Team:BYU Provo/Notebook/Metabolism/mayjune

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BYU 2014 Notebook

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May 6, 2014

Nano-dropped the Promoter plasmids that were recently purified. Almost all of the plasmids were over 100ng/ul concentration.

May 8, 2014

Set up PCR to amplify Bla gene. This is the iGEM link that eventually needs to be changed to *Q5 protocol link Used psB1A3 plasmid as template with primers BI374 and BI375.

May 9, 2014

Ran a gel to check PCR products. Looks great! Did PCR-up of PCR product using This is the iGEM link that eventually needs to be changed to *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

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. This is the iGEM link that eventually needs to be changed to *Restriction digest protocol

Week of May 17th

May 13, 2014

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 This is the iGEM link that eventually needs to be changed to *Sigma-Aldrich plasmid prep protocol according to the Sigma-Aldrich kit, for new pIG91.

May 15, 2014

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.

Week of May 24th

May 20, 2014

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 ProtocolThis is the iGEM link that eventually needs to be changed to *Taq PCR protocol

May 21, 2014

Ran a gel to verify PCR. Colonies 1, and 4-7 look good. Set up overnights.

May 22, 2014

Did plasmid preps on overnights from colonies 4-7.

Nanodropped:

  • 206.1 ng/ul 260/280: 1.89
  • 215.4 ng/ul 260/280: 1.89
  • 400.2 ng/ul 260/280:1.85
  • 170.0 ng/ul 260/280: 1.84
  • 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. This is the iGEM link that eventually needs to be changed to *Restriction digest protocol

    RD 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.

    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….

    Week of May 31st

    May 27. 2014

    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. This is the iGEM link that eventually needs to be changed to *Restriction digest protocol

    Because 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 This is the iGEM link that eventually needs to be changed to *ligation protocol

    Ligation mixtures were each transformed into DH5alpha competent cells according to This is the iGEM link that eventually needs to be changed 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

    May 29, 2014

    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

    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

    Plasmid Preps were ran on the overnight samples.

    June 5, 2014

    Plasmids were nano-dropped.

  • Lig 274M-1 415.2ng/ul and 260/280: 1.86
  • Lig273M-8 515.6ng/ul and 260/280: 1.86
  • 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

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

    June 12, 2014

    Set up Q5 PCR for genes NorB, NosZ according to the Q5 PCR protocol This is the iGEM link that eventually needs to be changed to *Q5 PCR protocol using P.Aeroginosa as template and NorB forward and NorB reverse primers, BI335 and BI336 respectively.

    June 13, 2014

    Restriction digest of PCR products of NorB and NosZ according to restriction digest protocol This is the iGEM link that eventually needs to be changed to *Restriction digest protocol After incubation for 2 hours restriction digest was treated with CIP to prevent vector from religating

    Transformation

    Transformed ligation mixes into competent DH5alpha according to transformation protocol This is the iGEM link that eventually needs to be changed to *transformation protocol except used 4ul of ligation mix instead of 2.