Set up restriction digests on New Plasmid (pIG91+BlaGenepIG102), and promoter plasmids to build final construct with promoter and the bla gene together.

RD 5-222M, 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

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.

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.