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BYU 2014 Notebook Edit September October

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2 September 2014

Alpha Amylase: Based off the sequencing results from the latest round of colony PCR of alpha amylase, it looks like colony 3 contained a mutated PstI restriction site. From the streak plate and overnight was started. (JB)

3 September 2014

Alpha Amylase: Performed a plasmid prep of the colony 3 mutant following the SpinSmart High-copy plasmid purification. I took the prep and measured the plasmid concentration by nano drop and it only read around 20 ng/uL. This is definitely too low to get any sequencing from. I started a new overnight from the colony 3 plate streak. (JB)

Aii: I obtained colonies on all of the plates that I used to transform the Aii and TolB into E. coli, which I hope is a good sign. My next step in the process is to do colony, which I finished today. I used the plasmid specific primers.(CZ)

Dispersin: Today I did the spinsmart plasmid purification protocol on the on the cells that were overnighted last Wednesday and Pelleted on Thursday. (JM)

4 September 2014

Alpha Amylase: Performed a plasmid prep of the colony 3 mutant again. This time I got a much larger pellet of cells from the overnight. The concentration was around 60 ng/uL however. Not sure why I have been getting such low plasmid concentrations lately. (JB)

5 September 2014

Aii: Today I am running my PCR products from Wednesday out on a standard agarose gel. I have a long series of strong bands at the correct size, so I will be purifying two of those colonies and using them for sequencing. (CZ)

Alpha Amylase: Today I started four overnights from the alpha amylase mutant colony 3 streak. Hopefully we will be able to obtain a sufficient concentration of the plasmid since the last few rounds of plasmid prep have yielded unusually low concentrations. (JB)

Dispersin: I took my purfied plasmid of pET15b and PIG97 and set up a restriction enzyme digest using the EcoR1 and Pst1 restriction sites.(JM)

6 September 2014

Alpha Amylase: Today I combined the four overnights down and pelleted them. (JB)

8 September 2014

Alpha Amylase: Today I performed a plasmid prep following the SpinSmart High-copy plasmid purification protocol and materials. Despite the four combined liquid cultures I was only able to get a concentration of 114 ng/uL. This should be sufficient enough though for sequencing. I prepped for sequencing of the mutant colony using the forward and reverse pSB1C3 primers and the new internal primer that was created a few weeks ago. We can also start assaying the amylase in pet15b to determine the pre-mutant efficiency of the protein and later assay the mutant amylase to be sure that the mutation does not decrease the efficacy of the protein. I tested the pH of the biofilm samples to see if we would need to add buffer to them so that the protein would not denature if the pH was hostile. Luckily, the pH range for all of the samples was in the 7.1-7.5 range so we will not need to add any buffer when we start sequencing. (JB)

Dispersin: After digesting I quickly took 5 microliters from each and ran them out on a gel. I was pleased to find bands of the appropriate size for the PIG97 plasmid with the cut out Dispersin B gene as well as a large band for the pET15b plasmid. So I used the full reaction mixtures and ran them out on a low melt gel. Unfortunately they didn't show up under a low power UV light so I needed to use the high power imager in order to remove the appropriate sections from the gel. I then set up a ligation reaction. (JM)

Aii: Today was mostly spent in growing up overnights of my bacteria from two colonies, Plate 2, numbers 1 and 2. These were the two brightest bands on the gel, and were of the correct size. I also worked on the fundraising for our tickets. (CZ)

9 September 2014

Aii: My colonies had grown up fairly well after 24 hours. I spun them down, and also did some work with Jared's Dispersin overnights and plates.

10 September

Alpha Amylase: Today a majority of the time was spent up in Salt Lake City at the University of Utah retrieving N. multiformis from Ananda Shankar, a graduate student at the university. He was very generous and gave us a culture that he had been growing for the past couple of months, frozen stock, and very specific instructions on how to make the media needed for the N. multiform is to grow.

