory components. See what we eventually went with on our human practices page link here when we've decided.

Wednesday

The day started with gel electrophoresis of the PCR fragments that were made yesterday. As can be seen in the lab book, our genomic extraction appears to have been successful. However, we haven't managed to make our vector properly. Another day of PCR and gel electrophoresis, I fear...

Paul and Emmy inoculated cells from the streaked plates in liquid medium- hopefully these will work.

Prototyping of the hardware that we are hoping to use progressed to foam. Andreas is hoping to make the structure look more professional-like by spraying it with various faintly toxic (yet shiny) aerosols.

In the afternoon James Brown gave an inspiring talk, leading to us redefining our modules and goals. We also have a clearer idea on what our individual roles are.

Thursday

PCR with more controls and repeats, with slightly tweaked PCR parameters, resulted in yet more failure. The primers probably need to be redesigned.

Tom gets even deeper into construct design, with some light primer design for some of the simpler constructs to break up the day. With help from Orr from Jim A's lab, we were able to identify some constitutive promoters that we could use in the ratiometric construct. We further discovered that potentially all of the components that we need for the construct are in the iGEM 2012 Spring Distribution Kit! So we could start straight away once we have got the primers.

Our final designation or the team also occurred today, with us formalising our plans for the Summer in terms of the modules we are hoping to undertake. If all goes well, you should be able to see our modules under the project page.

Friday

Charlie had a brief chat with Dr. Vijayraghavan about potential applications of our project. She offered some useful advice given her proximity to the Bangalore and Caltech teams. See what she had to say in the human practices page link!.

Our newly inoculated bacteria shine ever brighter, with their induction by arabinose creating more successful results than before. In particular, Andreas and Emmy took them down to the dark room and tested if Andrea's electronics were sensitive to the light levels given off by the cultures. And surprisingly enough, it worked! A consistent 5-6% increase of the original base reading. The change was reliable and repeatable. The only worry is the time period for which light is produced by the bacteria after induction; full luminesence only takes place about 5 hours after the inducing arabinose is added.

Construct design by Tom continued, with the construct for the ratiometric emission by YFP and CFP based upon inducer concentration being made in a couple of hours. Difficulties persist with the construction of the lux operon for bacillus however. There really isn't enough data in the literature to make the RBS's properly - something which we may aim to rectify.

Lastly, the primer redesign was successful. Unfortunately, primer ordering is hampered by a lack of any add to basket button on the Sigma-Aldritch. Even more unfortunately, this week seems to be holiday week for the whole of the department, and everyone who could help is away. Something may have to be done over the weekend.