Team:Glasgow/Weekly Report/Week 3

Week 3

Wet Lab

• In the lab this week, we have been working on assembling some of the biobricks we require.
• The ligations of GvpA to pSB1C3 and GvpC to pSB1C3 were successfully transformed into Top10 and DH5 alpha cells. This was confirmed by restriction digests (using EcoR1 and Pst1) of miniprepped DNA taken from cultures of transformants.
• Creation of switch biobrick
  An important step completed this week was to ligate our recombinase switch into a plasmid vector. The switch was ligated into a pSB1C3 containing RFP; to achieve this, both the switch and the vector were digested using EcoR1 and Spe1, the desired fragments were then ligated – this resulted in the removal of RFP from pSB1C3. Thus, our switch biobrick was created.
• The switch was also ligated into two other pSB1C3 vectors: both with GFP but each with a different RBS – either 0034 or 0032. This was done by digesting the switch with EcoR1 and Spe1, and the vectors with EcoR1 and Xba1, allowing the insertion of the switch downstream of GFP and result in the formation of the scar sequence between the switch and RBS.
• This switch ligation to the vectors was successful, and then transformed into Top10 cells. We confirmed this with a restriction digest (EcoR1/Pst1) of miniprepped DNA from transformation cultures (see gel 10).
• Manipulation of GvpA and GvpC
  Before GvpA and GvpC can be ligated to the switch, we have to ensure that GvpA and GvpC are sufficent to produce gas vesicles. To do this, they will be inserted into a plasmid upstream of a promoter – in this case, the J23100 promoter. They will each be ligated separately. GvpA was inserted upstream of the J23100 by digesting the GvpA with Xba1 and Pst1, and the promoter vector (J6100z) with Spe1 and Pst1. This would cause a new sequence to be formed between J23100 and RBS of GvpA. The transformation of the ligation between J23100 and GvpA was successful, and confirmed by restriction digest as before (EcoR1/Pst1).
• Next, GvpC had to be inserted upstream of GvpA. To do this, GvpC/pSB1C3 was digested with Xba1 and Pst1, and J23100/GvpA was digested with Spe1and Pst1 – again, creating a new sequence between GvpA and RBS of GvpC. This ligation was set up at the end of the week.
• MotA isolation
  We also isolated the MotA gene. PCR was used to isolate MotA from E.coli strain DS941, the isolated MotA could then be used further. Restriction digests, followed by ligations, were set up to insert MotA into 2 plasmids pSB1C3 and the plasmid containing promoter J23100

Dry Lab

• Alterations were also made to the models; for example, we modelled the buoyancy of the bacteria with different gas vesicles volumes, as well as modelling their buoyancy in the absence of flagella.
• Design work began on the logo, and new pages were added to the wiki.

Admin and Outreach

• A big focus of this week was to prepare for the “Meet the Expert” event in the Glasgow Science Centre (5th and 6th of July).
• We made some Cartesian Divers (see picture below), and water buckets filled with bubbles – operated by a bike pump. These simple visual aids were crated to provide a simple explanation of our project, clear to both adults and children.
• For the event, we also designed a poster complete with an explanation of iGEM and synthetic biology, an outline of our project, and a comic strip. A short questionnaire was also drafted in an attempt to understand the public's perception of our project.