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.
• 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
  • pTG002-pTG006 (the five export constructs to test) 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 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 overnight
• 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 are promising: GFP is significantly expressed 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

• Attempted to PCR out pKS001 backbone using primers with overlaps for Gibson assembly.
• PCR product was DpnI digested and PCR purified. A gel was then run to check for presence of product
  • Product was not present in most of the lanes. It was at this point that we realized our primers were designed bind and extract the backbone from pWW1523, not pKS001, which the other groups in the lab were using. Thus, the primers for the most part didn't bind to pKS001, as expected.
  • Protocol will be repeated on pWW1523 in lieu of pKS001 tomorrow

• 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 (strain confirmed to contain pAA001) was set up for experimentation with more inducer concentrations later this week

Thursday, 7/17/14

• Miniprepped the overnight liquid cultures prepared yesterday.
• Samples of the minipreps were shipped out for sequencing with the sequencing primers used for PCR

• pWW1523 backbone was PCRed out with overhangs for Gibson assembly
• PCR product was DpnI digested, PCR purified, and then run on a gel to confirm its creation

• agrBCDA Reception System:
  • Transformed 2 cultures of JM109 cells with pAA008 & pAA009 and pAA008 & pAA010 respectively.
  • Cultures were plated on Kan + Cm plates
• Combinatorial Promoters:
  • A second experiment was set up for the jwAA001 strain (expressing pAA001), testing more inducer concentration combinations:
    • ([IPTG] in μM, [aTc] in ng/mL): (0,0); (5,25); (50,25); (500,25); (5,50); (50,50); (500,50); (5,100); (50,100); (500,100)
  • 6 liquid cultures carrying pAA003 were miniprepped, and the resulting plasmids were shipped for sequencing.

Friday, 7/18/14

• Analyzed Sequencing Data. Successful clones created for pTG004 & pTG005. Need to redo 2, 3, & 6.

• pWW2149 backbone was PCRed out with overhangs to be used in Gibson assembly.
• Gibson assembly of pMB001 - pMB006

• Combinatorial Promoters: Collected data from yesterday's experiment testing different inducer concentrations run overnight.
• agrBCDA Reception System:
  • Yesterday's transformations failed. So transformations were redone with 1 hour incubation in SOC medium before plating.
  • Redid PCR of pAA008 & pAA009 backbones, hoping to get higher yields for more Gibson assembly product.