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Team:BYU Provo/Notebook/Metabolism/mayjune
From 2014.igem.org
BYU 2014 Notebook |
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Week of May 3rd
April 29, 2014
--CS-- Today we tried to get started but realized that we actually didn’t have anything that we needed. Dr. Grose could not find any P. aeruginosa PAO1 here at BYU, so we sent an email to Dr. Romesberg at The Scripps Institute requesting DNA from them. We also discovered that the antibiotic plasmid BioBricks that we need to transform are actually not in the iGEM kits that we already have, so we sent emails to the teams that submitted them last year to try to acquire the plasmids from them.
May 1, 2014
--CS-- Today we sent out some more emails to people regarding P. aeruginosa PAO1 DNA samples. Since we hadn’t heard anything from Dr. Romesberg, I sent an email to Stephen Lory at Harvard (www.pseudomonas.com says that he is the go-to guy for P. aeruginosa PAO1 samples) and Bri sent one to somebody else. Julie and I also wrote descriptions of our primers on the BYU database spreadsheet that Dr. Grose sent us. Bri also contacted the iGEM Registry people to try to track down the antibiotic BioBricks we need.
Week of May 10th
May 6, 2014
--JR--Nano-dropped the Promoter plasmids that were recently purified. Almost all of the plasmids were over 100ng/ul concentration.
--CS-- We are still trying to get ahold of people for P. aeruginosa PAO1. Yesterday Bri sent the guy from www.pseudomonas.com an email and today Dr. Grose sent him one. Hopefully we will get a response from him in the near future about how we can go about getting our hands on some of that bacteria. Besides that we worked on the promoter library we have. We transformed the remaining plasmid from J23100, J23108, and J23110 into DH5α and plated that bacteria according to the protocol that we have. We also used the NanoDrop to measure the DNA concentrations of the plasmids that had already been isolated from the plasmid preps that we did as a class. All of the plasmids had pretty good concentrations and 260/280 ratios.
May 7, 2014
--CS-- Today we planned on doing plasmid preps for the J23100, J23108, and J23110 bacteria that we cloned yesterday but only the J23110 bacteria ended up growing and expressing RFP (which is the plasmid that we actually already have isolated— somebody had temporarily misplaced it). J23108 had a few colonies grow but none express RFP, and J23100 had nothing grow at all, so we decided to just refrigerate them and see what Desi thinks about them tomorrow.
May 8, 2014
--JR--Set up PCR to amplify Bla gene. Q5 protocol link Used psB1A3 plasmid as template with primers BI374 and BI375.
--CS-- We were planning on doing plasmid preps today for J23108 but realized when we got here that we needed to inoculate the bacteria overnight in order to do those. So Julie picked a colony and grew some bacteria up from it and will take care of the plasmid prep tomorrow (Bri and I will both be gone).
May 9, 2014
--JR--Ran a gel to check PCR products. Looks great! Did PCR-up of PCR product using GenElute Protocol Kit linkRan restriction digests on vectors and insert. Chose the IGEM plasmids containing a strong, and medium-strong promoter strength respectively)
May 10, 2014
--JR--Restriction digests were run on the following samples: Promoter part plasmids BBa_J23102, and BBa_J23118, and Bla gene PCR product. SpeI HF, and XbaI were used with NEB buffer 4 in this protocol for each of the three samples. Restriction digest protocol
Week of May 17th
May 13, 2014
--JR--Figured out that we didn’t want to use the promoter vectors that I digested. We tried using pIG91 from the freezer, but the RD gel did not show any DNA present. Also did Sigma-Aldrich plasmid prep protocol according to the Sigma-Aldrich kit, for new pIG91.
