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From 2014.igem.org

BYU 2014 Notebook Edit May June

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Week of May 3rd

--CB-- /--TR-- This week in the lab we started our experiment to remove the sera gene from N multiformis. We attempted to gain access to the genomic DNA of N multiformis by boiling the organism for 5 min to lyse the cell, this technique has worked on similar organisms in the past and due to its simplicity we opted for this approach. After boiling we added our primers and proceeded to perform PCR. Then run our PCR product on a gel. We did this by adding our DNA samples (both forward primers and reverse primers) and a DNA ladder of known size to an agarose gel that had been stained with ethidium bromide and electrophoresing for 30-40 min. After isolating our band of DNA we purified it using a freeze and squeeze method. This method involves excising a band of DNA from the agarose gel and the gel slice cut into small pieces and placed into a micro centrifuge tube. This tube is then placed in a -20C freezer for 20 minutes then removed and immediately centrifuged at 12,000 for 5 minutes at room temp. Agarose debris is will be forced to the bottom of the cup and our now purified DNA is floating on top ready for removal.

Week of May 10th

May 5, 2014

--CB TR-- We finished out the freeze and squeeze experiment. We took the agarose gel from the freezer that held our PCR product and centrifuged it for 5 min at max speed the added to the following protocol for a freeze and squeeze to get our purified product. Freeze and Sqeeze 2ul fragment one from PCR 2ul fragment two from PCR 4ul 5x buffer 4ul 5x enhancer .5ul dNTP .5ul primer on left side of left homology block .5ul primer on right side of right homology block .2ul Q5 enzyme QA 20uL ddH2O Because Desi said that doing a freeze and squeeze was probably an unnecessary step we also ran a normal where we do not do a freeze and squeeze, rather we run straight to the SOEing part of it. SOEing protocol 1ul fragment one from PCR 1ul fragment two from PCR 4ul 5x buffer 4ul 5x enhancer .5ul dNTP .5ul primer on left side of left homology block .5ul primer on right side of right homology block .2ul Q5 enzyme QA 20uL ddH2O We got our SOEing product and ran it on a gel (picture taken and to be included once we scan it). We also performed a restriction digest and plasmid prep.

Week of May 17th

May 13, 2014

The planned schedule Week 1 Ligation, Transform Week 2 Conjugate (long time) • Grow the transformed ecoli (s17) • Grow N multiformis • Mix together, turn off the lights, but on some Barry White and wait Week Work on getting funding * experiment.com • Fancy black card man• Week4 Select for knock out with Kanamycin Select with sucrose Grow in serine rich, low, and no environments Thursday: Today we took the product from our ligation and transformed it. (protocol to be added) We made 3 tubes Tube 1: 200uL s17, 1uL of 93.1 ng/uL mini-prep pSR47s Tube 2: 200uL of our ligation. Tube 3: 300ul of LB, 90ul ddH2O, and 1ul of S17 • S17 is the bacteria that makes our suicide plasma grow. All 3 tubes were placed on a shaker at 37 degrees C. Tuesday: Today (Tuesday) we did ran a gel of the plasmid prep (low melt) of the plasmid digest and the PCR digest (pics to be uploaded) We also did a mini-prep with the pSR47s plasmid. And a ligation

Week of May 24th

The past week I have spent approximately 6+ hours researching funding opportunities for our team and estimating costs. Opportunities include • Rathnau Instituut Grant • Departmental Funding • Crowd founding initiatives o I have started to build a profile on Experiement.com • Various Donors Verified our clones via colony PCR We did this by taking samples from 8 colonies to test for our clone. Alongside the 8 colonies we also ran a negative control (just the plasmid with no insert) and a positive control (our original soeing product) Protocol: 16.5 DDH2O 2.5 REdtaq Buffer 1 DNTP 1 Primer A 1 Primer B 1.25 Redtaq 2 Boiled template (colony) These we PCR’d and wil check on Thursday.

Week of May 31st

I spent a ton of time this week writing the Rathenau Instituut grant… like LOADS of time. And after it was run through the class refiners fire I think it came out pretty good. I also ran a gel of our new cloning PCR to verify that our TAQ pcr worked. Tuesday morning we found that the PCR we ran last week failed. We are now rerunning the clone verification using regular TAQ polymerase instead if RedTAQ polymerase. Another group (one of the Cameron’s) also had their RedTAQ PCR fail. By cross checking with a different polymerase we hope to verify whether or not the polymerase is to blame for the failed experiment. When boiling template we used 50 uL of ddH2O with our template.

