In the Lab
At the end of the day, an iGEM team’s project is made or broken in the lab. And at CLSB, if you were to walk along the science corridor to the small, unassuming lab that is Mr Zivanic’s, in the months leading up to Jamboree, be it before school or after, during term time or while the students are meant to be off school, you would undoubtedly find it bustling with activity. For this is where the iGEM team made our home over the last year. This is where we developed from a team that marvelled at the accuracy of our micropipettes and struggled to put on microbiology lab coats to one that routinely performed gel extractions with ease, and confidently recorded the growth rate of our cyanobacteria. We came from humble beginnings, but by soldiering on past cells that demanded -80ºC freezers and ligations that refused to yield any results for three weeks in a row, by coming in at the crack of dawn and leaving after the sun had long since set, by sacrificing our well earned summer rest while our friends went off on holiday, we have achieved more than we could ever have hoped for.
Proof of Concept
Our genetic modification of Synechocystis had two main aims. First, we planned to increase the rate of electron export from the cell. We anticipated that this would increase the efficiency of our biological photovoltaic (BPV) cell and increase the growth rate of Synechocystis. Previous studies have noted that the primary problem with Synechocystis is its slow rate of growth so we thought that speeding this up would be an important advance. We also believed that improving the rate of growth of Synechocystis would lead to improvements in the potential generated by the BVP cell. We designed and carried out proof-of-concept experiments to test these two hypotheses.
In order to increase the growth rate of Synechocystis sp. PCC 6803, we overexpressed the CmpA gene (which codes for a bicarbonate transporter) by linking it to a strong constitutive promoter. Our proof of concept here was simple: we measured the rate of growth in Synechocystis both before and after transformation. We originally intended to dilution plate and then count the number of bacterial colonies but Synechocystis PCC sp. 6803 grows so slowly that this proved to be very difficult. Instead, we decided to use a colorimeter to measure how much light (at 680nm) was absorbed by the broth. We then had the UCL team confirm our results by measuring the optical density of the broth using a spectrophotometer.
Our results show that the growth rates in the exponential phase of growth differ significantly. By focusing on the exponential part of the growth curve, we calculated the maximum gradients to be 0.016 for untransformed and 0.034 for transformed Synechocystis PCC sp. 6803. As the transformations took a long time, we were unable to set up multiple cultures derived from colonies from multiple transformations plates and so could not obtain numerous repeats.