Team:Glasgow/Weekly Report/Weeks 9and10

Week 9

Wet Lab

• A construct containing the switch with MotA was transformed into DS941 strain with the MotA gene deleted. The ability of this switch construct to restore swimming in the bacteria will eventually be tested.
• The Reverse RFP/Switch/GFP construct and Reverse MotA/Switch/GFP were digested using EcoRI and XbaI to allow ligation into pSC101BB. The resultant ligation will be transformed next week.
• The synthesized (altered) FliC was successfully ligated into pCR 2.1 vector. This was accomplished by digesting using NdeI and ClaI. The ligation was then transformed into TOP10 cells.
  The success of the transformation will be tested by singly digesting the isolated DNA with EcoRI, SpeI and PstI. This will be compared to the original FliC to determine whether or not the synthesized FliC can be inserted into pSB1C3.
• The plasmid containing the J23100 promoter with GFP and the BB0034 RBS was successfully transformed into DH5α. These cultures will later allow for the comparison of J23100/GFP and Switch/GFP to establish how efficient the Switch promoter is after adding the intervening sequences and att sites between the promoter and coding sequence.
• The plasmid containing the J23100 promoter/GvpA/GvpC was successfully transformed into the DS941 strain with the deleted MotA gene. There is potential to use these transformants for a floating assay to try and determine how well the Gas Vesicle genes can prevent the cells from sinking.
• A growth curve of cells containing the pZJ7 plasmid with the araC promoter/GvpA/GvpC was established; this appeared to show that exposure of the cells to arabinose does little to effect the growth of the cells.
  A floating assay of these cells was also set up by resuspending two populations of cells in NaCl (cells exposed to arabinose and cells not exposed to arabinose). Images of the cells will be taken over time.
• A restriction digest of PhiC31 Integrase with RBS was set up using EcoRI and PstI to eventually allow the ligation of the construct into the pZJ7 vector. This would allow the ability of the switch to be tested in vivo.
• The glass bead experiment – mentioned previously – was repeated using different salt concentrations and measuring the movement of the beads over a certain length of time. This allowed the production of graphs which could potentially be translated to give a model of E.coli floating in solution.

Dry Lab

• A list of many of the constructs created was compiled. This then allowed the BioBrick compatible constructs to be uploaded to the iGEM registry page.
• More people involved in synthetic biology/water treatment etc. were contacted to try and gather more opinions as to how well our tool would work in specific applications.

Week 10

Wet Lab

• MotA
  It was found by setting up swimming assays using swarm plates that MotA is not sufficient to rescue the DS941 strain with the MotA deletion. MotA on its own cannot restore swimming to the cells. It was then thought that the MotA construct may require MotB to rescue the phenotype.
  Over the course of this week: MotB was isolated by PCR, the product was then digested with XbaI and PstI to allow its insertion upstream of MotA. J23 promoters of varying strengths (for example J23116) and MotA constructs were digested with SpeI and PstI to allow a ligation between the vector and MotB.
  The resultant ligation was then transformed into DH5α and DS941. If the transformations are successful, the isolated DNA could then be transformed into the non-swimming strain to try and rescue the phenotype.
• Reverse RFP/Switch/GFP
  The Reverse RFP/Switch/GFP in pSC101BB was sequenced and revealed no anomalies. The plasmid was then transformed into DS941 cells containing the PhiC31 Integrase in p15a-BAD (pZJ7) plasmid. This transformation should allow the integrase reaction to occur in vivo.
• The transformation of Reverse RFP/Switch/GFP in pSC101 BB into DH5α was unsuccessful.
  The transformation of the Reverse MotA/Switch/GFP in pSC101 BB into DH5α was also unsuccessful.
• Fluorescence
  Solutions of cells were set up to allow the relative fluorescence of the J23100 promoter constructs to be compared with the Switch constructs. J23100/RFP was compared to Reverse RFP/Switch/GFP which was not exposed to integrase (fluoresces red) and J23100/GFP was compared to Reverse RFP/Switch/GFP exposed to integrase (fluoresces green). The analysis of the fluorescence levels revealed that the J23100 construct were far more fluorescent than the Switch constructs.
• This analysis also suggested that there may be some GFP expression from the Switch construct when the Switch was not exposed to integrase.
• FliC
  The original FliC in pCR 2.1 vector was transformed into DS941 and DS941-Z1 strains in which FliC had been deleted. Swimming assays carried out with these transformants revealed that the FliC in pCR 2.1 was able to restore swimming in the deletion strains. Further development of this revealed that the more strongly FliC was expressed the better the swimming phenotype of the transformants.
• This suggests that FliC works better than MotA to rescue the non-swimming strains and may be a good candidate for use in the Switch construct.
• The synthesized FliC was successfully ligated into pSB1C3 by restriction digest with EcoRI and PstI. This serves as strong evidence that the two amino acid changes in the hypothetical FliC BioBrick have been altered and corrected to allow FliC to be BioBrick compatible.
• he synthesized FliC in pSB1C3 was then successfully transformed into TOP10 (confirmed by miniprep, digest and gel electrophoresis). An oligo was then designed to allow a promoter and RBS to be inserted at the beginning of the synthesized FliC gene. This would then allow the ability of the synthesized FliC to rescue the deletion strains to be tested.

Dry Lab

• More work was done using the glass bead experiment to try and determine if the experimental set up allowed for the observation of different speeds of the glass (silica) beads.
• More random walk model was done to try and quantify the randomness of movement by swimming.