Team:Glasgow/Weekly Report/Weeks 3and4

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.

Figure 1: Empire Biscuits (supplied by Aimee)

Week 4

Wet Lab

• The working of the switch
  The switch works! An in vitro reaction, using purified phi c31 integrase was carried out on 2 plasmids, one containing our switch and GFP with 0034 RBS, and the other containing GFP 0032 RBS. Various concentrations of integrase were used (8, 4 and 2 μl/μl), including a control containing no integrase.
• The change in switch was confirmed by restriction digest with HindIII and Pst1. The plates were also visualised to confirm the presence of GFP, and for further confirmation of the correct switch, it was purified and sent for sequencing.
• GvpA and GvpC
  More work was done on GvpA and GvpC. A restriction digest was set up to insert GvpC upstream of GvpA in the pSB1C3 plasmid vector. This was accomplished by digesting GvpA/psB1C3 with Spe1 and Pst and GvpC/pSB1C3 with Xba1 and Pst1. These fragments were then ligated and transformed into empty Top10 cells. The J23100/GvpA/GvpC ligation was also transformed into Top10 cells.
• Attempts were then made to confirm that GvpC had been successfully inserted upstream of GvpA, by digesting with EcoR1 and Pst1. Unfortunately, the gels were difficult to interpret.
• MotA
  The ligation of MotA into the two separate plasmid vectors were confirmed by restriction digest. The ligations were then transformed and the DNA purifed and sent away for sequencing.
• Modification of plasmid pzJ53B
  This is a low copy number plasmid with kanomycin resistance, which could eventually be used as a vector for the integrase switch. A restriction digest was carried out on pzJ58B (NcoI and BstBI) to remove GFP, gp3 and attL/attR sites. This meant that a MCS (containing an iGEM prefix, suffix and a Kpn1 site to separate them), could be ligated into the vector. The addition of the Kpn1 site will allow for easy insertion of genes into the plasmid vector at a later date.

Dry Lab

• Steps have been taken to create a gif/video of the random walk model and the floatation model, to compare the two methods of transportation over time.
• Plans have been made for the monitoring of bacterial upward movement (when the gas vesicles are working!). This will be done by quantifying the amount of light scattered at layers in the fluid, to be detected by a webcam and analysed using appropriate software. Parts were ordered for this, and other components acquired and tested.

Admin and Outreach

• During the weekend, we attended the “Meet the Expert” event at the Glasgow science centre. There was a lot of interaction with the public, and we had a lot of interesting conversations with the public about our project. The displays were a big hit with the children.
• The dialectic society was contacted in an attempt to organise a debate on the subject of synthetic biology/GM.
• The wiki front page is under development, and some changes made to the existing pages.