Team:Wageningen UR/Notebook/Sophieke
Here you can find the notebook from Sophieke.
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
Designing primers to amplify napB from Pseudomonas aeruginosa PA01 genome, containing overhangs of pSB1C3 backbone. Designing primers to amplify homology arms up- and downstream nasT gene of Pseudomonas putida EM42 , containing overhangs of integrative vector pGNW. Started with the PCR amplification of the first gene, napB, from the genome of P. aeruginosa and ligating it into the plasmid backbone pSB1C3. This was followed by transformation of the plasmid into competent Escherichia coli, screening of colonies, miniprep and sending plasmids for sequencing. In parallel, the nasT gene of P. putida was amplified with homology arms for pGNW, ligated into this vector and transformed into E. coli DH5α λpir. Colonies were screened for the plasmid and a positive colony was cultured. P. putida EM42 was transformed with the plasmid through conjugation.
The other genes of the nap operon from P. aeruginosa were PCR amplified, as well as all the genes from the nap operon of Cupriavidus necator H16. Only the amplification of napC from C. necator was not successful. All the genes were ligated into pSB1C3, the plasmids were transformed into E. coli. The colonies containing the plasmids were grown, miniprepped and the plasmids were sent for sequencing. napA from P. aeruginosa and napB from C. necator were wrong. P. putida colony containing the pGNW-nasT plasmid was cultured and transformed with pQURE, leading to homologous recombination. Colonies were screened for knock-out of nasT, but no successful knock-out yet.
Reamplified napA P. aeruginosa and napC C. necator from genome. Ligated the genes into pSB1C3, transformed into E. coli. Transformed P. putida containing pGNW-nasT plasmid again with pQURE plasmid and screened colonies for knock-out. Still not successful.
Screened transformed E. coli for napA P. aeruginosa and napC C. necator. Positive colonies were cultured, miniprepped and plasmids sent for sequencing. This was also done for more colonies containing pSB1C3-napB C. necator. napA contained a mutation and was therefore reamplified with newly designed primers to remove the mutation and subsequently ligated into pSB1C3. All the nap genes from P. aeruginosa, exept for napA, and C. necator were amplified from pSB1C3 plasmid with primers containing ribosome binding sites and overhangs compatible with pSEVA22 backbone. Reamplified genes from C. necator were ligated together in pSEVA22 containing the J23100 promoter. P. putida containing pGNW-nasT plasmid was transformed again with pQURE plasmid and screened colonies for knock-out. This time successfully. Positive colonies were grown on LB to remove pQURE. Primers were designed to amplify nap genes from the genome of Pseudomonas stutzeri JM300.
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
E. coli was transformed with pSB1C3-napA P. aeruginosa and colonies were screened, miniprepped and sent for sequencing. This time, napA was correct. E. coli was transformed with pSEVA22-p100-napEDABC from C. necator and the resulting colonies were screened, but no positive colonies. The nap genes from P. stutzeri were amplified from the genome and ligated into pSB1C3. The resulting plasmids were transformed into E. coli, colonies were screened and cultured. The first experiment with P. putida ∆nasT was performed, by growing the knock-out and the WT P. putida on nitrate as only nitrogen source.
napA from P. aeruginosa was amplified from pSB1C3-napA and ligated into pSEVA22 containing the J23100 promoter together with the other nap genes from P. aeruginosa to form the operon. E. coli was transformed with the resulting plasmid and colonies were screened, but no positive ones. E. coli was again transformed with pSEVA22-J23100-napEDABC from C. necator and the resulting colonies were screened. Colonies containing the plasmid were cultured, miniprepped and the plasmids were sent for sequencing. The cultured E. coli colonies containing the plasmids with the nap genes from P. stutzeri were miniprepped and the plasmids were sent for sequencing. The correct genes were amplified and ligated together into pSEVA22 + J23100 promoter. The nitrate and nitrite assay protocols were tested by making standard curves with nitrate and nitrite solutions. It took a few tries to find the optimal strategy. Primers were designed for the nap genes of Paracoccus denitrificans DSM 413.
The nap genes from P. aeruginosa were ligated again into pSEVA22 + J23100 and E. coli was transformed. Colonies were screened, but no positive one. The pSEVA22-J23100-napEDABC from P. stutzeri was transformed into E. coli as well and colonies were screened. Colonies containing the plasmid were cultured, miniprepped and the plasmids were sent for sequencing. P. putida ∆nasT was transformed with the correct pSEVA22-J23100-napEDABC from C. necator, the colonies were screened and a correct one was cultured. The nap genes from P. denitrificans were amplified from the genome and ligated into pSB1C3. E. coli was transformed with the plasmids and the resulting colonies were screened. Correct colonies were cultured, miniprepped and the plasmids sent for sequencing. All nap gene sequences were correct.
P. putida ∆nasT was transformed with the correct pSEVA22-J23100-napEDABC from P. stutzeri, the colonies were screened and a correct one was cultured. The culture was miniprepped and the plasmid sent for sequencing. The correct plasmid was transformed into P. putida ∆nasT. The nap genes of P. denitrificans were amplified from the pSB1C3 plasmids and ligated together into pSEVA22 + J23100. E. coli was transformed with the resulting plasmid and colonies were screened. Correct colonies were cultured and miniprepped and the plasmids were sent for sequencing. Experiment was performed with P. putida ∆nasT and WT growing on different nitrogen sources to see if nitrate or nitrite is consumed. A preliminary experiment with P. putida ∆nasT containing nap from C. necator and P. stutzeri was performed.