Sequencing data also came back today for the amylase mutant plasmid prep. (JB)

Dispersin: Today I transformed DH5Alpha cells with 7 microliters of my ligation mixture. Given that the bands were difficult to see and cut out however, I also began 4 overnights (two for each) of the PIG97 and pET15 colonies in case the slices I cut from the gel do not have the DNA concentrations necessary to have a successful ligation and subsequent transformation. Lastly I used the DNA gel extraction kit on my gel slices so that if my transformation is unsuccessful I can check and see if I have the necessary DNA by running 5 microliters out on Friday. (JM)

Aii: Today I purified the 2 overnights that I did on Monday and spun down yesterday. I used the SpinSmart High-copy plasmid purification protocol, and then Nanodropped the results. It turns out that in both tubes there was a very low count of DNA (approximately 20 ng/uL). Nonetheless I submitted both tubes for sequencing.(CZ)

11 September

Alpha Amylase: Today I spent some more time reviewing the sequencing results. It has been a bit difficult going through and piecing everything together to see if there are or are not any additional mutations to the alpha amylase gene. The coverage was very good and very accurate, but there was a section of the internal primer sequencing that was a bit spotty. There were extra base pairs sometimes, a missing base pair others, or a base pair that could not be read. But the mutation is there! (JB)

12 September 2014

Alpha Amylase: Since there was the spotty section of sequencing where none of the primers picked up the section I prepped two more instances for sequencing, one using the amylase specific forward primer and the other using the internal primer again. (JB)

Dispersin: Unfortunately my ampicilin plate didn't have any colony growth which means that the DH5Alpha cells likely did not pick up the pET15b plasmid which has amp resistance. Which means my ligation probably didn't work. After running out 5 microliters of my extracted DNA from Wednesday I was unable to see any DNA at all, which confirmed my suspicions that my ligation was unsuccessful. It seems I didn't extract it properly from the low melt on Monday. So today I again ran the spinsmart plasmid purification procedure on the overnights. Monday morning I will digest them and in the afternoon run them out on lowmelt. Hopefully this time by doubling the colonies contributing the plasmid the concentration will be high enough to not run into the same difficulties purifying the vector and insert for ligation. (JM)

Aii: I redid my overnights for the same colonies. We believe that our LB+CAM has broken down, but I had colonies this time that will hopefully have a higher expression rate. (CZ)

15 September 2014

Alpha Amylase: Sequencing came back and all the rounds of sequencing combined show a complete amylase with signaling sequence for multiformis, a mutated, no longer functioning PstI site, and no additional mutations! This means we can start assaying amylase pre- and post-mutation of the PstI restriction site!

The rest of my time was spent preparing the growth media for multiformis following Ananda's protocol he shared with us. There were some issues with the pH however. It was supposed to be 7.2-7.4. It was reading around 7.8 and when I added the Na₂CO₃ it made the pH even more basic. Will need to discuss what to do about this. (JB)

Aii: I got my sequencing results back, but it doesn't look like anything turned up, which is worrisome. There a few explanations for that result. There could have been a problem with our LB+CAM broth. Our incubator has also not been working at the correct temperature. Lastly, the plasmid prep kid is old, and we think there may be problems with that as well. However, I grew up new overnights to try to fix the problem. (CZ)

Dispersin: I was sick today but I called in to have Cam digest my plasmids but when he tested them with a nano-dropper they had a minimal amount of DNA too low for any work to be done. (JM)

16 September 2014

Alpha Amylase: Today not much was done for amylase as I spent most of the time trying to fine-tune the pH of the multiformis media. I did so, as well as pellet down the old cells that Ananda gave us and transfer it to this new media. Also, as multiformis is a very sensitive bacteria and likes to flock together it is important not to re-suspend repeatedly or with a lot of force. I made this mistake, but luckily you could still see clusters of the bacteria, so hopefully it will not upset them too much. (JB)

Left: multiformis media

Right: Pelleted multiformis

17 September 2014

Alpha Amylase: We need to move alpha amylase into the conjugation plasmid pIG111 in order to be able to test it in N. multiformis. However, we only have around 48 uL of purified, mutated amylase plasmid and we need to have enough for the transformation into pIG111, for the iGem registry, and for our own personal stock. To address this I transformed 5 uL of the purified, mutated plasmid into DH5α. After, I will be able to have a greater stock of purified plasmid and will be able to meet all the requirements for amount of plasmid needed for all these assignments. (JB)

