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.
    1. pAA008: insert contains agrC (receptor) with 4 SH3 domains (part of scaffold system)
    2. 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).
    3. 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

• Colonies were picked from the 4 plates that grew colonies of the 5 incubated overnight (attempting to clone plasmids pTG002-pTG006) and resuspended in 10 μL water.
• Colony PCR was run on the colony resuspensions.
  • Results appear to suggest transformation of: 1 colony with pTG003 (agrBD with His-tag on C-terminus of ligand agrD), 2 colonies with pTG004 (lamBD), 4 colonies with pTG005 (fsrB with His-tag on N-terminus of ligand), and 1 colony with pTG006 (fsrB with His-tag on C-terminus of ligand)
• Liquid cultures were prepared for the most promising colonies as screened by colony PCR and incubated overnight

• Reattempt to clone pAA009 & pAA010 for the Agr system.
  • Redid PCR of pAA009/pAA010 backbone. PCR product was then DpnI digested and PCR-purified.
  • Gibson assembly of pAA009 and pAA010 using the PCRed backbone and the respective AgrA geneblock
• Combinatorial Promoters:
  • Colony PCR run on 6 colonies transformed with pAA003.
  • Liquid cultures were prepared for those colonies that were deemed promising based on colony PCR screening.
  • Additionally, jwAA001 liquid cultures with inducers (aTc & IPTG) had been allowed to continue growing last night. Fluorescence measurements were repeated this morning and appeared to confirm the AND-gate logic we had desired
  • New overnight liquid culture of jwAA001 was set up for experimentation with more inducer concentrations later this week

Thursday, 7/17/14

Friday, 7/18/14