Synthetic nitrification in Pseudomonas putida
Synthetic nitrification in P. putida
Here you can find the notebook from Thomas.
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
Conducted literature research and prepared growth curve and toxicity experiments.
Continued with preparations, and prepared M9 minimal medium for the first time.
Carried out growth experiments with P. putida EM42, and refined cloning strategy further.
Conducted the first toxicity test with hydroxylamine, nitrite, and nitrate.
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
Carried out another toxicity test with a higher resolution of concentrations tested.
Attempted to conduct a test to see if hydroxylamine or nitrite degrade without the presence of bacteria. Unfortunately, pre-cultures failed to grow in M9 minimal medium.
Streaked now stock plates and repeated the test of last week, to try and solve the problem.
Started with my cloning approach, running my first PCR (overlap extension). Also amplified gblocks to perform Gibson assemblies with. Growth problems with P. putida EM42 still not solved.
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
Repeated several PCRs that failed last week, using different parameters this time. Also ran some gradient PCRs to solve the problem. Poured some plates with antibiotics.
Tried my first Gibson assembly! No luck, though. Also still wrestling with some PCRs, especially backbones for some reason. Still troubleshooting growth problems with P. putida EM42 in M9 minimal medium.
Tried 30 mM acetate instead of 110 mM acetate in my M9 minimal medium; this seemed to do the trick! P. putida EM42 grows well now. Repeated toxicity experiments with this new concentration. Also tried some more Gibson assemblies, repeating the ones that failed and using newly amplified PCR products to attempt new assemblies.
Toxicity experiment results form last week were lost due to a malfunctioning plate reader. Conducted quite a few colony PCRs. Many Gibson assemblies were incomplete. Made heat-shock competent cells. Started another toxicity experiment.
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
Some Gibson assemblies appeared to be successful, judging by the colony PCR results. Miniprepped some transformants and sent the plasmids for sequencing. Also, finally generated toxicity data for P. putida EM42 on hydroxylamine, nitrite, and nitrate.
One of the four plasmids that I wanted to construct is complete! Both cytochrome plasmids are also constructed but contain mutations that would result in no or incomplete gene expression. Tried to fix these by ordering primers that anneal to the "incorrect" region, amplifying the entire plasmid, and performing blunt-end ligation.
Went on vacation!
Still busy PCRing fragments that have not yet yielded a successful amplicon. Continued trying to fix the two plasmids that had mutations.
Finally completed the final plasmid, and sent it for sequencing. Performed a lot of colony PCRs this week.
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
Many of the colony PCRs were inconclusive, so I had to repeat them with different primers and parameters. Sent the (hopefully fixed this time) plasmids for sequencing.
Two out of four plasmids were now complete! Two still needed fixing. Decided to put the genes on these plasmids under an inducible promoter instead of a constitutive promoter. Back to PCRs and Gibson assemblies!
Assemblies worked in one go; sequencing results were also perfect straight away! It's amazing how fast cloning can go when you're not trying to construct something that exerts negative selection pressure on your cloning vector.
Transformed P. putida EM42 with all three plasmids sequentially, first with a single transformation followed by a double transformation. This worked surprisingly well.
Wetlab - October
- Week 22: 4th of October - 8th of October
Unfortunately, obligations for other iGEM deadlines meant that I was not able to analyze the nitrifying activity of the P. putida EM42 NC2 and NCX strains.