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
Nitrate and nitrite assays were performed to see if Nap from C. necator and P. stutzeri work. As a new strategy for the nap operon from P. aeruginosa, napE, napF and NapD were ligated together and napA, napB and napC were ligated together without backbone. These ligation products were PCR amplified and ligated in the backbone. Subsequently, the ligated plasmid was transformed into E. coli and colonies were screened. Still no correct colony could be identified, therefore, we gave up on Nap from P. aeruginosa. In parallel, the nos genes were amplified from the genome of P. stutzeri and ligated into pSB1C3. The resulting plasmids were transformed into E. coli, colonies were screened and cultured. The correct nap genes from P. denitrificans were reamplified and ligated into pSEVA22 + J23100.
The experiment with P. putida ∆nasT and WT growing on different nitrogen sources to see if nitrate or nitrite is consumed was repeated and in the same experiment P. putida ∆nasT containing the Nap from C. necator and P. stutzeri were tested for nitrite production. Nitrate and nitrite assays were performed. The pSB1C3 plasmids containing the nos genes from P. stutzeri were checked with sequencing and subsequently, the genes were reamplified and ligated into pSEVA22 + J23100. The ligation product of nap genes from P. denitrificans with pSEVA22 + J23100 was transformed into E. coli and subsequently colonies were screened. Correct colonies were grown, miniprepped and sent for sequencing.
Holiday
Holiday
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
The ligated product of the nos genes and pSEVA22 + J23100 was transformed into E. coli. Only one colony was grown, which was screened with colony PCR. Unfortunately, the correct plasmid was not present.
E. coli was again transformed with the ligation product of the nos genes and pSEVA + J23100. Sequencing of pSEVA22-J23100-napEDABCGH from P. denitrificans were checked and a two mutations were found. New primers were made to remove these mutations from the operon. The operon was reamplified with the new primers in three parts and these were ligated again in pSEVA22 + J23100. The ligation product was transformed into E. coli.
E. coli colonies were checked for pSEVA22-J23100-nosRZDFYLTatA or pSEVA22-J23100-napEDABCGH with colony PCR. Correct colonies were grown, miniprepped and the plasmids were sent for sequencing. Sequencing showed correct nap operon from P. denitrificans, but mutation in the nos operon from P. stutzeri. The nap operon from P. denitrificans was transformed into P. putida ∆nasT. The accessory genes for Nos were amplified from the genome of P. stutzeri and were ligated into pSB1C3. The ligation product was transformed into E. coli.
The nos operon was reamplified to remove the mutation and ligated again in pSEVA-J23100. The resulting ligation product was transformed into P. putida. Colonies were screened and correct ones were sent for sequencing. Unfortunately, again mutations were seen. Therefore, the nos operon was ligated into pSEVA22 with two weaker promoters, namely BBa_R0051 (pR) and J23118. These ligation products were transformed into E. coli. The transformed E. coli colonies were checked for the presence of the accessory genes for Nos. Correct colonies were cultured, miniprepped and plasmids were sent for sequencing.
The transformed E. coli colonies were screened for pSEVA22-pR-nosRZDFYLTatA or pSEVA22-J23118-nosRZDFYLTatA. Correct colonies were cultured, miniprepped and plasmids were sent for sequencing. Again, mutations were found in both operons. The correct accessory genes for Nos were reamplified and ligated into pSEVA83-J23100. The ligation product was transformed into E. coli and the resulting colonies were screened. Correct colonies were cultured, miniprepped and plasmids were sent for sequencing.
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
The nos operon was again ligated into pSEVA22 containing the pR or J23118 and the resulting products were transformed into P. putida. Colonies were screened but no correct colonies were found. This was repeated, but again no luck. An experiment with P. putida ∆nasT containing the Nap originating from P. stutzeri, P. denitrificans and C. necator was performed to determine if the Nap enzymes are working. Nitrite assay was performed to check nitrite production. The plasmid containing the correct nos accessory genes was transformed into P. putida ∆nasTand a correct P. putida was cultured.
The nos operon was ligated into pSEVA22 containing the J23105 promoter and the resulting product was transformed into P. putida. The colonies were screened and correct ones were cultured, miniprepped and plasmids sent for sequencing. Again mutation. The nos operon was ligated into pSEVA22 containing a rhamnose inducible promoter. The ligation product was transformed into P. putida. Experiment with the 3 Nap enzymes was repeated. The nap operon from P. denitrificans was amplified from pSEVA22 and ligated in the pGNW backbone. The ligation product was transformed into E. coli.
E. coli containing the pGNW with the nap operon from P. denitificans was screened and correct colonies were cultured. After miniprepping the cultures, the plasmids were sent for sequencing. E. coli containing the correct plasmid was conjugated with P. putida ∆nasT. The knock-in op the nap operon in P. putida was completed. P. putida containing pSEVA22-pRham-nosRZDFYLTatA was screened and cultured. Plasmids were sent for sequencing. The accessory plasmid for nos was transformed in the correct strain. P. putida containing pSEVA22-J23100-napEDABCGH from P. denitrificans was transformed with pSEVA65-NirKV from P. stutzeri.
An experiment was performed with P. putida containing pSEVA22-pRham-nosRZDFYLTatA and pSEVA83-accessory growing with different concentrations of rhamnose to obtain the optimal concentration. An experiment was performed with P. putida containing Nap P. denitrificans on plasmid, in the genome and in combination with NirKV. Nitrite assays were performed to quantify the activities of the enzymes.
Wetlab - October
- Week 22: 4th of October - 8th of October
An experiment was performed with P. putida containing pSEVA22-pRham-nosRZDFYLTatA and pSEVA83-accessory growing in sealed bottles containing nitrous oxide. GC-MS was performed to quantify the concentration of N2O in the headspace of the bottles at multiple time points.