Week Eight

Monday, 8/4/14

• Ran a colony PCR on 4 colonies picked from a CARB plate that had been plated with a 2nd Round-the-horn reaction.
• A gel was run on the products, and a liquid culture was set up for the one colony suggesting it contains a properly formed pTG004-noFLAG plasmid.

• Reattempted transformation of Anton's B2 pTet_GFP GG-assemblies into JM109. Plated on Cm plates & incubated overnight.

• Transformed JM109 cells with the pAA009 & pAA010 Gibson assembly products from Friday.
• Transformed DH5α-Z1 cells with the pAA002 Gibson assembly products from Friday.

• We began characterization of a constitutive promoter family designed by Berkeley iGEM 2006 (biobrick parts BBa_J23100-J23118). We transformed JM109 cells with the re-suspended plasmids.
• Some parts were not included with the kit mailed to us, so we might request those at a later date.

Tuesday, 8/5/14

• Miniprepped the liquid culture of pTG004-noFLAG incubated last night. However, the final DNA conc. was too low to send for sequenceing, so the liquid culture was grown up again for another try tomorrow.

• Ran colony PCR on colonies transformed with A2 (pTet_GFP from Anton).
• Set up liquid cultures with carbenicillin for picked colonies.

• Reran colony PCR using the new primers that we ordered.

• Only 1 colony was resulted from yesterday's transformation of pAA009. Set up a liquid culture of this colony.
• Ran gradient PCR on pAA009 backbone using 6 temperatures from 50-70°C.
• DpnI digested pAA009 backbone for 2 hours and stored at 4°C overnight.

• Picked 6 colonies from the plate transformed yesterday with pAA002 and set up liquid cultures.

• Miniprepped transformed liquid cultures grown up last night

Wednesday, 8/6/14

• Redid miniprep of pTG004-noFLAG liquid culture.
• Sent sample for sequencing
• Transformed that pTG004-noFLAG miniprep into DH5α-Z1

• Set up overnight liquid culture of pMB006-transformed colony that looks promising based on yesterday's colPCR

• Ran a colPCR on the single colony that obtained from Monday's transformation, but didn't get the right band.
• Ran a gel on the pAA009 backbones that we PCR'ed yesterday, confirming bands that were around the size.
• Did Gibson assembly with the backbones for pAA009/10, and then transformed DH5alpha-Z1 cells with them.

• Ran a colony PCR on colonies from earlier transformations. 5 of then were of correct length.
• Shipped out colPCR products for sequencing.

• Set up and ran TXTL reactions on the plasmids containing the family of constitutive promoters.

Thursday, 8/7/14

• Set up liquid culture of 1 colony of DH5α with pTG004-noFLAG (transformed yesterday).

• Induced expression of pTG004 in pTG004-FLAG constructs by adding varying amounts of aTc to 3 separate liquid cultures (MOPS media).
  • Concentrations of aTc induction: 0 nM, 250 nM, 500 nM

• Miniprepped overnight liquid culture of pMB006. Shipped to Eurofin for sequencing.

• Ran colony PCR on yesterday's transformations. No successes.

• Analyzed sequencing results, suggesting that 2 of the pAA002 colonies have desired plasmid.
• Started overnight liquid culture for experiments on the pAA002 combinatorial promoter.

Friday, 8/8/14

• Re-DpnI-digested PCRed backbones created on July 30th, by adding 2.5 uL 10x CutSmart Buffer and 0.5 uL of a newly ordered stock of DpnI and incubating for 6 hours.
• PCR-purified DpnI digests, yielding much lower DNA concentrations from what was measured on July 30.
• Ran Gibson Assemblies of pTG002/3/6 using the new backbones & appropriate geneblock inserts
• Assemblies were transformed into JM109

• Took pTG004-FLAG liquid cultures induced with aTc and prepared the samples via the LCMS protocol with acetonitrile & vacufuging.
• Set up 3 more liquid cultures but with pTG004-noFLAG (sequencing-verified) with varying aTc induction (0 nM, 250 nM, 500 nM).

• Again tried transforming A2 plasmid (pTet_GFP) from Anton into JM109. Plated on carbenicillin plate.

• Analyzed sequencing results. They were messy and revealed nothing about the sequence.
• Resuspended colonies and grew overnight cultures for pMB001, 2, & 5

• Tested B83 combinatorial promoter (within pAA002 plasmids) in tbAA002 cells using IPTG & aTc induction.
  • ([IPTG] in μM, [aTc] in ng/mL): (0,0); (5,0); (50,0); (500,0); (0,25); (5,25); (50, 25); (500,25); (0,50); (5,50); (50,50); (500,50); (0,100); (5,100); (50,100); (500,100)