Aii: Today was mostly spent on fundraising things. I pulled my overnights out of the incubator yesterday, but when I did the plasmid prep, we didn't get a much higher cell count (it was 8 ng/uL before, and 40 ng/uL now). This time I'm going to make three tubes of bacteria and purify them on one filter to get a higher count. (CZ)

Dispersin: Due to the failure of my plasmid preps, I again set up overnight Colonies, because I cant do anything until those cultures grow I also helped Cam with his overnights.

18 September 2014

Alpha Amylase: The transformed colonies grew up very well last night. I started two overnights in 5 uL of LB + cam for a plasmid prep tomorrow and digest and ligation into pIG111.

Additionally, things are looking a little brighter for our N. multiformis we have growing. Yesterday it looked like the celled that had been pelleted and transferred to fresh media we clumping up and not looking like the small, white flocks we saw when we picked it up from Ananda. However, today they had separated out more and were forming more of those white flocks Ananda instructed us to be looking for. As well, the frozen culture I started yesterday is looking good. There were one or two small flocks that appeared to have formed since yesterday when I started this batch. (JB)

Aii and Dispersin: I did another plasmid prep for both my three test tubes of Aii, as well as Jared's pET15b and pIG 97. We are hoping that the plasmid prep kit is still working correctly. (CZ)

19 September 2014

Alpha Amylase: The plasmid prep turned out alright. We made new LB + cam, so maybe we are still getting low yields due to something in the kit. From the two overnights I was able to get a concentration of 132.7 ng/uL. This is good enough so I went ahead and used all of it for a restriction digest so I can ligate into the pIG111 conjugation plasmid for testing in multiformis. I followed the regular protocol for this, however, I substituted the 14 uL of ddH₂O in the insert protocol for 14 uL of alpha amylase. There was only ~30 uL of the pIG111 so I used the normal recipe for that procedure. The old plasmid prep of the amylase mutant will be used for personal stock and for sending to the iGem parts database. Also, there are several far more significant flocks in the freezer stock of multiformis and it is appearing more and more like the batch Ananda gave us that had been growing for a while. (JB)

Dispersin: I ran a nano-dropper test on the purified plasmid Cam prepped for my from my overnights on Thursday unfortunately he placed each overnight in a separate binding column. So my pET15b plasmid concentration is low in both tubes, only 18ng/ul and 14ng/ul. I will just have to fill a larger low melt well with as much as I can from both solutions and hope the end concentration I get from cutting out the band will be enough. I also ran a PCR reaction using the B1307 and B1308 primers which target 200 bp up and downstream of the insert site of the PIG97 plasmid using some of my purified PIG97 plasmid so that I will have the Dispersin B gene along with all the standard igem restriction sites to work with in the future should I need it. (JM)

20 September 2014

Alpha Amylase: Ran the the restricted pIG111 and mutated alpha amylase with signaling sequence on low melt gel. The alpha amylase came our really strong, but the pIG111 was far fainter. I combined the two for a ligation reaction and will leave it overnight. (JB)

21 September 2014

Alpha Amylase: Performed a transformation into DH5α with 3 uL of the ligation (pIG111 + mutated alpha amylase w signaling sequence) and plated it on a chloramphenicol LB plate. (JB)

22 September 2014

Alpha Amylase: I missed the fact that the pIG111 plasmid has ampicillin resistance, not resistance to chloramphenicol. I redid the transformation using the ligation piece and plated it on the correct type of plate.

