From 2012.igem.org

Latest revision as of 01:04, 27 September 2012

Welcome to the NRP UEA iGEM 2012 Wiki Lab Book

Please choose the relevant link to access our diary of that week!

Week Zero | Week One | Week Two | Week Three | Week Four | Week Five | Week Six | Week Seven | Week Eight | Week Nine | Week Ten | Week Eleven | Lab Protocols | Experiments

Week 1

The first week of work was an exciting time for the NRP UEA iGEM team as we officially started our iGEM project! We spent the week largely focusing on planning out our project and taking part in some human practices activities before we can officially begin lab work. We were able to take part in the University of East Anglia School of BIO Colloquium, have talks on our project from PhD students working on similar topics, visit a fellow iGEM team and take part in a radio interview.

Day 1 (09/07/12)

Today the University of East Anglia School of Biological Sciences hosted its annual colloquium, an event where members of faculty gather together to discuss news over the year, give talks on their research and present posters. We were able to listen to many great talks on the research that has been going on in UEA and view the posters on display, where we were able to find out which researchers in the school were looking into nitric oxide and other topics relevant to our project, with a view to working with them in the future. We also spoke to representatives from Bioline, Qiagen, Fisher Science and Star Labs about the products they sold which we would likely require later on in the project; a few representatives even spoke about possible sponsorships and working with the iGEM team more closely, and we were able to leave with a few business cards giving us a few avenues to pursue.

Day 2 (10/07/12)

Today was a day largely involving meetings and planning ahead of the start of the project the following day. We met in the morning to discuss the project and we confirmed that we would be going into the lab the next day to transform E. coli with some earmarked BioBricks. Richard Bowater and Richard Kelwick told us that they had been able to synthesise a hybrid bacterial and mammalian, nitric oxide-sensitive promoter using PyeaR and NS2E9 elements in two orientations which would be the starting point of our BioBrick. We then looked at BioBricks produced by past teams to decide which BioBricks would be relevant to our project and therefore we would be transforming into the chassis; we decided on using the nitric oxide sensor developed by our advisors, and two BioBricks associated with bioluminescence.

We also looked at deadlines for iGEM and began to draft our project outline for the deadline later that week. Richard Bowater also informed us of the COSHH forms we would be signing ahead of the lab work the next day and uploaded them to our Google Drive account so we could read over them and ensure our first day of lab work was completed with a high degree of safety. We then looked at the finances and began to discuss our trip to Amsterdam as well as organising the purchase of a Promega card for the university's Promega vending machine. The rest of the afternoon was then spent discussing our film, logo and future human practices events; we also found out that our radio interview would be in just 2 days!

In the afternoon we sat in on a talk from a representative from the Biotechnology and Biological Sciences Research Council (BBSRC) on funding for research and how changes are being made to grants. We learnt that the BBSRC wanted their grant applicants to consider the 'impact' of their research (both in a scientific and economical sense) as part of their applications for funding; following this talk we too will be looking at what impact our research may make as part of our work on the ethics and future applications of our work.

Day 3 (11/07/12)

Meetings

Our original rabbit-based logo

Today was our first day of lab work, however, before we could do that there were more meetings to be had and decisions to be made! We discussed the concept logos that our artist, Amy Congdon, had produced. Many of them contained the image of a rabbit, an image synonymous with UEA's campus, and an image found on this very wiki. We felt perhaps that because our logo would be very much our public image, and that outside of the iGEM world a logo baring a rabbit prominently may give the wrong impression of synthethic biology to the public, that the rabbit aspect of the logo may be better as a smaller piece of the image, or even non-existent in it. We did, however, believe that the "N=ORWICH" part of the logo was extremely important and clever in representing our project on nitric oxide , and this can be seen in the finalised version of the logo all over the wiki!

We also focused on the information we wanted to present in our interview on STAR Radio the next day. As we were told we would be on both the farming and the business portions of the show, we focused on how to discuss Synthetic Biology in the senses of both agriculture and business. Further to this, we ensured that we had a clear and precise definition of synthetic biology in order to help put the idea across to the radio show's listeners and hopefully publicise the branch of science and help dispel fears about it.

In the afternoon we had our end of day lab meeting where we built on the project outline for Sunday's iGEM deadline and contacted Cambridge's iGEM team to discuss a visit and meeting while we were in Cambridge for the radio interview.

Labs

Today was our first day in the labs and we began by transforming NEB 5-alpha Escherichia coli with three BioBricks we had picked out as being applicable to our project in order to characterise them and investigate how far we would be able to use them in our project. When choosing the BioBricks we looked at those that had been used in previous years to either sense/report on nitric oxide specifically, or those that been used in sensing/reporting on physiologically relevant molecules that we could adapt for nitric oxide sensation. The three BioBricks we used were:

. [http://partsregistry.org/Part:BBa_K381001 BBa_K381001]; produced by Bristol in 2010, a nitrite and nitrate sensitive promoter (PyeaR) using GFP to report nitric oxide presence. PyeaR is usually repressed by NsrR, however when nitrates are converted to nitric oxide, NsrR is sequestered thus preventing its repression of PyeaR and allowing the production of GFP. We can therefore, utilise this function in order to show levels of nitric oxide via GFP expression.