--CS-- So today we continued work on our primers and Julie’s antibiotic part. We forgot to do plasmid preps over the weekend of the bacteria that we had grown up so we grew up some new bacteria from a colony on our J23108 and J23110 plates. To do so we used a plastic swab thing to pick a colony off the plate and add it to 5 ml LB+Amp in an incubation tube (yellow cap) and put that on a shaker in the 37°C incubator room overnight. For the antibiotic, Julie had already ran a restriction digest for the β-Lactamase part (BLa) and two different promoter plasmids. We then ran the BLa on a low-melt gel and cut out the band of our digested DNA. Since Julie cut the promoter plasmids with XbaI and SpeI we put 1 μl of calf intestine alkaline phosphatase in the digested vector and let it incubate for a while. We then realized that the promoter plasmids are in the J61002 plasmid and not the pSB1C3 plasmid, which is the plasmid backbone required for BioBrick parts. Some of the plasmid is in the freezer (called pIG91 in the plasmids II box) and the prophage group is also growing up more of the plasmid in some E. coli. The prophage pros had pelleted the bacteria, so Julie and Bri did plasmid preps to isolate the plasmid.
May 14, 2014
--CS-- Today we did the plasmid preps of the J23108 and J23110 bacteria that we grew up overnight. I also added 1 μl CIP to the restriction digest of the pIG91 vector for an hour and ran it on a low-melt gel. When we went to cut out the DNA though there was no DNA in the well for our plasmid (the ladder had DNA though so the gel worked fine), so it appears that the freezer sample of pIG91 DNA must not have been a good sample. We will try the procedure again with some of the pIG91 plasmid that was isolated yesterday.
May 15, 2014
--JR--Assisted in setting up new RD of pIG91.
Assisted Nano-dropping the recently purified pIG91 plasmisds, all concentrations were approximately 100ng/ul.
Assisted in setting up ligation of digested pIG91 (treated with CIP), with the digested Bla. Both were digested using SpeI HF and XbaI. This is so that we can then have our part in the iGEM registry plasmid to allow for quick and easy future cloning.
--CS-- Today we redid the restriction digest of pIG91 using some of the plasmid that Julie and Bri isolated on Tuesday. We followed the protocol and used the XbaI and SpeI enzymes. We added 1 μl CIP to the digest an hour before running it on a low-melt gel at 80V for about 50 minutes. We then checked the concentrations of the J23108, J23110, and pIG91 plasmids that we had isolated during the week. Everything checked out pretty well in terms of concentrations and purity.
Week of May 24th
May 20, 2014
--JR--Set up colony PCR. Chose 8 colonies from the transformation and streaked colonies onto another plate. Colonies 1-7 were white colonies. We chose colony 8 as a red colony to act as a negative control. Made a master mix (10x) the Taq PCR ProtocolTaq PCR protocol
--CS-- Today we did the colony PCR for the transformed BLa E. coli that Julie had made at the end of last week. We followed the given protocol and used 7 white colonies and 1 red colony (definitely not recombined) in the PCR. Realizing that I would need a ton of the iGEM backbone plasmid (pIG91) for all of the different genes that I would be making standard parts for, I also transformed 2 μl of the previously isolated pIG91 into thawed DH5α according to the given protocol and grew it up in 5 ml LB+Cam overnight in the 37°C incubator shaker.
May 21, 2014
--JR--Ran a gel to verify PCR. Colonies 1, and 4-7 look good. Set up overnights.
--CS-- Today I did the plasmid preps of the pIG91-transformed DH5α that I grew up yesterday according to the protocol in the kit. I also confirmed that there was DNA in the products by measuring the DNA concentration with the NanoDrop. All of the samples had concentrations about 300 ng/μl and 260/280 ratios near 1.8 so they looked great. I recorded the concentrations on the tubes and put them in the freezer. I also ran our PCR products from our colony PCR on gels. Our colonies 1, 4, 5, 6, and 7 all had bands, meaning that these colonies were a success in terms of cloning and transformation. Julie also started overnight liquid cultures of these colonies from the streak plates that she made.
May 22, 2014
--JR--Did plasmid preps on overnights from colonies 4-7.
Nanodropped:
--JR--Set up restriction digests on New Plasmid (pIG91+BlaGenepIG102), and promoter plasmids to build final construct with promoter and the bla gene together.
RD 5-221M, restriction digest of pIG102 cut with EcoR1 and Xba. Restriction digest protocolRD 5-222M, and RD 5-223M
Same as above except: Enzymes used are EcoR1 HF and Spe1 HF Buffer 4 Used Two reactions, one containing promoter plasmid J23104 (5-222M), and the other J23111 (5-223M) Ran Low Melt Gel at 90V for 46 min.
