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From 2014.igem.org

BYU 2014 Notebook Edit September October

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Week of September 6th

September 3, 2014

--CS-- Today I checked the sequencing results from all of last week's sequencing. The samples that I submitted for nirS (3-5) and norC (3-2 and 3-4) all turned out great! The results showed that the promoters were inserted into the plasmids upstream of the gene sequences, just as we wanted them. I also checked the norB mutagenesis results. It appears that they did not work properly, so we'll have to try those all over again. The PCR hasn't been working properly, so I think I should try starting over from there instead of transforming and everything all over again. Julie and I also talked and decided that Julie would take over with constructing the ginormous plasmid containing all four of the denitrification genes since she is pretty much done with her antibiotics stuff now.

Week of September 13th

September 8, 2014

--CS-- Today I reviewed where everything is at right now with the denitrification project. I transformed 2 μl and 4 μl of the DpnI-digested nosZ into DH5α, letting it incubate at 37°C for 1.5 hours, and then plated it onto LB+Cam plates, putting them in the old 37°C incubator overnight.

September 9, 2014

--CS-- The nosZ plates that I grew up overnight didn't have any colonies, so I will have to start the mutagenesis reaction all over since it hasn't worked for the past few times. Today I did a new colony PCR reaction for the norB mutagenesis reactions since the past few times haven't really worked.

September 10, 2014

--CS-- Today I ran an analytical gel of the norB PCR products. My gel turned out great this time around! Here is the image:

I will be able to sequence a few of these this time and hopefully will be able to tell if the mutagenesis actually worked or not since all of the other tests failed to have very reliable results.

I also went back to old plates and picked a colony for nosZ from before mutagenesis but after successful cloning to use for plasmid preps prior to redoing the mutagenesis reaction. I also whipped up a new P. aeruginosa PAO1 stock plate just in case we need that anymore the rest of the semester since the stock plates we have are getting old. I also contacted Brother Lee about getting some Durham tubes made up.

September 11, 2014

--CS-- The nosZ overnight appeared a little faint, so I let it grow up another day to make sure I have plenty of stuff to work with for plasmid preps.

September 12, 2014

--CS-- I didn't have a ton of time today so I pelleted down the nosZ overnights and put them in the freezer. I also prepared norB samples for sequencing by putting 2 μl of the PCR product from earlier in the week with 1 μl of both the forward and reverse primers in PCR tubes and submitting them to Desi.

September 13, 2014

--CS-- I reviewed the sequencing results for norB. The first site appeared to be fixed conclusively, as shown by the image below that has all 4 of the sequences matching the desired mutated sequence at the top. The second site though is still inconclusive; the sequencing results were very uncertain in the region of the mutagenesis site, as shown below.

So it appears that I should probably resubmit the samples to see if I can get better results for the second site. But so far things are looking positive!

Week of September 20th

September 15, 2014

--CS-- Today I first helped Julie out by transforming the ligations that she had made for norB and nosZ with the promoters. I then tried to round up some Durham tubes but was unsuccessful; I arranged, however, to have some made for us tomorrow. I completed the plasmid prep of the nosZ that I had grown up last week using the kit. I then took some of this and did a single-site mutagenesis PCR with Dr. Grose. I also submitted some of the purified nosZ plasmid for sequencing with the internal primers. I also submitted norB samples 4-7 with the vector reverse and gene reverse primers again since the last time they had failed to show anything in the sequencing results.

September 16, 2014

--CS-- Today I did the DpnI digest of the PCR product from the single-site mutagenesis that Dr. Grose and I did yesterday. I added 1 μl of DpnI to the PCR product, mixed briefly, and incubated at 37° for 1 hour. I then transformed 2 μl of this into DH5α, letting it incubate at 37°C for 1.5 hours, and then plated it onto LB+Cam plates, putting them in the old 37°C incubator overnight.

September 17, 2014

--CS-- I got a pretty good amount of colonies on my nosZ plate today for a mutagenesis so I went ahead and did the colony PCR of 16 of these samples, streaking out plates of the picked colonies as I went. When these were done I ran an analytical gel of the PCR products. The images are shown below:

These gels showed that my PCR did not work since all of the primers are all down at the bottom of the gel. I am pretty certain that I added everything to the PCR mix, so it seems that these colonies not only lack the mutagenic nosZ but also lack the pSB1C3 plasmid altogether since I used the vector forward and reverse primers (307/308). This seems strange since I grew them up on LB+Cam plates, so I will consult Desi or Dr. Grose about this. While waiting for everything I also did a massive cleanup of all of my samples in the fridge and freezer to discard anything that is not needed anymore. We also received the knockout E. coli for testing our denitrification genes in vivo so I streaked the different strains out on LB and put them in the 37°C incubator overnight.