Week of June 7th

This week started out a little bit rough. We found that not only did Tanner forget to pull the plate from the incubator (again) I foolishly left the gel I ran Thursday in the electrophoresis dish without taking a picture (because I was under the delusion that that would be okay) and so our product diffused all over the place so this week we are re-re-doing out clone . We poured a gel, placed in 8 colonies and are running it now. If this works we hope to jump into the conjugation with N. multiformis. So bad news, it didin’t work. The bands we saw from our + control and our colonys showed at 600bp instead of the 1000bp where they shold have been. We are not certain why this happened but our best guess would be that we cut out the wrong bands and placed them in our plasmid. So now we are going to re do out SOE to get new SOEing product that we will remove from a gel and put into our plasmid. Redo of SOEing. 1ul fragment one from PCR 1ul fragment two from PCR 4ul 5x buffer 4ul 5x enhancer .5ul dNTP .5ul primer on left side of left homology block .5ul primer on right side of right homology block .2ul Q5 enzyme QA 20uL ddH2O

Week of June 14th

June 10, 2014

--JR--Looked at iGEM webpage, tried to figure out some code things….

--CS-- So I talked to Dr. Grose about my colony PCR results and since there were some solid bands for norB and nosZ but the bands were not in the spot they are supposed to be (both appear to be shorter than they should be), I did another colony PCR with the DNA from the colonies. This time I used the pSB1C3 forward and reverse primers (307 and 308) to check since those primers are known to work and the primers appeared to have caused the problem with my PCR (there was a laddering pattern observed in the gel, suggesting that the binding wasn’t very good). I then ran the PCR products on a gel. My gels looked like this, with 4 DNA ladders spread throughout the 8 selected norB colonies, 7 of the selected nosZ colonies, and a norC positive control from a previous experiment that had worked properly:

All of these bands are for rather short sequences, suggesting that the pSB1C3 vectors closed in on themselves instead of adding the genes like we hoped they would during the incubation. I used CIP during the restriction digest, so this should not have been a problem but apparently it was.

June 12, 2014

--JR--Set up Q5 PCR for genes NorB, NosZ according to the Q5 PCR protocol Q5 PCR protocol using P.Aeroginosa as template and NorB forward and NorB reverse primers, BI335 and BI336 respectively.

--CS-- Since my restriction digest appeared not to work, I started everything all over again today for norB and nosZ. Julie and I used Q5 to amplify these genes from the P. aeruginosa genome after putting a colony in 50 μl water and boiling it to release the DNA. The gel of the PCR looked something like this:

The first lane after the ladder is norB, which looks right since it is a 1400 bp gene. Then there were 3 bands that showed up for nosZ in the right lane. Since one of these bands is where it should be, we will continue with the restriction digest though and use the low-melt gel to pick the portion we want if these extra bands show up there too.

June 13, 2014

--JR--Restriction digest of PCR products of NorB and NosZ according to restriction digest protocol Restriction digest protocol After incubation for 2 hours restriction digest was treated with CIP to prevent vector from religating

Transformation

--JR--Transformed ligation mixes into competent DH5alpha according to transformation protocol transformation protocol except used 4ul of ligation mix instead of 2.

--CS-- Today Julie and I ran a restriction digest of norB and nosZ using XbaI and SpeI enzymes according to the protocol. We added 1 μl CIP 1 hour prior to running the low-melt gel to prevent self-annealing of the backbone. We then ran the low-melt gel and cut out the bands that contained our genes and the vector. Here is an image of the low-melt gel:

The pIG91 digest turned out really well but the other ones were a lot fainter. We then melted the cutout bands and did ligations according to protocol. Julie then transformed these into bacteria and plated them on LB+CAM and put them in the 37°C incubator overnight.

Week of June 21st

June 17, 2014

--CS-- Today I ran the colony PCR of the transformed bacteria that Julie had grown up last week. The gel of my PCR products looked like this:

The image on the left has 7 norB colonies with a negative control (no insert) and the image on the right has 7 nosZ colonies with a negative control. Bet you can’t tell which lane is the negative control! So it appears that this round of cloning didn’t work either. I went through the whole procedure with Julie and we were unable to figure it all out, so we think it might be a primer problem but we really have no idea.

