- Preliminary WT Toxicity experiments (Toluene, Benzyl Alcohol, Benzaldehyde)
- Developed P. putida culturing technique
June 1, 2017
- Seeded BBa_K1966004 in LB/CM
June 2, 2017
- Developed genetic construction plan and designed primers
- Growth with IPTG gradient. Protein extract for SDS PAGE
June 5, 2017
- SDS PAGE and staining
June 6, 2017
- Destaining, incorrectly identified xylR
June 8, 2017
- Interview with Dr. Hazen to discuss bioremediation
- Genomic DNA extract (E. coli MG1655, P. putida NRRL-4067)
- Transformed (heat shock) Pu promoter BBa_K1031803 (Peking 2013) and plated. Preliminary test of RFP expression with BBa_K1966004 (2016 Pu construct)
June 9, 2017
- Met with Dr. Loffler’s lab manager, Cindy regarding the acquisition of genomic DNA
- PCR (MarA,RobA, SoxS, NdH, ttgA, ttgB, ttgC, PBC) ; testing annealing temps and mastermix
- Plated BBa_K1031803 colonies grew. Took 2 colonies, sampled for colony PCR. Frozen stock. Liquid culture in CM
June 10, 2017
- Plasmid extracted
June 12, 2017
- PCR (MarA, SoxS, Rob, AcrA, AcrB)
- PCR of Pu insert. Ran gel of PCR product and colony PCRs: correct ~1070 bp. Gel extracted PCR. Grew BL21DE3 for comp cells. PCR encompassing xylR and backbone failed.
June 13, 2017
- Gel Extract (MarA, SoxS, Rob, AcrA)
- Clean and Concentrate (MarA, SoxS, Rob, AcrA)
- PCR (AcrA, AcrB)
- PCR (2x DMSO) of BBa_K1966004 backbone and xylR sequence: failed on gel. BL21DE3 comp cells made.
June 14, 2017
- PCR (TolC, EmrA, EmB, PBC)
- PCR (PBC)
- Segmented backbone with xylR for 3 piece Gibson. Correct. Gel extracted
June 15, 2017
- Gel Extract (TolC, EmrA, EmrB)
- PCR (PBC, pSB1C3, AcrB, Rob)
- 3 Piece Gibson: CM piece, xylR piece, new Pu piece. Transformed into Top10.
June 16, 2017
- Plasmid Extract (pSynth12)
- Gel extract (Rob, AcrB)
- PCR (PBC, pSB1C3)
- Took colonies, frozen stock, colony PCR, liquid culture. Called it PK.XR
- Developed standard preparation protocol
- Transferred aldehyde stocks to anaerobic aliquots
- Updated IGEM primer database
- Transformed Bba_J364000 from registry for positive GFP control.
June 23, 2017
- Developed standard preparation protocol
- Transferred aldehyde stocks to anaerobic aliquots
- Updated IGEM primer database
- Transformed Bba_J364000 from registry for positive GFP control.
- Saw green pellet on plasmid extract of Bba_J364000. Colony PCR reaffirmed. BL21DE3 transformation. Characterized PK.XR with IPTG gradient 0-.5 IPTG-- no success.
June 24, 2017
- Transformed Bba_J04450 and Bba_K741002 for future GFP, RFP positive controls into Top10.
June 25, 2017
- Red and green colonies. Liquid cultures, frozen stocks.
June 26, 2017
- Genomic DNA extract (P. putdia B8)
- Discussed IGEM safety forms
- Wrote IGEM project descriptions
- PCR ((ttgA, ttgB, ttgC, ttgD, ttgE, ttgF, ttgG, ttgH, ttgI)
- Plasmid Extract Bba_K741002 and Bba_J04450 then transformed to BL21DE3.
June 27, 2017
- Characterization PK.XR at .1 mM IPTG, 1 mM m-xylene. RFP, GFP to frozen stock.
July 5, 2017
- PCR'ed xyl-A,M, B from BBa_K1966003, PCR'ed PK.XR into 2 backbone pieces
July 6, 2017
- Transform Top10 CDP (CRISPR digestive plasmid or “CDP”)
- CDP digest (KpnI-HF and XhoI)
- Updated IGEM primer database
- Developed CRISPR strategy
- Gel check of PCR - success, extracted. Gibson Assembled (prematurely) xyl-A,M, B into PK.XR in the sfGFP position. Transformed T10. Characterization PK.XR IPTG gradient 0-1.5 mM with1 mM xylenes.
July 7, 2017
- Clean and concentrate (CDP digest)
- Received PBC (from Neel)
- Seeded WT/GFP/RFP in flasks for HITES outreach. Took PK.XR AMB plate and stored in cold room.
- PCR colony check (plasmid inserts)
- Destaining, no xylR
July 15, 2017
- Plasmid Extract (AcrZ, AcrR, TolC)
July 16, 2017
- Digest (pMarA, SoxS, Rob, EmrAB, AcrR, AcrZ, TolC)
- Single enzyme digests, enzyme varies depending on RE redundancies
July 17, 2017
July 18, 2017
- Transform BL21DE3 (AcrR, AcrZ, TolC)
- Plated B. cepacia ATCC 25416 and P. aeruginosa PAO1 at Ripp lab
- 30˚C testing of PK.XR (P. putida grows at lower temperature) M9
July 19, 2017
- Glycerol Stocks (AcrR, AcrZ, TolC)
- Plasmid extract (AcrR, AcrZ, TolC, MarA, SoxS, Rob, EmrAB)
- PCR (plasmid inserts)
- Inoculated liquid cultures of B. cepacia ATCC 25416 and P. aeruginosa PAO1 at Ripp lab
- Characterization 30˚C, no GFP. Capped
July 20, 2017
- Genomic DNA extract at Ripp lab (B. Cep ATCC 25416 and P. Aeru PAO1)
- CDP Plasmid Extract
- Digest CDP and MarA (Kpn1-HF, NdeI, XhoI; Xba + SpeI)
- PCR (MexA, MexB, OprM, MexX, MexY)
July 21, 2017
July 22, 2017
- PCR (MexA, MexB, OprM, MexX, MexY, AcrB)
July 24, 2017
- Gel extract (MexA, MexB, OprM, MexY)
- Clean and concentrate (MexA, MexB, OprM, MexY)
- Tried uncapped, aerobic-- need Oxygen for GFP. Protein extraction. SDS Page. Seeded DH5 alpha for interlab into flask for comp cells
July 25, 2017
- PCR (AcrB, MexB)
- Destained SDS PAGE. GFP there in overgrown samples, no xylR anywhere. Realized translation rates were low via Salis Lab
July 26, 2017
- Ligation (CDP + MarR)
- Interlab Transformation/ showing underclassmen how to transform.
