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BYU 2014 Notebook Edit July August

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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 according to restriction digest protocol . 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 with NEB buffer 4 and BSA.

July 24, 2014

--CS-- Today I worked some more on the team wiki.

Week of August 2nd

July 28, 2014

--JR--Treated our plasmid restriction digest with CIP to prevent plasmid from religating to itself. Ran restriction digests out on low melt gel. Bands all visible, however NosZ was very faint. This band was right next to the plasmid digest, and band was in the same position, which aided in cutting out the band from the low melt.

July 29, 2014

--CS-- Today I worked some more on the team wiki.

--JR--Melted restriction digests and set up ligation reaction according to ligation protocol. Used 5ul of vector for the NosZ gene since band was very faint, and reduced water content to 4.5ul. All others according to protocol with respective genes and our digested pIG91. Ligation was allowed to run 2+hrs. Transformation was then set up according to transformation protocol.

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.

Whole-genome analysis of the ammonia-oxidizing bacterium, Nitrosomonas eutropha C91: implications for niche adaptation.

Week of August 2nd

July 30, 2014

--JR-- Set up colony PCR reactions. Multiplied and did master mix 45X's the single Taq protocol . Used primers BI307 and BI308 for the plasmid forward and reverse. We chose to use these primers just to ensure that we would get PCR product, as it has often not worked at this stage before. Then those that we get product on we plan to do further checks to confirm proper directionality of our genes.

Week of August 9th

August 4, 2014

--JR--Set up and ran a gel on our colony PCR products. NorC looks great! NirS worked, NorB appears to have worked. NosZ may have worked, but is in question. We will be setting up a few more colony PCRs to check the working ones for directionality. Then we plan to do some sequencing of these plasmids.

--CS-- Today Julie and I ran the gels from the colony PCR that she did last week. And we got some great results! The images are below:

The image on the left is the gel of nirS, norB, and norC in that order with a DNA ladder, nirS 1-8, and norB 1-5 on top and a DNA ladder, norB 6-8 and norC 1-8 on bottom. The image on the right is the gel of nosZ 1-16 except 9 with DNA ladders intermixed. There were great bands at the appropriate places for all of the bands except for nosZ, which had one band show up kinda close to where it should have. We talked to Dr. Grose about our results and she said that we should first do a directionality PCR (vector primer on one end and gene primer on the opposite to test that they are inserted in the proper direction since we used the compatible XbaI and SpeI sites for inserting the gene into the vector) and then proceed in sequencing vectors that have the gene inserted in them in the proper direction. So we made some great progress!

I also continued reading the article I referenced last time. I still need to look at it more thoroughly though to figure out exactly what the new bacteria do in terms of denitrification.

August 6, 2014

--JR--Set up some more colony PCR to check directionality of colonies that produced bands. PCR this time was done with the pSB1C3 MCS Forward primer (BI307) and was done with each genes respective primer. Chose colonies 4,5,7,8 of NirS; 1,2,3,5,8 of NorB, and 1,2,3,7 of NorC. Did according to Taq protocol.

August 7, 2014

--JR--Ran a gel for the PCR's set up yesterday. Looks like we have NirS, NorB, and NorC in the plasmid AND in the right direction! Denitrifying here we come! Also, set up overnights for the colonies which were indicated by the PCR to contain the insert.

August 8, 2014

--JR--Purified plasmids from positive colonies.

Week of August 16th

August 11, 2014

--JR--Nanodropped plasmid preps today. They had extremely low concentrations, which I suspect is a result of me not letting the elution reaction incubate long enough before centrifuging. However, this is a good thing because I overnighted the wrong colonies anyways. Thus I restarted and made sure to set up overnights of the correct colonies.

--CS-- Today I got caught up with Julie on the progress of the denitrification gene cloning process. She started overnights today and I will take over with plasmid preps tomorrow before submitting them to Desi for sequencing.

Week of August 23rd

Week of August 30th