Team:Wageningen UR/Notebook/Anemoon
Here you can find the notebooks from Anemoon.
Modeling
- September
- October
- November
- December
- January
- February
In this month, I did the following: getting introduced to Python, CobraPy, constraint-based modeling in genome scale metabolic models (for mono- and co-cultures) and searching for genome sale metabolic models (Paracoccus denitrificans and Methylococcus capsulatus)
I tried automatic gap-filling on the P. denitrificans GSMM (which failed) and started the manual gap-filling process. I also started working on the particle scale of the biofilter model.
I finished the manual gap-filling of the P. denitrificans GSMM. I continued working on the particle scale and started working on the biofilter scale of the biofilter model.
I finished the bases of the biofilter model (one model combining the three different scales).
I prepared the P. denitrificans and M. capsulatus GSMMs for community FBA (cFBA) and performed cFBA with SteadyCom (in Matlab). I also incorporated a sensitivity analysis: the model now determined biofilter productivity and efficiency
I runned my model with different conditions to get the final results
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
Amplified Pseudomonas aeruginosa nirM & nirC from the genome. Ligated it in pSB1C3 and transformed into Escherichia coli. Screened colonies with colPCR.
Screened colonies for P. aeruginosa nirM & nirC were sequenced and correct.
Amplified P. aeruginosa nirS,F,D,L,G,H,J,E,N from the genome. Ligated it in pSB1C3 and transformed into E. coli. Screened colonies with colPCR. (it would take some more time for the complete nirJ sequence to come in).
Amplified P. aeruginosa nirQ,O,P and norC,B,D from the genome; all but nirP were succesful. Ligated it in pSB1C3.
Transformed P. aeruginosa nirQ,O and norC,B,D in E. coli. Screened colonies with colPCR and sent selected colonies for sequencing: all correct.
P. aeruginosa nirS,F,D,L,G,H,J,E,N were sequenced and correct.
Amplified Pseudomonas stutzeri norC,B,D & nirK,V from the genome. Ligated it in pSB1C3 and transformed into E. coli. Screened colonies with colPCR and sent selected colonies for sequencing: all correct.
P. aeruginosa nirS,M,C,F,D,L,G,H,J,E,N & nirO & norB,D were amplified from pSB1C3 for operons in pSEVA.
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
Amplified P. stutzeri nirS,T,B,M,C,F,D,L,G,H,J,E,N & nirQ,O,P from the genome. All but nirJ,E succesfully. Ligated it in pSB1C3 and transformed into E. coli. Screened colonies with colPCR and sent selected colonies for sequencing (partly in week 6 as there we not enough stickers): all correct.
Amplified P. stutzeri nirJ,E succesfully with a second PCR. Ligated it in pSB1C3.
Attempted to amplified P. aeruginosa nirP from the genome two times: second time seemed succesful. Ligated it in pSB1C3.
Amplified P. aeruginosa nirS,F,H & nirQ & norC from pSB1C3 for operons in pSEVA.
Transformed P. aeruginosa nirFDLG & nirHJEN in pSEVAb-22, nirSMC in pSEVAb-65 and norCBD in pSEVAb-65; all with PJ23100 promotor.
Tested nasT strain for streptomycin resistance: it did not have it.
ransformed P. stutzeri nirJ,E in E. coli. Screened colonies with colPCR and sent selected colonies for sequencing: all correct.
Transformed P. aeruginosa nirP in E. coli. Screened colonies with colPCR and sent selected colonies for sequencing: nirP missing/very incomplete. => need to reasses amplification from genome.
Transformed P. aeruginosa nirSMC, nirFDLG, nirHJEN & norCBD in E. coli. No colonies grew. => need to redo ligation: ligated nirSMC, nirFDLG, nirHJEN & norCBD. These were transformed in E. coli. Only nirFDLG could be sent grown and sent for sequencing.
Amplified P. stutzeri nirK,V & nirS,T,B,M,C & norC,B,D & nirQOP from pSB1C3 for operons in pSEVA: nirS,B,H,K & norC,B unsuccesful (this week). nirFDLG and nirQOP were ligated into pSEVAb-22 and -43 with PJ23100 promotor, respectively. These were transformed in E. coli. The colonies were screened with colPCR. Only nirFDLG could be grown and sent for sequencing.
Amplified P. stutzeri nirS,B,H,J,E,K,Q & norC,B from pSB1C3 for operons in pSEVA. These subunits were used for ligation: nirSTBMC in pSEVAb-65, nirHJEN in pSEVAb-22, norCBD in pSEVAb-65, nirQOP in pSEVAb-43, and nirKV in pSEVAb-65, all with PJ23100 promotor. They were transformed in E. coli. colPCR was used to screen, only positive colonies of nirQOP, nirHJEN and norCBD were found and grown for sequencing.
