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.
We decided not to perform the 3A assembly as we felt that, although more difficult and involving more steps, standard assembly would allows us more versatility in our approach and we would learn more doing it. Below are the techniques that we have used throughout our project in order to make the relevant parts, introduce them into replicative plasmid for Synechocystis PCC6803 and transform this cyanobacterium. These methods comprise of:
- Amplifying the parts and mini-prepping the plasmids
- Restriction digests, PCR clean-up and gel extraction for insertion into pSB1C3
- Ligation reactions into pSB1C3 and transformations
- Mini-prepping of new parts for submission and to use for digests for the next step
- Restriction digests, PCR clean-up and gel extractions for insertion into pDF-lac
- Miniprepping the amplified pDF-lac plasmid and transformation of Synechocystis PCC6803
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 PCC6803. 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 gBlock.
Transformation was carried out using this protocol.
Plasmids containing the parts were mini-prepped using this protocol.Back to the strategy Back to the top
Restriction digests, PCR clean-up and gel extraction for insertion into pSB1C3
Gel electrophoresis - Here is the link to the original expereiment. For our experiment we have adjusted the steps very slightly. Firstly we used 1% agarose gel at 100V for 30 minutes instead of the given timings. Also we imputed the SybrSafe dye and dissolved 5ul in 100ml of the gel before it solidified.
PCR purification - Here is the link to this experiment we did.Back to the strategy Back to the top
Ligation reactions into pSB1C3 and transformations
Ligations - Here is the link to this specific experiment we did.
Mini-prepping of new parts for submission
We used the same protocol as previously. However this proved difficult, as colonies grew very slowly with two constructs that contained the J23119 promoter. Figure 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 7. shows the mini-preps from the three constructs/ parts. We ran 1ul of 1ug/ul ladder alongside 3ul of the three mini-preps. The photo is bad quality and was more obvious on the gel. We have repeated this process 4 times and concentrated the plasmids from all these mini-preps in order to submit it. It is also obvious that the first two parts that contain J23119 promotor had less growth in LB broth and, hence, less plasmid mini-prep. This is why we decided not to use J23119+cmpA for transforming Synechocystis, but to just use cmpA and a promoter already present in pDF plasmid
The mini-prep procedure was repeated several times and the plasmid DNA was concentrated from all of these submissions.Back to the strategy Back to the top
Restriction digests, PCR clean-up and gel extractions for insertion into pDF-lac
Miniprepping the amplified pDF-lac plasmid and transformation of Synechocystis PCC6803
pDF-lac plasmid was miniprepped using the same technique as already desribed.
Synechocystis PCC6803 culture received as a streak on a BG11 + glucose plates. Culture was immediately restreaked on another plate under sterile conditions and a liquid stock set up. Liquid stock was set up using 200ml X1 BG11 solution and 5 loops of the original culture from the streak plate. Liquid culture set up under 26ºC with 2000 lux.
Once the culture was in the exponential phase of growth, 20ml of it was centrifuged for 30min at 4000rpm and the sediment was washed with 20ml of fresh BG11 twice (resuspended, centrifuged and repeated). Final sediment was resuspended in 1ml of BG11 medium. This was aliquoted into 100ul and 25ul of the miniprepped plasmid was added, Mix was left in the light for 4 hours, after which it was transferred into a dar incubator at 30ºC overnight. Following this, full volume was plated onto BG11 plates with the mix of streptomycin and spectinomycin and left for 4 days.