Team:Wageningen UR/Notebook/Jenny
Here you can find the notebooks from Jenny.
Modeling
- September
- October
- November
- December
- January
- February
- March
Made and tested a toymodel based on data from literature.
Built and simulated the full model.
Performed the sensitivity analysis of the full model to identify which parameters are important for formaldehyde sensitivity.
Built and tested two model extensions and improved the sensitivity analysis.
Built and simulated 10 model extensions.
Tested the model extensions and combined the model extensions. Did sensitivity analysis one the most promosing ones.
Added volume to one of the model extensions and simulated cell division.
Wetlab - May
- Week 1: 10th of May - 14th of May
- Week 2: 17th of May - 21th of May
- Week 3: 24th of May - 28th of May
- Week 4: 31st of May - 4th of June
Introduction labwork and make competent DH5alpha cells.
Transformed E. coli with LuxI and LuxR biobrick plasmids. Isolated the plasmids.
Sequenced the biobrick plasmids. Isolated plasmids containing the GFP and RFP genes. Many PCRs with overhangs to add promoters to the genes.
More PCRs and used GoldenGate (GG) to make a AHL producing plasmid.
Wetlab - June
- Week 5: 7th of June - 11th of June
- Week 6: 14th of June - 18th of June
- Week 7: 21st of June - 25th of June
- Week 8: 28th of June - 2nd of July
Send the AHL plasmid for sequencing. There was a mutation in the promoter. Used Goldengate to make the negative control plasmid which contains GFP under control of the lux pR promoter.
Send the NC plasmid for sequencing. Used GG to make another AHL producing plasmid with a different promoter, sequencing did not give good results.
Sequenced inserts and used GG to make the NC, reporter and PC plasmids with GFP.
PCR backbones and isolate NC, reporter and PC plasmids.
Wetlab - July
- Week 9: 5th of July - 9th of July
- Week 10: 12th of July - 16th of July
- Week 11: 19th of July - 23th of July
- Week 12: 26th of July - 30th of July
Sequencing of NC, reporter and PC plasmids and tried to make another AHL production plasmid in which LuxI and RFP are in one operon.
Sequencing of AHL production plasmid, inserts were mutated. Made competent K12 cells.
Transformed K12 with the NC, reporter and PC plasmids.
Started making a AHL production plasmid in which RFP and LuxI are under control of the rhamnose inducable promoter. Did a growth experiment in 15mL tubes to see if AHL medium induces the reporter strain.
Wetlab - August
- Week 13: 2nd of August - 6th of August
- Week 14: 9th of August - 13th of August
- Week 15: 16th of August - 20th of August
- Week 16: 23th of August - 27th of August
- Week 17: 30th of August - 3th of September
Tried new PCR's for the Rhamnose promoter. I did a plate reader experiment to test the PC, reporter and NC plasmid. Used LB as medium and conditioned medium (CM) was ON culture of the PC strain which was filter sterilized.
Made the AHL production plasmid with the Rhamnose promoter.
Made empty plasmids to use as negative control for the plate reader experiments.
Repeated the plate reader experiment in M9. This gave growth issues.
Made NC, reporter and PC plasmids where GFP was preplaced by GFP. Did a growth experiment to test if supplementing the M9 medium with LB would solve the growth issues.
Wetlab - September
- Week 18: 6th of September - 10th of September
- Week 19: 13th of September - 17th of September
- Week 20: 20th of September - 24th of September
- Week 21: 27th of September - 1st of October
Transformed EM42 (P. putida) with the NC, Reporter and PC plasmid containing RFP. Repeated the plate reader experiment of 4/8 with 20% LB, 80% M9 as medium.
Made NC, reporter and PC plasmids where the lux pR promoter was replaced by the lux pL promoter. Plate reader experiment of last week was repeated with different CM concentrations, and a plate reader experiment was done to test the constructs containing RFP.
Plate reader experiment was done with the constructs containing the lux pL promoter. A coculture experiment was done on a petridish with the PC and Reporter strains.
The coculture experiment was repeated and the double knockout putida strain from Marta which is unable to grow on glucose was transformed with the constructs containing RFP. The luxpL contructs were put in a different backbone. K12 was transformed with both the RFP and lux pL constructs.
Wetlab - October
- Week 22: 4th of October - 8th of October
The coculture experiment on the petridish was repeated with a coculture of P. putida and E. coli. A coculture experiment with Putida and E. coli was done in erlenmeyers, which showed that the P. putida glycerol stock was contaminated as it grew on glucose. A plate reader experiment was done with the E. coli strains containing both the RFP and lux pL construct and AHL medium from P. putida.