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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.

--JR--Plan for designing and constructing denitrification plasmid: Digest NorC at Xba and PstI so to insert it in front of NirS. Then, we need to get promoters in front of NorB and NosZ. The plan is to digest each of those plasmids at EcoRI and XbaI sites so as to insert the pomoter J23111 in front of each of the genes.

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.

--JR--Set up restriction digests for denitrification genes. according to RD Protocol NosZ and NorB were digested with EcoRI and XbaI, expected digest sizes are 4000 and 3400 base pairs respectively. NorC was digested with XbaI and PstI, expected digest size of 500bp. NirS was digested with SpeI and PstI, with expected digest size being approximately 3700bp.

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.

--JR--

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.

September 18, 2014

--CS-- Today I submitted norB samples 4-7 for sequencing. I added 2 μl PCR product to 1 μl reverse primer; for each sample I did one reaction with the gene reverse primer and one reaction with the vector reverse primer.

Week of September 27th

September 22, 2014

--CS-- Today I reviewed the sequencing results from the norB samples that I submitted last week. They did not have very confidant outputs, and when I aligned the sequences to the desired sequence it showed that the site was not actually mutated. These results were certainly not completely reliable due to low quality reads, but they suggest that mutagenesis did not work at the second site. The image of the sequencing alignment is shown below:

Neither the gene nor vector reverse primers worked. Skip suggested that instead of trying sequencing over and over again I could do a PstI digest of the purified plasmid and run it on a gel to determine how many cut sites there are in the whole plasmid based on the number of bands. To try this out I picked some colonies for nosZ (1, 5, 6, and 9) and norB (4, 5, 6, and 7) and put them in 5 ml LB to grow up overnight cultures in the 37°C shaker. I also realized that although we had successfully clones nirS and norC into the pSB1C3 we had not prepped those for submission as BioBricks, so I picked some colonies from the plates that had the proper clones of those genes and grew up overnights of those as well using 5 ml LB.

September 23, 2014

--CS-- Today I started by doing the plasmid preps of the overnight cultures I made for all 4 genes. I then checked the DNA concentrations and purity of these using the Nano-Drop. The nirS and norC concentrations were in the twenties, so I will have to redo those. All but 2 of the other plasmid preps resulted in concentrations in the hundreds and 260/280 readings near 2, and the 2 were above 50 ng/μl so they should be alright. I set up the norB and nosZ plasmids for PstI digests based on our restriction digest protocol. For the samples that had more than 100 ng/μl, I mixed 5 μl of the samples with 5 μl buffer, 38 μl ddH₂0, and 2 μl Taq polymerase. For the samples that had less than 100 ng/μl, I mixed 10 μl of the samples with 5 μl buffer, 33 μl ddH₂0, and 2 μl Taq polymerase. I then put these on the shaker in the 37° incubator overnight.

September 24, 2014

--CS-- Today I ran the analytical gels of the PstI digests. The image of my gel is below:

For some reason whenever I print or save the images of my gels, they never look as good as what I actually see on the screen. Unfortunately I had already tossed my gel, but I reviewed the results with Dr. Grose and she pointed out that since the band would be under 200 bp for either of the genes if the PstI sites were still in the genes I probably wouldn't be able to see the small bands anyway. So since this test did not really confirm anything Dr. Grose suggested that I just try the sequencing again to check the mutagenesis. I submitted all 4 samples from both genes to Desi with the vector reverse primers for sequencing.

September 26, 2014

--CS-- Today I reviewed the sequencing results from my norB and nosZ samples. It looks like my norB is now properly mutagenized! The nosZ sequence still has the PstI site in it though unfortunately. The sequencing results were pretty good (everything has some HQ data and some were even as high as 20%, a significant improvement to the results with 0% HQ that I've had recently), so I think that I will just do plasmid preps for sequencing from here on out and not even bother with trying the colony PCR product. The images of the alignments are shown below, norB on the left and nosZ on the right, with the PstI site highlighted:

I also tried playing around with the wiki formatting some to try making our pages look better but wasn't really successful with anything, so I just left it as it originally was.