Week Five

Monday, 7/14/14

• Redid Gibson assembly of comX constructs to reattempt cloning
• Constructs transformed into JM109, plated on carbenicillin, and incubated overnight

• Grew liquid cultures of E. coli transformed with pWW2149+pWW1521 and pWW2149+pWW1523 (colonies had been allowed to grow on agar plates over the weekend)

• Set up overnight liquid cultures of DH5α-Z1 transformed with pAA001 (with pTet-pLac combinatorial promoter)
• Transformed DH5α-Z1 with Gibson product (aimed at assembling pAA003--containing pBAD-pTet combinatorial promoter) created Friday.

Tuesday, 7/15/14

• ComX System
  • Ran colony PCR again on overnight colonies with reassembled comX constructs (3 colonies picked from each of the 5 plates)
  • Ran a gel on the PCR products. Resulting gel.
• agrBD, lamBD, & FsrB Systems
  • PCR assembly of backbones with overhangs needed for assembly of agrBD, lamBD, & FsrB export systems
  • DpnI digestion of PCR product for 2 hours and then PCR purification were run to remove any non-backbone fragments of DNA
  • The five export constructs to test [link to images of the 5 plasmids we constructed] were assembled via Gibson using the PCRed backbones and geneblocks (containing the inserts) that arrived yesterday afternoon
  • 1 μL of each of the 5 Gibson products was then transformed into JM109. The bacteria were then plated on 5 carbenicillin plates & incubated @ 37°C overnight

• Testing the Scaffold System
  • Created glycerol stocks of the overnight liquid cultures (with bacteria transformed with pWW2149+pWW1521 and pWW2149+pWW1523) and stored them at -80°C
  • The remainder of the overnight cultures were then grown in clear MOPS media
  • Different concentrations of arabinose [specifically what concentrations?] were added to separate aliquots of the cultures in a 96-well plate
  • GFP fluorescence data was collected from these colonies in a plate reader over [how many hours post-induction?]
• Construction of lam & fsr Reception Systems
  • PCRed pKS001 template to extract vector backbone with overhangs for later Gibson assembly

• Began experiments on bacteria transformed with pAA001, testing different inducer concentrations:
  • ([IPTG] in μM, [aTc] in ng/mL): (0,0); (0,100); (100,0); (100,100)
  • Preliminary results promising: GFP significantly expressed [include link to the graph] only at 100 μM IPTG, 100 ng/mL aTc.
• Began assembly of agr reception system:
  • PCRed backbones for pAA008, pAA009, and pAA010 plasmids to include overlaps to be used in Gibson.
    • pAA008: insert contains agrC (receptor) with 4 SH3 domains (part of scaffold system)
    • pAA009: insert contains 2 components: GFP regulated by a P2 promoter and agrA (response regulator) linked to an SH3 peptide (other part of scaffold system).
    • pAA010: identical to pAA009 but does not contain SH3 peptide/scaffold at end of agrA
  • DpnI digested PCRed backbones and ran a gel to confirm presence of backbone. pAA008 backbone appears to have been successfully constructed, but the backbone for pAA009/pAA010 apparently failed (need to redo).
  • Gibson assembly of pAA008 backbone with geneblock containing modified agrC sequence

Wednesday, 7/16/14

Thursday, 7/17/14

Friday, 7/18/14