From P. aeruginosa, nirSMC, nirHJEN, norCBD were ligated in pSEVAb-65, pSEVAb-22, pSEVAb-65, respectively, all with PJ23100 promotor. They were transformed in E. coli. colPCR was used to screen, but no positive colonies were found.
The grown P. aeruginosa nirFDLG colonies were send for sequencing twice; sequencing quality was poor and no conclusions could be taken.
Performed toxicity analaysis of sodium nitroprusside (SNP) on Peudomonas putida in M9
From the P. stutzeri nirQOP, nirHJEN and norCBD colonies, only the nirQOP colonies grew. Their miniprepped DNA was send for suquencing: quality of the sequencing result was poor.
Regrown from glycerol stock and made new miniprep for sequencing for P. stutzeri nirQOP & nirFDLG and P. aeruginosa nirFDLG. Again, the quality of sequencing was too poor to take any conclusions.
A piece of dna slightly wider than P. aeruginosa nirP was amplfied. nirP was amplified from this piece of DNA. nirP was ligated in pSB1C3 and transformed in E. coli. The colonies were screened and some were send for sequencing: sequence was correct.
More colonies from plates from week 6 and 7 were screened for P. aeruginosa nirHJEN, nirSMC & norCBD and P. stutzeri nirSTBMC, nirHJEN, nirKV & norCBD. Both a positive colony for P. aeruginosa and P. stutzeri nirHJEN were send for seqeuncing.
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
P. aeruginosa nirP was amplified from pSB1C3 for operons in pSEVA.
Amplified the suspected P. aeruginosa nirFDLG & nirHJEN and P. stutzeri nirFDGL & nirQOP operons from the new minipreps to send PCR amplification of the operons for sequencing. nirHJEN seemed to be missing in the colony.
Ligated P. aeruginosa nirS,M,C & nirQ,O,P & nirH,J,E,N & norC,B,D and P. stutzeri nirS,T,B,M,C & nirH,J,E,N & nirK,V & norC,B,D with pSEVAb-65 (nirS+ or nirKV and nor) or pSEVA-22 (nir heme); and checked whether the ligation was correct by PCR amplification and agarose gel electrophoresis. All but both nor operons gave a band in the correct size. The bands were cut out of the agarose gel and used in a new ligation with the pSEVA backbones,
Did first preliminary test with SNP and nitrite assay to see whether nitric oxide is detected and how much is released: it is detected, however, I need a better standard curve of sodium nitrite next time.
Sent the amplifications of the operons for sequencing
The checked and gel cut ligations were transformed in E. coli. The colonies were screened and only for P. aeruginosa nirQOP and nirSMC positive colonies were found and send for sequencing: both showed that the sequences were not complete for these operons. A very often occuring band size was present for nirKV which was smaller than both subunits but bigger than only one. Therefore, dna of such a colony was send for sequencing: instead of nirKV it contained a lysine specific E. coli permease. So, I decided to reamplify nirK,V from pSB1C3.
New ligation of P. aeruginosa norCBD & nirHJEN and P. stutzeri norCBD & nirSTBMC was performed and checked with PCR amplification. All but nirSTBMC contained the right size band and were transformed directly in P. putida. A new ligation and check were performed for nirSTBMC as I discovered I had used the wrong dilutions before; this time the right size band was present.
The amplification of both nirFDLG and the P. stutzeri nirQOP PCR amplification showed that nirQOP was correct except for one point mutation; and that the new minipreps made in week 8 for P. aeruginosa and P. stutzeri were switched (confirmed via Blast). Therefore, they were once more miniprepped and sequenced with the correct primers. The P. stutzeri nirFDLG was correct except nirD was missing and a curcuma gene from Maria was there. P. aeruginosa nirFDLG quality was too bad to be sure.
• This week I was on holiday
• This week I was on 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
From the transformed colonies from week 10, only norCBD colonies grew. Screening showed that they did not contain the full operon.
I found that nirE is not essential in P. putida. Because of this and the difficulties in cloning, I decided to no longer add this subunit in the heme operons. Therefore, I reamplified the nirN to get the right ends for this approach.
From both strains, norCBD and nirHJN were newly ligated. Also, the nirKV with newly amplified subunits.
I checked the ligation of week 13, and found only nirHJEN (2x) and nirKV contained the right bands. These ligations were used to transform P. putida (nirHJN) or E. coli. No positive colonies were detected for nirKV.
