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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 J23101 in front of each of the genes. Set up overnights to obtain necessary plasmids.

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--Ran a low melt gel on digested products, cut out respective bands. Set up ligation reaction according to protocol. Ligation with previously digested J23101 and NorB and NosZ digests each respectively. Also a ligation was set up with the NorC and NirS digests done yesterday.

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.

--JR--Did colony PCR. The NirS/NorC ligation appears to have worked, but discovered I have used the wrong NorB and NosZ for my plasmids which needed to be digested. Disappointing setback. Set up some new correct overnights.

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.

--JR--Did plasmid preps for NorB and NosZ

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.

--JR--Set up restriction digest again for NorB and NosZ with EcoRI and XbaI according to protocol.

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!

--JR--Ran lowmelt on digests. Set up ligation reaction.

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.

--JR--Set up transformation according to protocol for NorB and NosZ, with Cameron's help.

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.

--JR--Took out plates from incubator. Picked colonies for colony PCR

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.

--JR--Set up new overnights of NorB and NosZ.

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.

September 20, 2014

--JR--Did plasmid preps on NorB and NosZ. This time they had good concentrations. ~150ng/ul each.

Set up restriction digests again according to protocol. With EcoRI and XbaI.

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.

--JR--Set up ligation reaction for promoter J23101 and NorB and NosZ respectively, according to protocol.

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.

--JR--Set up colony PCR. Results didn't look great. No product. Went back to check plasmid concentration from NorB and NosZ--they weren't good, explaining why these transformations wouldn't have worked right. Set up some more plasmid preps.

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.

--JR--TransformedNorB and NosZ ligations.

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.

--JR--Set up colony PCR to verify transformants. This time it appears 2-3 of the NosZ transformations worked, and all the NorB colonies picked appear to have worked also! Hooray! Finally! Set up overnights on 2 of the NosZ working colonies and 2 of the NorB working colonies.

September 27, 2014

--JR--Did a plasmid prep on NorB and NosZ. Still getting relatively low plasmid concentrations. Approximately 50ng/ul. I even documented which buffers I used, using a different combination on duplicate plasmid preps, no apparent difference was made. Set up restriction digests using such plasmids. Digested the NorB+Promoter plasmid using EcoRI+XbaI, and the new NosZ+Promoter at EcoRI+SpeI.

Week of October 4th, 2014

September 29, 2014

--JR--Ran digests out on gel. The bands looked good (brightness), but the sizes did not appear to be what I expected. I expected the NorB digest to be approximately 3400bp as it would be the psB1C3 plasmid with the norB gene and promoter (minus the RFP gene), and then the NosZ digest we wanted the ~2000bp product as we just wanted the NosZ gene and promoter from the plasmid. However it appears that both products looked as though they were around 3500bp, with the NosZ products appearing larger than the NorB.

--CS-- Today I looked over my sequencing results a little bit more. Dr. Grose and I then did another mutagenesis reaction using her kit for nosZ since the sequencing results showed that the unwanted PstI site has yet to be mutagenized. I also did some planning to figure out what all I need to take care of this week in order to be able to submit all of the genes as BioBricks next week. It's getting close but hopefully we can make it all work out!

September 30, 2014

--JR--Did plasmid preps for NorB and NosZ, got decent, but not great plasmid concentrations ~50ng/ul. Set up restriction digests for these plasmids. This time set it up in a way that would digests NosZ so as to produce to differently sized bands. Digested NorB using EcoRI and SpeI so as to produce a ~1400bp fragment which will be cloned into the digested NosZ plasmid, which will be digested using EcoRI and XbaI producing a ~4000bp fragment. Things went well until I ran these out on gel. All the products looked to be the same size, but yet again the ladder looked weird. Not sure if there is a problem with the ladder or the LM gel.

--CS-- Today I did a DpnI digest of the mutagenesis PCR product from yesterday (~25 μl) by adding 1 μl DpnI and 3 μl 10X CutSmart buffer (total ~29 μl). After incubating at 37°C for 1 hour I transformed 2 and 4 μl of it into DH5α. After an hour and 20 minute recovery period I then plated this transformed bacteria onto LB+Chloramphenicol plates and incubated them overnight in the 37°C incubator.

October 1, 2014

--JR--Resetting up restriction digests. According to previous plan as stated in September 30.

