Team:CLSB-UK/Parts

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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.

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

Parts created by CLSB-UK team

We have created the following parts for our project:

Table 1. Table of parts created by the CLSB-UK team.

BBa_K2078000

Parts available for transforming Synechocystis PCC6803 are very rare, which is one of the reasons why this chassis is not used very often. One of the limiting factors in the growth of cyanobacteria is their ability to acquire carbon dioxide, but there is currently no part coding for any elements of the bicarbonate ion transport system. This is why we wanted to make a part that would code for one element of it, by cloning cmpA gene out of the Synechocystis PCC6803 genome. However, having performed necessary calculations we found out that we couldn't successfully clone this gene and would have to order it as a G-block from IDT. having done that, we amplified it using PCR and used this insert to create two parts as shown below.

First of these parts simply contains cmpA gene with a suitable RBS and we hope that other teams working with cyanobacteria in the future will find it useful.

Figure 1. Cloning strategy used to produce BBa_K2078000 part. cmpA was ordered from IDT as a gBlock together with the forward and reverse primers. It was amplifies using PCR thermocycler.

Figure 2. pSB1C3 plasmid map showing BBa_k2078000 part.

BBa_K2078001

Parts available for Synechocystis PCC6803 chassis are few and far between, so we struggled to find an available promoter that was suitable for our project. This is why we opted for an E.coli promoter in the hope that it would work in Synechocystis, too. Whilst the promoter chosen is a consensus sequence in E.coli, we are hoping that, once tested in PCC6803, it will work well as a constitutive promoter.

Figure 3. Cloning strategy used to produce BBa_K2078001 part.

Figure 4. pSB1C3 plasmid map showing BBa_k2078001 part.

BBa_K2078002

The main aim of this part was to check if amilCP could be used as a reliable reporter protein in Synechocystis PCC6803. If the colour of the chromoprotein were to be visible, it would allow for this to be an extremely useful tool in the work with cyanobacteria.

Figure 5. Cloning strategy used to produce BBa_K2078002 part.

Figure 6. pSB1C3 plasmid map showing BBa_K2078002 part.