A new ligation was attempted with earlier checked ligations of NirSMC, nirSTBMC, and nirQOP from P. aeruginosa. They were screened in week 15, and none of the colonies were positive.
The P. stutzeri nirXDLG plasmid was used to amplify the nirDLG part; so the ligation could go from a four to 2 piece ligation.
From this week on I started with a new cloning strategy. The Golden Gate Assembly was not too successful, and I decided with my supervisors to try overlap extesion PCR for the cargo of the plasmids. The PCR product would be ligated in the plasmid backbone via Golden Gate.
Tested the release of nitric oxide from SNP (this time with standard curve nitrite) and the stability of nitrite in the incubator with the nitrite Assay.
Pieces of the P. stutzeri nirQOP were amplified to remove the point mutation.
Performed toxicity analaysis of sodium nitrite on P. putida in M9
The weeks prior I designed a NO-responsive biosensor. This week I amplified the correct parts and genes from Cupriavidus necator, and ligated them.
I ligated the presumably P. stutzeri nirQOP.
I started to PCR amplify the fragment of both strain's norCBD and nirKV for the overlap extension PCR, and performed the first overlap extension PCR.
I transformed the biosensor and the P. stutzeri nirQOP in E. coli. The biosensor gave positive colonies, but sequencing showed that the PCR to amplify norR created a mix of norR and another gene. The sequencing quality was poor, and due to timeconstraints parts of plasmids of different colonies were amplified and combined into one new plasmid. Positive colonies were also found for nirQOP and sequencing showed the point mutation was removed successfully.
The extension overlap PCR from week 16 gave right bands for nirKV and P. aeruginosa norCBD. These pieces were ligated and transformed in P. putida. Only nirKV gave positive colonies in screening. Sequencing showed te sequence was correct.
Preperation PCR and overlap extension PCR was performed for the subunits of P. aeruginosa nirFDLGHJN & nirQOP & nirSMC. For both norCBD's the overlap extension PCR was repeated. Onlyy nirSMC and nirQOP gave right bands.
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
P. aeruginosa nirSMC and nirQOP were transformed in P. putida. Only nirQOP gave positive colonies. Sequencing showed the sequence was correct. nirSMC was newly transformed.
For both norCBD's a two step overlap extension PCR was performed: first get norCB, then norCBD.
(preparatory and) overlap extesio PCR of P. aeruginosa nirFDLGHJN was performed. Only P. stutzeri norCBD was successful. This P. stutzeri norCBD and the P. aeruginosa norCBD from week 17 were ligated and transformed in P. putida. The olonies were screened and only on P. stutzeri norCBD seemed positive.
The newly ligated biosensor was transformed in P. putida. No positive colonies were found. A new transformation was done.
A growth experiment with the P. putida pnirKV was performed.
I screened the biosensor colonies; the positive colonies contained a point mutation introducing a stop codon.
Sequencing showed that the P. stutzeri norCBD was missing big parts of norB. Both P. stutzeri and P. aeruginosa norCBD were ligated and transformed in P. putida; but with a different backbone: pSEVAb-22. Two positive P. stutzeri colonies were sequenced after screening. One missed parts of norB, the other had bad sequencing quality;; however, presumably the end of norB not present.
The newly transformed nirSMC was screened and sequencing of a positive colony showed it was correct.
P. stutzeri was newly ligated, but with two different promotors: PJ23100, and rhamnose inducible. These ligations were transformed in P. putida. Sequencing results of positive colonies showed only the plasmids with the rhamnose inducible promotor were correct.
Parts of the biosensor were amplified and then ligated to remove the point mutation.
Tested the release of nitric oxide from SNP (this time with standard curve nitrite) with higher concentrations in the incubator with the nitrite Assay.
The correct plasmids or Nir and Nor were transformed in different combinations (sometimes together with the Nap from Sophieke). They were screened to test if the transformation was usccessful.
P. putida was transformed with the biosensor. No colonies grew.
I performed a Rhamnose analysis: test growth of the strains with norCBD.
I performed a combined experiment with Sanne and Sophieke where we used nitrite and nitrate assays to test different strains created. (Interesting for me are the Nap + Nir strains)
Wetlab - October
- Week 22: 4th of October - 8th of October
I performed a combined experiment with Sanne and Sophieke where we used sealed aerobic bottles and GC to test different strains created. (Interesting for me are the Nor and Nap+Nir+Nor strains)
The strains with combinations with nirQOP were screened; and all did not posses this plasmid, but streptomycin resistance