Difference between revisions of "Team:CLSB-UK/Experiments"

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.

Amplifying the parts and mini-prepping the plasmids

We used the following parts for our project:

• J23119 – a consensus sequence of an E.coli promotor in order to test if it is working in Synechocystis. As there aren’t many cyano parts available we thought it would be useful if this promoter worked and could be used for constitutive expression later on.
• K592025 – amilCP chromoprotein in order to test if the promoter is working. We understand the difficulty with a purple protein in cyanobacteria, but we also wanted to know is this reporter protein is useful.
• We used two other parts K1172501 and K1172303, but since we couldn’t track down a suitable RBS for these, we didn’t end up making a part with them.
• Empty pSB1C3 plasmid for the submission of the new part.
• CmpA was ordered from IDT with RBS and Bio Brick prefix and suffix synthesised as a G-block.

Transformation was carried out using this protocol.

Plasmids containing the parts were mini-prepped using this protocol.

Restriction digests; PCR clean-up and gel extraction

Table 1. Restriction digests for new parts.
Figure 1. What we expected to see on the electrophoresis gels from the parts where we had to gel-extract the insert.
Figure 2. Results we attained from our electrophoresis gel.

Ligation reactions and transformations

Table 2.Ligation for new parts.
Figure 3.The picture shows the transformation plates after 24 hours with colonies growing on all of them. Negative control (just a vector and no insert) had no growth.

Mini-prepping of new parts

We used the same protocolFigure 4. the photo of the streak plate with J23119+K592025 showing that even after 24 hours there is only faint expression of amilCP as this is a consensus sequence promoter.

Figure 4.The growth of the first part in the LB plate after 18 hours.
Figure 5.The pellet from the LB broth after the same length of time. There is no noticeable colour on the plate yet, but there is after pelleting.
Figure 6.The growth on a plate after 24 hours that just shows some purple colour appearing.