Team:Glasgow/Weekly Report/Weeks 7and8

Week 7

Wet Lab

• FliC
  As mentioned in week 6, the miniprepped FliC transformant DNA was digested with EcoR1 and Pst1. The gel image of this digest revealed to topisomerase reaction was unsuccessful; this may be due to the insertion of a primer dimer in place of the insert.
• Integrase
  During this week, work was done to convert φc31integrase into a biobrick. In order to insert φc31integrase into pSB1C3, both the gene and the vector were digested with Xba1 and Pst1. The ligation of φc31integrase into pSB1C3 will allow for the insertion of a RBS. The resultant construct was then digested with EcoR1 and Alwn1. PSB1C3 was also digested – using Alwn1 and Xba1.
• In order to complete an in vivo integrase reaction, the φc31 integrase needs to be inserted into a plasmid with an inducible promoter, pZJ7 was therefore selected as it contains an arabinose-inducible promoter, however pZJ7 contains an EcoR1 resisitance gene.
• Site directed mutagenesis was carried out and the DNA was transformed into DH5α and Top10. The resultant DNA isolated from the transformants was digested with EcoR1 and revealed that site-digested mutagenesis was successful in DH5α #2.
• Also, to increase the success of the in vivo integrasenreaction, the switch has to be inserted into a low copy number plasmid – in this case pSC101BB (previously known as pZJ53B). A problem with plasmid, however, is a Spe1 site within the ori. For this plasmid to be biobrick compatible an attempt must be made to remove this site.
• Promoters
  Last week, four different strength promoters were transformed into DH5α; the DNA from these transformants was then isolated and restricted using Spe1 and Pst1 to remove RFP from the plasmid. The desired fragment was then extracted for later use.
• Ligations
  The four different promoters were then ligated to MotA, and then transformed into DS941. This will later be used to determine which promoter gives the best expression level of MotA to rescue the MotA KO strain.
• In order to carry out the ligation, MotA/pSB1C3 had to be digested with Xba1 and Pst1 to extract the desired MotA fragment.
• A similar method was used to ligate the GvpA/GvpC to the four different promoters. This would help to determine at what level of expression will gas vesicles be produced in a way to cuase floating. The GvpA/vpC PCR product was restricted with Xba1 and Pst1 for ligation into the promoter plasmids (digested with Spe1 and Pst1).
• Switch/RFP/GFP
  Over the course of the week, work was done on the source of the reverse genes/switch/GFP constructs. The reverse RFP/switch/GFP/pSB1C3 constructs were transformed into Top10 and DH5α, the DNA was then isolated and digested with HindIII and Pst1. This will give a pattern of the unflipped switch which can then be compared to switch exposed to integrase – therefore acting as additional proof that the switch promoter has changed direction.
• MotA
  Work was also done this week to switch off the expression of MotA in the MotA/switch/GFP construct by exposing it to integrase in vitro. The flipped switch was confirmed by restriction digest, and later the resultant transformants will be observed in a non-swimming strain to determine if any MotA activity (therefore swimming) is seen.
• Again, to confirm that the switch has flipped in response to integrase: three colonies exposed to 8uM integrase and three which were not exposed were selected and digeted with HindIII and Pst1. The in vitro reaction is not completely efficient as one of the samples has failed to switch – this can be confirmed by looking for presence of GFP.

Dry Lab

• The previous experiment which was being used to simulate bacteria in solution showed that the silicone beads fall extremely slowly.
• The next experiment was set up using denser glass beads, however, it was discovered that using paper as a diffuser resulted in slight blotches in the observation photographs.

Admin and Outreach

• The team signed up to participate in the Glasgow Science Centre's Explorathon to explain the project and also introduce aspects of synthetic biology to the public.
• As well as this, many individuals involved in synthetic biology and water industries were contacted to obtain some more options about the application of the project.
• Work was also done on the logo, and team photos were taken.

Week 8

Wet Lab

Dry Lab

Admin and Outreach