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Team:BYU Provo/Notebook/Metabolism/julyaug
From 2014.igem.org
BYU 2014 Notebook |
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Week of July 5th
June 30, 2014
--BRK--I chose one colony of E.coli from four separate LB and CAM plates containing transformed bacteria with EreB and a promoter of either 101, 108, 111, or 119. These four colonies were streaked on four separate LB, CAM, and Erythromycin plates and grown overnight at 37C.
--CS-- We got our new reverse primers in today so I first checked all of the sequences to make sure they were correct and then ran Q5 PCR for all 4 genes. Julie then ran a gel of the PCR products. It looked like this:
The lanes are, from left to right, nirS, the DNA ladder, norB, norC, and nosZ. The most prevalent bands in each lane appear to be in the appropriate places based on length so it looks good to continue.
July 1, 2014
--BRK--The streaked colonies grew well on the LB/CAM/Erythromycin plates, indicating that the plasmid with the EreB gene had been successfully transformed. I took pictures of all the plates to compare the growth and use in the future to determine the effect of the different promoters.
--CS-- Today I did PCR cleanup, the restriction digest (XbaI and SpeI), the low-melt gel, the ligation, and the transformation of all 4 of the denitrification genes since we got the new reverse primers yesterday. My low-melt gel looked like this:
The lanes are, from left to right, the DNA ladder, nirS, norB, norC, nosZ and pIG91. I put my plates in the 37°C incubator overnight.
July 2, 2014
--CS-- Today we went to the wastewater reclamation facility in Park City. I learned a lot about the entire treatment process; I almost wish that we had gone to see the facility even earlier on to get a better idea of what actually happens. The visit gave me some more hope about the feasibility and importance of adding the denitrification pathway to N. multiformis. At first the worker who gave us the tour made it seem that the majority of the ammonia was converted to nitrogen during the activated sludge carousel stage, but the algal bloom observed in the final effluent stage and the high proportion of nitrite/nitrate released in the effluent show that there certainly is a need for improvement in nitrate removal. The readings they took in March (they provided us a copy of their report sheet) showed that of the 34 mg/l ammonia that enter the facility, about .5 mg/l ammonia and 25 mg/l nitrite/nitrate leave the facility. This shows that although the ammonia is removed very effectively (99%) there is still quite a bit of room for improvement in removing the nitrate from the effluent to reduce the occurrence of eutrophication. We got some pictures of the water leaving the facility where algae was growing all over despite the rather low flow of water. Another thing that I learned was that the conditions of the activated sludge carousel might actually be in favor of forcing N. multiformis to do both nitrification and denitrification since it might not be too favorable from a metabolic standpoint. As the waste water enters the carousel it is aerated to activate it. As it moves around the carousel though the oxygen supply is depleted, resulting in a portion of the carousel in which the conditions are anoxic. Denitrification occurs during this stage. Here is a diagram:
This change from activated to anoxic conditions works well with our project since, as Dr. Breakwell discussed when I met with him last week, this will help convince our engineered N. multiformis to undergo both nitrification (aerobic) and denitrification (anaerobic) since the different pathways are favorable under different oxygen conditions.
July 3, 2014
--BRK--Our team went to the wastewater treatment plant to learn more about the entire treatment process. I collected the biofilm and effluent water run-off from the plant to bring back to the lab for testing.
Week of July 12th
July 7, 2014
--BRK---Searched for a process to detect antibiotics in water. A few processes were discussed in a powerpoint written by Dr. Rosenfeldt from the University of Massachusetts. http://k12s.phast.umass.edu/sess/emerging/
Week of July 19th
July 14, 2014
--CS-- Today we moved the lab equipment and supplies over to the new building.
July 17, 2014
--CS-- Today I did colony PCR for all 4 of the genes using the pSB1C3 forward and reverse primers (307 and 308). And the PCR worked great! The only problem is that all of the bands showed up under 500 bp, meaning none of the colonies had their genes in them. So it appears that we need to go all the way back to the start again. I didn't bother saving an image because it was very clear that all of the bands were too short.
July 19, 2014
--CS-- Today I worked on the team wiki.
Week of July 26th
July 21, 2014
--CS-- Today I worked a little bit on the team wiki. I also helped Julie get things set up for the PCR since we are going to have her run through the cloning process with all the genes one time to see if she can get things to work. And then there was a chemical spill alarm in the building. After that I finished up the preliminary safety sheet for our team.
--JR--Set up a Q5 PCR for 5 denitrification genes: NirS, NorB, NorC, NosZ. Set up according to Q5 PCR protocol with the following primers for each gene respectively NirS, BI333, BI334; NorB BI335, BI336; NorC BI337, BI338; NosZ BI339, BI340; Negative control with primers BI339, and BI340, but with no template.
July 23, 2014
--CS-- Today I worked some more on the team wiki.
--JR--Did PCR cleanup according to the GE cleanup kit protocol for the 5 denitrification genes. Then set up restriction digest of the genes. Used all 50 ul from the PCR reactions, also used 50ul for the plasmid digest as well. Decreased the water amount in reaction to compensate for added DNA content. Used XbaI,and SpeHF
July 24, 2014
--CS-- Today I worked some more on the team wiki.
Week of August 2nd
July 29, 2014
--CS-- Today I worked some more on the team wiki.
July 24, 2014
--CS-- Today I selected colonies from the plates that Julie made for the colony PCR. I added each colony to 50 μl water and boiled that; I also streaked out these colonies on new LB+Cam plates. I then started looking into the genomes of Nitrsomonas eutropha and Nitrosomonas europaea to see if they already have denitrification genes in them since we might be switching over to one of them as our chassis. I did some BLASTn comparisons and none of the 4 denitrification genes from P. aeruginosa PAO1 came up with any hits to either genome. I did BLASTx too though and although nits and nosZ did not have any hits with either of the new bacteria, both norB and norC matched the genomes. The norB gene had E values of 8e-128 and 3e-126 for N. europaea and N. eutropha, and the norC gene had E values of 3e-51 and 2e-50 for N. europaea and N. eutropha. It appears from these results that the two new bacteria that we might switch over to actually have some of these genes. I found a paper in PubMed about N. eutropha but I did not have enough time to look through it thoroughly. I will go back and try to find more papers to figure out whether or not these bacteria already do denitrification.
