Team:Glasgow/Weekly Report/Week 4

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.