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 TOPO-TA cloning 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, pBAD33 was therefore selected as it contains an arabinose-inducible promoter, however pBAD33 contains an EcoR1 site in the Cm 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 integrase reaction, the switch has to be inserted into a low copy number plasmid – in this case pSC101BB. 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.

Figure 1: Lots and lots of Flapjack cookies (baked by Martin)

Week 8

Wet Lab

• FliC
  Work was done to replace a fragment of the fliC gene in plasmid vector pCR2.1 with a synthesised segment. The synthesised segment is missing Pst1 and SpeI sites within the gene – unlike the original fliC. pCR2.1/fliC is cut with Nde1 Cla1 so that it can be ligated to the synthesised fliC segment. EcoR1 was used to determine in which orientation the gene was inserted.
• The DNA was also cut wth Pst1 to determine which orientation FliC had inserted into pCR2.1. Samples K and L were then taken to be eventually tested for their ability to rescue the FliC KO strain.
• pBAD33-based plasmids
  Another check was carried out on the mutated pBAD33 plasmid by digesting with EcoR1; this was to ensure the EcoR1 had a successfully been removed from its resistance gene and it would therefore be suitable for future use as a biobrick compatible vector. The mutagenesis was successful.
• Site-directed mutagenesis was also carried put on pSC101BB to alter a Spe1 site within the ori; the transformant has yet to be miniprepped and digested to confirm the success of the mutagenesis.
• The pBAD33 plasmid contains an arabinose inducible promoter and will eventually be used as the vector for φc31 integrase – this will allow for the inducible expression of the integrase and therefore a control on when our switch flips.
• The GvpA/GvpC PCR product was ligated into pBAD33 to allow for gas vesicles expression to be controlled. The ligation was successfully transformed into DH5α and confirmed by restriction digest with EcoR1 and Pst1. In order to carry out the ligation, pBAD33 was restricted with EcoR1 and Pst1, the same restriction digest was also carried out on GvpA/GvpC PCR product.
• Inducible promoter
  Another inducible promoter was selected for eventual use in initiating integrase expression. A tetracycline promoter was selected and then inserted into the vector know as J61002 in place of the J23100 promoter. To achieve this, the J23100 promoter had to be first cut out of J61002 with EcoR1 and Spe1. The Tet promoter (R0040) was then also digested with EcoR1 and Spe1, and then ligated into J6100Z downstream of RFP. The success of this ligation will be confirmed by restriction digest and is selected for through the use of two different antibiotic selections.
• Switch/RFP/GFP and fluorescence
  By observing the lack of colour (in white light) in colonies which contained reverse RFP/switch/GFP/pSB1C3, when compared to J23100/RFP colonies, it was thought that the presence of the att sites may be effecting the efficiency of the J23100 promoter in this switch. Therefore, the fluorescence of the switch RFP and GFP needed to be compared to the florescence of J23100/RFP and J23100/GFP. A construct contained J23100 promoter downstream of GFP was required.
• To accomplish this, the pSB1C3 plasmid containing GFP was digested with EcoR1 + Xba1, the J23100 promoter was inserted as an oligo with EcoR1 and Spe1 sticky ends. The resultant ligation was then transformed and ligation will later be determined by restriction digest.
• Work was done to attempt to insert the switch conformations into a low copy number plasmid that will be compatible with pBAD33-int (pSC101BB). Firstly, the switch constructs (Reverse RFP/switch/GFP and reverse MotA/switch/GFP) must be extracted from pSB1C3. In the case of MotA/switch, the construct was digested with EcoR1, Spe1 and AlmN1 in an attempt to better separate the desired DNA on a gel.
• The MotA/switch/GFP was also tested to determine whether or not the insertion of MotA had interfered with the switch's ability to respond to integrase. The DNA was exposed to 8uM integrase in vitro and then transformed into Top10 cells. Six transformants were then eventually chosen from short streak plates: two colonies with bright GFP (B) fluorescence, two colonies with intermediate (G) fluorescence and 2 with no (W) fluorescence. The DNA from these transformants was then digested with HindIII and Pst1.
• As expected, the “B” samples “switched” DNA whereas the “W” samples did not: the G samples seem to contain a mixture of both switched and non-switched DNA.
• Integrase
  Work has been done this week to ensure that φc31 integrase is biobrick compatible by removing an EcoR1 site from within the gene. As well as this, the φc31/pSB1C3 and gp3/pSB1C3 constructs were digested with EcoR1 and Xba1 to allow for the insert of a RBS (B0034) downstream of the genes.
• MotA
  As mentioned in the week 7 report, promoters of varying strength were ligated downstream of MotA. These constructs were then transformed into DS941 Δ MotA to observe whether or not the plasmids are able to rescue the KO swimming ability. Very little rescue was observed, the strength of the promoter appears to make no difference. Therefore, another component may be required to rescue the KO – for example, MotB.

Dry Lab

• Much work was done on how to best model gas vesicle movement. The gas vesicle experiement was repeated using varying salt concentrations to determine the effect changes in liquid density may have on the movement of the beads (and eventually the bacteria).

Figure 2: Very jammy Victoria Sponge Cake (baked by Robbie)