. [http://partsregistry.org/Part:BBa_K325909 BBa_K325909]; produced by Cambridge in 2010, a reporting BioBrick using L-arabinose to induce the pBADpromoter, which in turn activates the lux operon resulting in bioluminescence. We believe characterisation of this BioBrick may allow us to build a circuit along with nitric oxide-sensing BioBricks to result in a standard part that becomes bioluminescent in the presence of nitric oxide.

. [http://partsregistry.org/Part:BBa_K325100 BBa_K325100]; produced by Cambridge in 2010, a reporting BioBrick using L-arabonose to induce the pBAD promoter, which in turn activates luciferase and luciferin regenerating enzyme, resulting in bioluminescence. We believe characterisation of this BioBrick may allow us to build a circuit along with nitric oxide-sensing BioBricks to result in a standard part that becomes bioluminescent in the presence of nitric oxide.

Protocol on our first transformation can be found Here

We made an amendment to the original protocol that saw double the amount of cells being used in the experiment in order to maximise our number of transformed cells at the end. The agar plates with transformed bacteria on them were incubated over night.

Day 4 (12/07/12)

Cambridge

The NRPUEA Team meeting the Cambridge team

Today was the day of our road trip to Cambridge where we would be meeting the Cambridge iGEM team and visiting their labs as well as attending 'The Business Hub' program om STAR radio to give an interview on iGEM and synthetic biology. Before leaving the UEA Campus we finalised everything for the interview and gave mock interviews to Khadija and Pascoe, the two members of the team that would be speaking on air, in order to help prepare for the kinds of questions they may be asked. We also organised what our aims were for our meeting with Cambridge's iGEM team, and decided that as their project appeared to be of a similar like to ours that we would look into collaborating with one another.

We arrived at Cambridge's Downing Site, where their iGEM team were working in the Plant Sciences building, and were taken straight up to the lab to meet the team. In the lab we were introduced to their members and discussed our projects and the various problems we had encountered throughout the experimental side of the competition. Their team were a couple of weeks ahead of ours so it was interesting to hear their advice on certain aspects of the lab work; in addition we exchanged thoughts and constructive criticism on regards to each others projects. Ultimately we felt that both teams benefitted from the meeting and gain valuable information and experience.

Later in the day we traveled to STAR Radio 107.9/1 FM for our interview on their 'The Business Hub' show. The presenter met us and was extremely interested in the competition as well as synthetic biology as a whole. Khadija and Pascoe conducted the interview and did a fantastic job; the interview went very well and we were able to put across a lot of information on synthetic biology, iGEM and our own project. We hope that once the programme airs that we will receive feedback from members of the public.

Labs

After checking the plates of transformed E. coli we found that none had grown. We theorised that the chloramphenicol concentration of the agar might have been to high (50µg/ml) and decided to spend the rest of the week researching the growing conditions for the bacteria, the details of the cells we were using as well as compare various protocols of transformation used and developed by other iGEM teams in order to perform a successful transformation of E. coli again the next Monday.

Day 5 (13/07/12)

Human Practices

Our team artist, Amy Congdon, visited today in order to discuss our video to present at the Jamboree in Amsterdam as well as to discuss the design aspects of our project. The next Friday we will be going into a school in order to present about synthetic biology and the project, therefore Amy helped out the members of the team involved in that presentation to produce some equipment and props to aid with it. She also looked over our wiki designs and adapted the logo with our suggestions in order to produce a final draft of the logo that we all felt was the most suitable one. Amy's ideas on the video were extremely exciting and captured the entire team's imaginations as to our project and how we will be presenting it and we all began to anticipate starting to work on it. She also gave us some fantastic advice on the safety and ethics aspect of the project, as well as suggesting creative ways of addressing the issues involved with each.

Research

Those that did not meet with Amy instead focused on researching what had gone wrong with growing the E. coli and how it could be fixed, as well as looking at other BioBricks we could use and how to build the project on further once we had characterised some of the relevant BioBricks and produced our own. Having looked at previous protocols we felt that our agar plates contained too much chloramphenicol and therefore we decided it would be best to reduce the amount when trying to transform and grow the bacteria during the next week. We also looked at how previous teams had worked on creating data from BioBricks that sensed and reported on the presence of physiological molecules, and researched into how we could use expressions such as fluorescence or bioluminescence to determine the levels of nitric oxide present in an environment.

In the afternoon we were given a presentation by Sebastian Runkel, a PhD student at UEA whose research specialises in nitric oxide. He helped us to understand how nitric oxide was used in the body and what the major limitations and obstacles would be in working with it, obstacles and limitations we then went on to discuss and investigate how we could overcome them. Sebastian also spoke about using Salmonella as a potential chassis instead of E. coli due to its higher competency with nitrogen species, and we went on to discuss this as a possible path to investigate once we were able to produce a BioBrick in the E. coli chassis. This lead to new ideas on how our project might be utilised in the future.