--JR--Each of the Plasmids showed up, which was good for pIG102, where we want that to be our vector. Other RD only saw the plasmids and not the promoter part which we are trying to insert into pIG102….
--CS-- Today I helped Julie a little with plasmid preps of the overnights that she had made from our successful colonies. Since P. aeruginosa came in today, we streaked out some of the bacteria on a normal LB plate. We also isolated DNA directly from stock by sticking a tip into the bacterial stock, putting that in 50 μl water, and boiling the water; we also streaked out a plate using the tip that we pulled the bacteria out of the stock with. We then used PCR to amplify the 4 denitrification genes (nirS, norB, norC, and nosZ) in 4 separate PCR reactions. We used Q5 as the polymerase since it is high-fidelity.
May 23, 2014
--CS-- Today I took my streak plate of P. aeruginosa PAO1 out of the 30°C incubator, parafilmed it, and stuck it in the fridge. There were lots of colonies on the plate so things are looking good!
Week of May 31st
May 27, 2014
--JR--Set up RD of promoter plasmids J23101, and J23106 Used 3 RD enzymes because we will not be able to run on low melt and get out promoter part, so we want to ruin the plasmid the promoter is in so that it doesn’t relegate. Used Enzymes EcoRI, PstI, SpeI
Restriction digestst ran on promoter parts J23101, and J23106 according to restriction digest protocol. Restriction digest protocolBecause product we wanted from digest was so small, running a low melt gel would not be plausible. Thus we instead chose to inactivate enzymes thermally.To destroy enzymes reaction mix was incubated at 80C for 20 minutes, following restriction digest protocol and incubation.
Promoter digests and plasmid vector were each ligated together and incubated at room temperature according to ligation protocol ligation protocol
--JR--Ligation mixtures were each transformed into DH5alpha competent cells according to transformation protocol and then plated on both LB+Cam, and LB+Cam+Amp plates. Plates with both chloramphenicol and ampicillin anitibiotics we expect will select for successful transformants, those which have the standard plasmid backbone and a functioning promoter and beta-lactamase gene
--CS-- Today I ran a gel of the PCR products that I got from last week using a normal gel running at 175 V for about 20 minutes. I included a picture of my gel below:
--JR--All of my genes amplified as they should! They are all the appropriate length and have nice defined bands so I continued by using the PCR purification kit to clean up my DNA. I then ran restriction digests of my genes and the pIG91 vector with XbaI and SpeI, using 1 μl CIP to keep the vector from self-annealing. I loaded the digested genes onto a low-melt gel, ran it for about 45 minutes, and then cut out the sections that contained my desired genes. The bands were very faint in my low-melt gel though so I am not sure how well they will actually turn out. I then followed the ligation procedure in the protocol to insert my genes into the backbones. I let these go at room temperature overnight. I also transformed some promoter plasmids (J23102, J23104, J23111, and J23118) into DH5α and plated it out on some LB+Amp plates overnight to replenish some of the promoter plasmid stocks that Julie had used up in her experiments.
May 29, 2014
--JR--Took 8 colony samples from each of the LB+Amp+Cam plates and set up colony PCRs. Did according to Taq colony PCR protocol Used primer bIG307 pSB1C3 forward and bIG375 which is for Bla reverse.
Week of June 7th
June 3, 2014
--JR--Confirmed colonies via plating and colony PCR. Colony PCR showed colonies positive for colonies 1-4, 5, 6, and 8 for transformation of ligation 5-273M. However, on the plate only colony 8 showed up without RFP, thus I selected this colony as one that would be considered a final construct. Same process was repeated for transformed ligation 5-274M, where colonies 1, and 6 showed up on colony PCR, and where colony 1 was white—colony 1 was selected as a final construct. Overnights were set up and grown at 37C.
June 4, 2014
--JR--Plasmid Preps were ran on the overnight samples.
June 5, 2014
Plasmids were nano-dropped.
I also renamed these plasmids and submitted them to our BYU iGEM parts database.
Ligation273M-8 will be pIG105
Ligation 273M-9 will be pIG106
Week of June 14th
June 10, 2014
--JR--Looked at iGEM webpage, tried to figure out some code things….
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.
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.
