Team:CLSB-UK/Proof

• Project
  • Fuel Cell
  • Synechocystis
  • Future Research
• Lab
  • Strategy
  • Experiments
  • Notebook
  • Parts
  • Proof of Concept
  • Results
  • Demonstrate
• Human Practices
  • Human Practices
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  • Silver Medal
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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.

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  Strategy
• 
  Experiments
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  Notebook
• 
  Parts
• 
  Proof of Concept
• 
  Results
• 
  Demonstrate

Proof of Concept

Our genetic modification of Synechocystis had two main aims: to increase the rate of electron export from the cell, for increased efficiency in biological photovoltaic(BPV) cells, and to increase growth rate, in order to make working with Synechocystis easier. Previous studies have shown the rate of growth to be the main obstacle in working with SynechocystisPCC6803 and we believe that solving this problem would lead to improvements in the potential generated by the BVP cell, too. To this end we have designed and carried out proof-of-concept experiments to demonstrate these.

In order to increase exoelectrogenesis (rate of electron export), we planned to introduce two parts into Synechocystis - oprF and RibH(link to the two part pages here). Unfortunately we encountered some problems when working with the parts (mainly, we couldn't locate a suitable RBS to combine with them in order for them to be expressed in our chassis), so it wasn’t possible to test these in Synechocystis PCC6803. However, we did manage to create a biological photovoltaic cell with Synechocystis PCC6803.

In terms of increasing growth rate of Synechocystis PCC6803, 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 growth in Synechocystis PCC6803 both before and after transformation. However, when we originally planned this experiment we were faced with a problem: the actual method to use to measure growth. We originally intended to use dilution plating and count bacterial colonies, but Synechocystis PCC6803 grows so slowly that this was not an option. Hence, we decided to exploit the vivid blue-green colour of Synechocystis PCC6803 and use a colorimeter at 680nm to measure how much light(of a certain frequency) was absorbed by the broth. Simultaneously, we had the UCL team confirm our rates for us 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 slopes to be 0.016 for untransformed and 0.034 for transformed Synechocystis PCC6803. As the transformations took a long time, we were unable to set up multiple cultures derived from colonies from multiple transformations plates, in order to obtain numerous repeats.

Figure 1.Growth curves of Synechocystis PCC6803 untransformed and transformed with the pDF plasmid containing the cmpA gene.