Team:Wageningen UR/Notebook/Sanne
Here you can find the notebooks from Sanne.
Modeling
- September
- October
- November
- December
- January
- February
- March
Started by getting acquainted with nitrogen conversions, modeling, and MATLAB. Set-up ODEs, Gathered kinetic and experimental data and built the model in MATLAB. Additionally, a few test runs were done.
Everything was set up for model optimization, i.e. score function was written, parameter bounds were established. By testing 100,000 parameter sets and scoring the models, the system was collectively fit to agree with time-series data for two P. stutzeri strains.
As the model could not capture the full dynamics yet, we further refined the model. Parameter bounds were tweaked, the population volume was fixed at this stage. The model was run another 50,000 times and the best parameter sets were identified.
Model behavior was further analyzed. Again, parameter values were optimized through additional optimization rounds.
Volume simulation was added back to the model and optimized for. Relative sensitivity analyses were conducted. Furthermore, the model was further improved by adding a metabolite sink and a hypothetical metabolic switch.
In-depth analyses on model behavior were conducted. Identified parameters most influential in nitrous oxide accumulation. Read more about nitrogen conversions in general to explain model hypotheses.
Simulated the system for low ammonia concentrations, to predict what would happen in our biofilter. Simulated the system for different conditions and strains, to further validate the model.
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
Lab introduction, made competent E.coli DH5α, Then I annealed spacer (Sp.) 1-7+9 and performed restriction - ligation of the spacers into a pSEVA231-CRISPR plasmid. The cloning products were subsequently transformed into E. coli. Successful ligation was verified with sequencing.
Transformed P. putida with 8 pSEVA231-CRISPR plasmid, screening with colony PCR. Sp. 8 + 10-15 were annealed and ligated into the pSEVA231-CRISPR plasmid, transformed in E.coli and checked by sequencing. First growth experiment done with Sp. 1-7+9.
Spacer 14 was re-ligated and transformed in E.coli. Sp. 8,10-13+15 were transformed in P. putida. In parallel, qPCR primers were designed and ordered. Due to a contamination in growth experiment, all 15 spacers and non-targetting pSEVA231-CRISPR plasmids were again transformed into P. putida.
Transformed P. putida was screened and three glycerol stocks for Sp. 1-9 were made. Two growth experiments were conducted this week. The BAC was designed in detail, primers were designed and ordered.
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
Data was lost for one growth experiment due to the failure of the plate reader. This week, another two growth experiments were done with all spacers. We noticed that there was a lot of variability in biological replicates, even when grown on LB. Moreover, 3 different non-targeting constructs were transformed into P. putida.
Wells containing pSEVA231-CRISPR strains were screened for recombination events, which was not found. The complete Nap, Nir, Nor, Nos gene clusters were test-amplified with primers from the Synthetic Denitrification projects.
We attempted qPCR with the CRISPRi strains, but the cells did not grow well in our medium. By culturing the cells on different [acetate] we found that we needed to employ a lower concentration to promote cell growth. In parallel amplified the Nap, Nir, Nor, Nos operons and the Pcc1fos backbone in two fragments with platinum SuperFi II PCR master mix. All bands were at the expected size and were purified from gel. Subsequently, all 7 fragments were assembled by Gibson Assembly and directly transformed in E. coli EPI300 cells. In addition, the T7 promoter was added to the landing pad construct (pGNW-lox-Cre recombinase - lox) by means of Golden Gate cloning, after which it was transformed into E. coli DH5α.
Multiple colony PCRs of the E.coli EPI300 for presence of the complete BAC, by amplifying the fragment junctions with primers from the Synthetic Denitrification projects. The landing pad was sent for sequencing, and verified.
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
The growth experiment was conducted with Sp. 1, 2, 7-15. RNA isolation and cDNA synthesis were done for three biological replicates for Spacer 1 and the non-targeting pSEVA231-CRISPR plasmids.
Holiday
Holiday
Proposal writing and more iGEM work.
Wetlab - August
- Week 9: 2nd of August - 6th of August
- Week 13: 9th of August - 13th of August
- Week 14: 16th of August - 20th of August
- Week 15: 23th of August - 27th of August
- Week 16: 30th of August - 3th of September
Resolved issues with integrating the T7 polymerase at the attn7 site by assembling a new pGNW-lacI-T7 polymerase, which was transformed into E. coli and verified by sequencing. Subsequently, the pGNW was conjugated into P. putida ΔnasT. Further screening of the E. coli EPI300 cells for the presence of the complete BAC by amplifying the junctions with gradient PCR. We found that the majority of colonies contained all fragments, glycerol stocks were made. Several reactions were sent for sequencing, but unsuccessfully. In parallel, a qPCR was done with the cDNA for Sp. 1 and the non-targeting plasmid.
Conjugation failed, so the T7pGNW was electroporated into P. putida directly, screened and subsequently transformed with the pQURE plasmid. Eventually, P. putida ΔnasT :lacI T7 pol was generated. The landing pad was directly electroporated, but the transformation failed. In addition, a CRISPRi growth experiment with all spacers in technical duplicates was performed.
RNA was isolated for spacers 1,4,7,10 and the non-targeting plasmid. Subsequently, the obtained RNA was converted into cDNA. Succesful P. putida transformation of the landing pad. Subsequent Curing with pQURE failed.
The plate for the CRISPR growth experiment was screened for recombination events, which did not happen. qPCR for the Sp. 14 and the non-targeting construct was performed. Due to insufficient resources, the qPCR experiments were abandoned from this point on. pGNWs to knock-out terminal oxidases were designed, primers were ordered. The landing pad pGNW was conjugated into P. putida.
500 bp homologous arms at 5' and 3' of each terminal oxidase were PCR amplified and ligated into pGNW plamids. The constructs were transformed in E.coli DH5α and verified with sequencing. The
Wetlab - September
- Week 17: 6th of September - 10th of September
- Week 18: 13th of September - 17th of September
- Week 19: 20th of September - 24th of September
- Week 20: 27th of September - 1st of October
The P. putida landing pad pQURE was screened and the knock-in was obtained: P. putida ΔnasT :lacI T7 polymerase :lox - Cre recombinase - lox. The pGNWs to knock out the terminal oxidases were sent for sequencing and verified. A midiprep for the BAC was done and reactions verifying the presence of Nap, Nir, Nor, Nos were sent for sequencing, this time successfully.
More sequencing reactions for the BAC were sent, verifying successful assembly. Moreover, we screened for co-integrates for the terminal oxidases knock-outs. These were transformed with pQURE, but unsuccessfully. The BAC was conjugated into P. putida ΔnasT :lacI T7 polymerase :lox - Cre recombinase - lox. Subsequently, the presence of the Nap was verified with colony PCR.
Multiple colony PCRs were conducted to verify successful integration of the denitrification machinery (BAC) in P. putida. The final strain was called: P. putida :SD.
Five biological replicates for P. putida :SD were grown and both a nitrate and nitrite assay were done to test the activity of the denitrification machinery. The results of the nitrite assay suggested that the pathway was active. Later that week, CRISPRi plasmids and a Nos accessory plasmid were transformed into P. putida :SD.
Wetlab - October
- Week 18: 4th of October - 8th of October
An all-or-nothing experiment was conducted, P. putida :SD was grown on nitrate, nitrite, and nitrous oxide, and the accumulation of nitrous oxide was quantified in gas chromatography-mass spectrometry (GC-MS) experiment. Moreover, the effect of IPTG, increasing the expression of the pathway, was also included in this experiment. P. putida :SD + IPTG and nitrate did accumulate trace amounts of nitrous oxide, suggesting that Nap, Nir, and Nor worked.