Team:Westminster UK/Notebook
Labwork
Weeks 1 & 2
During the first week, our team was focused on conducting tests to assess the viability of our bacterial stocks. Using E.Coli (Strain: Top 10) from the previous Westminster iGEM team, we first attempted to create bacterial stocks. The same was applied for the selection of the Shewanella MR-1 strains that our team was planning to use. Tests to assess viability were primarily focused on transforming the organisms using plasmids containing fluorescent markers such as RFP. Initial results were not promising, but after testing different stocks, our team was able to identify the best performing organisms and these would be used for our project. While testing different plasmids containing different promoters, our team was also able to select which promoter best performed in Shewanella. This promoter would be used later in the project.
Week 3
During our third week in the project, our team was preparing to receive the Chlamydomonas that was sent to us by the Kaiserslautern iGEM team. We have mostly focused on troubleshooting issues that we have encountered while preparing the TAP growth media for Chlamydomonas. Once the Chlamydomonas was first inoculated, our team had to utilize different facilities in our university to accommodate the need for sunlight the organism. Once the Chlamydomonas cultures grew, they were kept in the fridge in order to be used in the MFC preparation.
Week 4 & 5
During the fourth week, our team had already established both Chlamydomonas and Shewanella stocks that were ready to be inoculated in a MFC. Our team worked on setting up stock solutions that were going to be used to prepare the MFCs; once the stock solutions were ready, the MFCs were prepared for the co-culture experiment. As a result of the experiment design, our team first connected the MFCs to the reading equipment without a resistor attached. The resistor was later introduced and the MFCs were left running until the data collection period as finished.
Week 6
Once the data as collected from the MFC experiment, the media in the MFCs were analysed using a microscope to perform cell counts. Our team has decided to focus on the ratio of the Shewanella and Chlamydomonas and assess which media had the most even distribution. The voltage reading was also analysed to see if there was a significant difference between the different MFCs and also to check which preparation was the closest to the control, which had the highest reading.
Week 7
Once the collaboration co-culture experiment with the Kaiserslautern team was completed, our team started to prepare for our final experiment focused on the improvement of the MTR pathway. During this week our team was getting started with ligation assays; using the best promoters from the viability experiments from week 1, our team started to ligate the biobricks that were going to be used for our experiment in the MTR pathway. After the ligations were done, then transformed E.Coli cultures were prepared in order to increase the amount of our desired plasmids.
Week 8 & 9
After bacterial stocks were cultured long enough, the conjugation assay with Shewenella was ready to be executed. Our team has prepared separate Shewanella stocks in order to prepare for the final MFCs assay. Once the stocks grew, they were poured in double antibiotic plates to select for the Shewanella that had absorbed the plasmid. The selected cultures from the double antibiotic plates were then inoculated so that stocks would be ready for the MFC assay. During the ninth week our team also started to prepare the MFCs and to build the anodes.
Week 10
During this week, our team poured the MFCs containing the transformed Shewanella. Each containing a different transcription factor for one of the enzymes in the Mtr pathway, our team also prepared MFCs containing Shewanella that had expressed TPHP, which was also of interest to our team. The MFCs were left running for data collection and further analysis.
Week 11
During this week, the data from MFCs was collected and analysed. Preliminary results indicated that one of the transcription factors had a considerable effect in the electric voltage, where all others were as distinct. The similarity between the performance of the control, compared to the Shewenella expressing TPHP also indicated that the expression of TPHP did not have a detrimental effect. The TodE assay has also been concluded during this period.