--CS-- Today I did colony PCR for 16 of the nosZ colonies that were on the 2 μl plate (the 4 μl plate didn't have any colonies grow) and 8 colonies from nirS and norC plates from a little bit ago since my last attempt at preparing BioBricks for those 2 genes didn't work out. After PCR I ran an analytical gel of the PCR products. My images showed that only one of the norC reactions worked. Since time is so short though I am just going to have to go ahead and do plasmid preps and see if sequencing works for those since my PCR didn't work (I suspect I'm not doing something right with the colonies when I'm putting them in the water to boil for template but I won't have time to figure out what exactly is going on). But anyway, so when I streaked plates out when I went through and picked colonies for the PCR I also added some to 200 μl aliquots of LB+Cam so I was able to go back to these and add them to 5 ml LB+Cam for growing overnight cultures.

October 2, 2014

--CS-- Today I did plasmid preps using the kit. I noticed that my pellets were rather small and Nano-Drop results ended up being pretty low. My nirS and norC overnights probably didn't work too well since they were from old plates, but since I have streaked them again they should hopefully work better if I need to do them again after today. As for my nosZ results, I'm not sure why they were as low as they were. They were certainly better than the other samples, but still not ideal. I think they might have not been able to grow long enough since I did my plasmid preps early in the morning but I'm not sure. I submitted some samples from each of the 3 genes to Desi for sequencing to make sure that I'm 100% sure that I picked the right colonies for nirS and norC and to make sure that the mutagenesis worked.

October 3, 2014

--CS-- Today I reviewed the sequencing results. The results confirmed that nirS and norC were indeed correct, so I just need to grow those up again to get a higher concentration of my plasmid for submission. The results showed that nosZ mutagenesis did not work again. The image comparing the sequencing results with the desired sequence is below:

The third nucleotide in the highlighted 6-nucleotide sequence showed up as a G in all of the sequencing results, and the results were decent quality, so it appears that the mutagenesis did not work again.

Week of October 11th, 2014

October 6, 2014

--CS-- Today I ran the sequencing results from last week by Dr. Grose and she suggested that I try some more plasmid preps since the reaction is just a low-efficiency one and we're bound to find a mutated one sooner or later. I set up 5 new overnights with 5 ml LB+Cam for other nosZ colonies that I hadn't prepped previously and Desi kindly agreed to set up sequencing reactions tomorrow. I also set up some overnights for nirS and norC since they had such low yields last time.

--JR--Set up overnights to do new plasmid preps. We decided to get a new kit as plasmid prep numbers have been consistently low for myself and others.

October 7, 2014

--CS-- Today I did plasmid preps of the nirS, norC, and nosZ overnight cultures that I grew up yesterday. I used the new kit that Dr. Grose got us and it made quite a difference. I got much higher yields, especially on the nirS and norC preps. I submitted the nosZ preps to Desi for sequencing.

--JR--Did plasmid preps. Still got relatively low concentration numbers. Between 40-80ng/ul.

October 7, 2014

--CS-- Today I reviewed the sequencing results from this last batch of nosZ samples. The sequencing quality was high enough to be reliable, and none of the results showed that the desired mutation actually occurred. Here is an image of the results compared to the desired sequence with the six nucleotides of the PstI site highlighted:

All of the sequences still have a G where we were hoping to see an A. Dr. Grose said that we should just keep checking colonies to see if something worked on one of them under the assumption that the primers and this specific reaction just have an abnormally low efficiency. I double checked the primers again too to ensure that they were correct; they were, so something else must be going on wrong in the reaction.

I also submitted all of my BioBricks to Jordan to prep for mailing in and submitted the information into the BioBrick submission pages and our parts page.

October 7, 2014

--CS-- Since none of our sequencing results have shown that the mutagenic PCR worked yet, Dr. Grose told me to keep making plasmid preps and sequencing them until we find a mutant. So today I started overnights for a bunch of nosZ colonies from the plate that I grew up last time we did the mutagenic PCR. I also fixed some things on our wiki.

October 8, 2014

--JR--Set up a restriction digest according to protocol. With NorB being digested with EcorI and SpeI, and NosZ being digested with EcoRI and XbaI.

Week of October 18th, 2014

October 13th 2014

--BRK--Added to the wiki page under Attributions. Digested plasmids from the EREB(111) overnight using plasmid mini prep kit and placed 50ul solutions in freezer.