Wet Lab :

  1. Preparation of media and plates
  2. Started cultures of B. subtilis

Human Practices :

  1. Collected strains from Dr Mokherjee & learned protocols for culturing and transforming B. subtilis

Wet Lab :

  1. Optimising biomass staining assay
  2. Preparing competent B. subtilis stocks

Wet Lab :

  1. Optimising biomass staining assay
  2. PCR for 5’ and 3’ HRs of ΔspoIIE construct
  3. PCR to add overhangs to pMiniMAD (getting incorrect size band, unsure why)
  4. Restriction digest to troubleshoot pMiniMAD issue
  5. Preparation of electrocompetent E. coli

Dry Lab :

  1. Introduction to HDock : Gathered subunits from AlphaFold databank for K-12 E. coli RNA polymerase (RNAP) - Played around with using the online software

Wet lab :

  • Optimising biomass staining assay
  • Ordered new primers for cloning pMiniMAD
  • Biosensor - shifting away from adrenaline, now focusing on expression of fusion protein

    • Wet Lab :

  • Final optimisations for biomass staining assay
  • Obtained correct size band for pMiniMAD with BbsI overhangs for digest & ligation

    • Wet Lab :

  • Couldn’t get digest & ligation for ΔspoIIE construct to work, ordered new primers for Golden Gate
  • Golden Gate with the new primers worked first time, confirmed we have the correct plasmid in our transformed E. coli by colony PCR
  • Transformed miniprepped plasmid into B. subtilis, re-inoculating subcultures multiple times to remove resistance gene

    • Wet Lab :

  • Cloning components for the engineered constructs from the genome of B. subtilis
  • ΔspoIIE B. subtilis strain is ready, including resistance gene removed
  • Doing biofilm formation assays and sporulation assays with the ΔspoIIE strain
  • Designed biocontainment system & ordered primers/DNA synthesis
  • E. coli biosensor transformant is ready, checking fluorescence in plate reader and with fluorescence microscopy

    • Wet Lab :

  • Cloning components for the engineered constructs from the genome of B. subtilis
  • Long-term biomass assays with ΔspoIIE
  • Spore CFU count assay – no colonies grew even at only 10-3 dilution, likely due to issues with the FeSO4 in the lab being oxidised; ordered new FeSO4
  • Oligo annealing for RBS and terminator sequences
  • Did Golden Gate with synthesised biosensor construct, which failed; the oligo annealed terminator is the most likely issue; re-did oligo annealing with thermal cycler (instead of heat block)
  • Golden Gate assembly with oligo annealed RBS and terminator failed; ordered new primers
  • Did genomic extraction from B. subtilis to clone some large fragments that we’re struggling to clone from colony PCR

    • Wet Lab :

  • Cloning components for the engineered constructs from the genome of B. subtilis
  • Spore CFU count assay with new FeSO4 showed ΔspoIIE was effective
  • Golden Gate assembly with oligo annealed RBS and terminator failed; diagnostic PCR and restriction digest suggests the oligo annealed parts are causing the issue (unsure why); ordered new primers to clone the RBS and terminator from the genome/distribution kit
  • Testing/optimising the pathogenic biofilm incidence assay with wild type B. subtilis 3610

    • Wet Lab :

  • Cloned RBS and terminator sequence from the genome/distribution kit
  • Golden gate with these RBS and terminator parts finally worked; assembled and transformed the constructs into E. coli, then into B. subtilis
  • Took confocal microscopy images of wild type biofilm; ordered microscopy dishes to look at the biofilms of the engineered strains & invasion of mVenus-expressing E. coli
  • Biofilm formation assays on stainless steel and PVC

    • Wet Lab :

  • Assembled and transformed the final few constructs
  • Biofilm biomass assays of engineered constructs
  • Pathogenic biofilm incidence assays with engineered constructs
  • Inducible biofilm formation assays with mstX construct
  • Did confocal microscopy with the engineered strains – unsuccessful, possibly due to user error or issues with the microscopy dishes