Team:Wageningen UR/Notebook/Marta
Here you can find the notebook from Marta.
Wetlab - March
- Week 1: 15th of March - 19th of March
- Week 2: 22nd of March - 26th of March
- Week 3: 29th of March - 2nd of April
An experimental plan was created and the auxotrophic E. coli KEIO strains were tested.
First growth experiment was performed. The concentration of Arg, Lys, Leu, needed to re-establish the growth of the respective auxotrophic strains was determined.
Second growth experiment was performed. The concentration of His, Tyr, and Trp need to re-establish the growth of the respective auxotrophic strains was determined. The Pseudomonas putida strain EM42 Δgts Δgcd was checked to contain the correct mutations via colony PCR.
Wetlab - April
- Week 4: 5th of April - 9th of April
- Week 5: 12th of April - 16th of April
- Week 6: 19th of April - 23rd of April
- Week 7: 26th of April - 30th of April
Creation of double mutants in E. coli via the λ-Red protocol. E. coli ΔhisD and E. coli ΔtrpD were initially selected and transformed with the pSC020 plasmid for λ-Red recombination.
Determination of the amino acids secretion of the E. coli mutants Δmdh, Δppc, ΔtrpR and ΔhisL. No growth of the biosensor strains could be detected.
Determination of the amino acids secretion of the E. coli mutants Δmdh, Δppc, ΔtrpR and ΔhisL. An improved protocol was used to maintain the correct nutrient concentration. 400uL Conditioned Medium : 100uL 5x M9. The mutant strains were also checked via colony PCR. E. coli ΔhisL did not show the expected results.
Test of carbon-source dependency of P. putida on E. coli.
Wetlab - May
- Week 8: 10th of May - 14th of May
- Week 9: 17th of May - 21th of May
- Week 10: 24th of May - 28th of May
- Week 11: 31st of May - 4th of June
Test of carbon-source dependency of P. putida on E. coli.
Based on the results obtained from E. coli ΔhisL (not real overproducing strain), the overproduction of Tyr was chosen as a second possibility to create a cross-feeding community of the two bacteria. Therefore, the amino acids secretion of the E. coli mutants ΔtrpR, and ΔtyrR was determined. The biosensor strains E. coli ΔtrpD, and E. coli ΔtyrA grew!
Tyr and Trp were selected as the amino acids to establish the cross-feeding community on. Creation of a P. putida strain ΔtyrA and ΔphhAB.
The pGNW plasmid was constructed with the correct homology regions to KO TyrA and PhhAB. Isolation of 500bp homology regions, golden gate in pGNW and isolation of correct colonies.
Wetlab - June
- Week 12: 7th of June - 11th of June
- Week 13: 14th of June - 18th of June
- Week 14: 21st of June - 25th of June
- Week 15: 28th of June - 2nd of July
Creation of a P. putida strain ΔtyrA and ΔphhAB: conjugation of pGNW in P. putida. Creation of overproduction plasmid SEVA64_trpE_S40F. The P_RBS_trpE fragment was assembled within the plasmid SEVA64.
Creation of overproduction plasmid SEVA64_trpE_S40F. Mini-prep of colonies containing SEVA64_trpE was performed. Colony PCR confirmed the cloning of trpE in SEVA64. Creation of a ΔtyrR ΔtrpD E. coli strain via λ-red protocol. Creation of a P. putida strain ΔtyrA and ΔphhAB: a P. putida strain ΔphhAB was obtained!
Creation of a P. putida ΔtyrA ΔphhAB. The KO of tyrA was attempted in P. putida ΔphhAB and in P. putida EM42. Creation of a ΔtyrR ΔtrpD E. coli strain via λ-red protocol. No positive results. Creation of overproduction plasmid SEVA64_trpE_S40F. The mutation of S40F was attempted via PCR.
A P. putida ΔtyrA was obtained! But no double mutant was found. Creation of a ΔtyrR ΔtrpD E. coli strain via λ-red protocol. No positive results. Creation of overproduction plasmid SEVA64_trpE_S40F. The mutation of S40F was attempted via PCR, the sequencing results indicated a deletion instead of substitution. Growth curves of P. putida EM42 and P. putida EM42 Δgts Δgcd on glucose and acetate. The growth curves for P. putida EM42 ΔphhAB and P. putida EM42 ΔtyrA in increasing concentrations of Tyr were performed. Growth curves of biosensor strains E. coli ΔtyrA and E. coli ΔtrpD on the conditioned medium from the overproducers P. putida EM42 ΔphhAB, P. putida EM42 ΔtyrA and E. coli ΔtyrR were performed.