June 19, 2014

--CS-- So things haven’t been working well for norB and nosZ. For some reason I constantly get gels that look like this:

The norB lane (the middle lane) seems to be blurry the majority of the time, and the nosZ lane (the right lane) seems to always have 3 bands in it. This makes it seem that the primers I have for these two genes are screwy, so I looked at my primer sequences again. Both of the primers have the correct sequences and have 24-25 bp of the gene start/finish in addition to the correct restriction digest sites and the extra 3 bp that help the restriction enzymes bind. I blasted the P. aeruginosa PAO1 genome with the primer sequences and found that there are actually quite a lot of sites in the genome that match the sequences. Besides the appropriate sites of the genes that the primers were designed for, these matches are much weaker though, having no more than 15 aligning nucleotides for the most part. I tried to look for some pairs of sites that would result in the extra bands found in the nosZ lanes, but I couldn’t identify anything obvious that would work for that. So I am unsure as to why norB always smears and nosZ always has 3 bands and why neither of them works. I can amplify both of them from the P. aeruginosa genome but somewhere after that things don’t work.

Week of June 28th

June 24, 2014

--CS-- Today I discovered the source of my cloning PCR problem. The reverse primers for all 4 of my denitrification genes were wrong! I don’t know how it happened but somehow the beginning of each of the gene sequences in the primers got switched around; the following shows what the primers are and what they should be:

nirS reverse (with SpeI site)

• Incorrect primer sequence: ccgACTAGTATCAGTACACGTCGTGCTGGGTGTT
• Correct primer sequence: ccgACTAGTTCAGTACACGTCGTGCTGGGTGTT

norB reverse (with SpeI site)

• Incorrect primer sequence: ccgACTAGTATCAGGCGGCCGCCTTGCCGCGCCGG
• Correct primer sequence: ccgACTAGTTCAGGCGGCCGCCTTGCCGCGCCGG

norC reverse (with SpeI site)

• Incorrect primer sequence: cggACTAGTATCAACCCTCCTTGTTCGGCGGCCA
• Correct primer sequence: cggACTAGTTCAACCCTCCTTGTTCGGCGGCCA

nosZ reverse (with SpeI site)

• Incorrect primer sequence: ccgACTAGTATCAAGCCTTTTCCACCAGCATCCGC
• Correct primer sequence: ccgACTAGTTCAAGCCTTTTCCACCAGCATCCGC

I compared the forward and mutagenic primers that we have to what I originally designed and they were all the same, so the problem only exists with these reverse primers. I went through the primers I originally designed too and double checked to make sure they were good for the genes and restriction sites, and they all seem to be alright in those regards. So it is interesting that norC and nirS actually worked despite the error. I sent Dr. Grose a summary of the primer issue and she will order some new primers so that we can get working on cloning the genes with the right primers next week.

June 25, 2014

--CS-- Today we prepared our presentations. We then gave them.

June 26, 2014

--CS-- Today I talked to Dr. Breakwell about our project to get some ideas about how to make the denitrification pathway work better in N. multiformis. He talked to me a lot about how the type of bacteria that N. multiformis is has a lot to do with whether or not adding the denitrification genes into it will work. We went through the redox reactions that are involved in ammonia oxidation (what N. multiformis already does) and denitrification (what we want N. multiformis to do) and found that the energy produced by the bacteria when using O2 as an electron acceptor (aerobic respiration) is more than that which would be produced when converting the nitrate all the way to nitrogen gas (which is anaerobic); this means that the bacteria will likely not prefer producing nitrogen over nitrate. He said that we would likely need to find a way of reducing the oxygen in the activated sludge as part of a cycle to promote the N. multiformis to do denitrification. He also said that knocking out the gene that converts nitrite to nitrate might work in forcing the pathway through to completion but we really don’t know until we try. He also said that having a constitutive promoter on the plasmid with all of the necessary genes might also do the trick; if the proteins are being made irrespective to the bacteria’s demands then the bacteria might just go ahead and use them to get more energy. I also ran my ideas for assaying the denitrification plasmid in E. coli and later in N. multiformis; he thought the Durham test would be a good test and also suggested we test the nitrate level of the broth before and after. Doing both will allow us to confirm if nitrate is being used up and if nitrogen gas is being produced as we designed. He also informed me that E. coli naturally denitrifies in anaerobic conditions when nitrate is present, so for testing our plasmid out in E. coli we will need to use a strain that is knocked out for denitrification genes.