I also looked at the multiformis we have growing (both what Ananda gave us that had been growing previously and from the frozen stock) under the microscope. It was far more difficult to find anything from the old batch (but that may have been due to just not taking sample from an area that had the flocks), but in the new batch you could see many cells that appeared to have somewhat of a kidney bean shape as Ananda described it, and they appeared to be flocculating together as we would expect to see from what we know of multiformis' growth patterns. Below are picture taken at x40 magnification. (JB)

Dispersin B: I ran a restriction digest on the 14ng/ul and 18ng/ul pET15b plasmid preps, as well as on the PIG97 plasmid prep. I used EcoR1 and the Pst1 restriction enzymes. After waiting 1.5 hours I filled a single low melt gel well with both the digested pET15b preps and another well with the digested pIG97. I was able to see bands of the appropriate size I(~1100bp for Dispersin B and ~3000+bp for pET15b) for both lanes under a low intensity UV light so I cut them out of the gel. (JM)

23 September 2014

Alpha Amylase: The transformations from last night did not work so we will have to skip the conjugation of amylase into multiformis. But we have plenty to do with the assay of amylase pre- and post-mutation so I will start that tomorrow. (JB)

24 September 2014

Alpha Amylase: Since no one else has been able to transform their pieces into pIG111, the conjugation plasmid, we needed to make more conjugation plasmid. What was left was a really low concentration (which means what had been used was very likely also low). I transformed the plasmid into DH5α and plated it on an ampicillin resistant plate and put the rest of the culture in 5 mL of LB + ampicillin to let it grow overnight. I still have my insert mut alpha amylase from the low melt gel cut, so once we have a good concentration of pIG111 plasmid and run it out on low melt gel, we will all be able to use that as vector and ligate our inserts into the conjugation plasmid.

I also checked the pHs of the media in which we have our old and new multiformis cultures growing up and they both appear to still be in the >7 range, so that is good (however the media in which the new stock was in was getting close to 7, so I will recheck the pH this weekend and then later next week to make sure it stays in the >7 pH range. But we have lots of cells growing it appears, so that is really good news! (JB)

Dispersin B: Today I ran a ligation reaction with my two gel slices and then transformed the ligation mixture into DH5alpha cells which were plated on plates with Ampicillin, the antibiotic for which resistance is conferred by the pET15b plasmid. (JM)

25 September 2014

Alpha Amylase: More issues with the pIG111 conjugation plasmid. I checked the temperature of the 35^oC incubator that the liquid culture was in and it was only reading 32^oC. I took it upstairs to Dr. Grose's other incubator and let it shake in there for the afternoon. Then I did the plasmid prep, followed all the instructions exactly, and nano-dropped the plasmid and it only showed a concentration of 24.9 ng/uL with a 260/280 reading of 1.92 and a 260/230 reading of 0.98 (indicating a contaminant that absorbs light at 230 nm wavelength). I did some sleuthing and have some theories. Yesterday we through out some old lysis buffer, so it shouldn't be that. But I talked to Julie and she mentioned that there were suspected issues with the resuspension buffer and so she had used another batch of it and had a good plasmid prep, but we have consistently had poor yields with the plasmid prep kit we have been using as of late. But then today she used the resuspension buffer and it gave good readings. The only things left I can think of are either the conjugation plasmid is a low copy plasmid or there is another ingredient that is having issues. Julie and I are pretty sure we used different wash buffers (we have two in the kit) and that is where the issue lies. I will talk to Dr. Grose and see if she has any further insight. (JB)

26 September 2014

Alpha Amylase: Dr. Grose said that sometimes if there is not enough ethanol in the wash buffer that the plasmid will not stick to the filter during the wash step. I used the kit upstairs and got a good concentration (96 ng/uL), but still low purity readings on the 260/230 reading. Dr. Grose said that might be due to not re-suspending all the cells or not lysing all the cells. I performed the restriction digest using the Xba and Eco restriction enzymes, ran it on low melt gel, and cut it out and aliquoted it out for those who will need it. The plasmid gave a really strong band under low powered UV, which is a good sign. (JB)

Dispersin B: I had Cameron pull out my plates yesterday, but I came in to check them. I have colony growth, which is exciting because it means that my restriction digest and ligation probably worked.(JM)

27 September 2014

Alpha Amylase: Today I performed the ligation of the alpha amylase mutant insert with the pIG111 vector. I will leave it overnight and perform the transformation into E. coli tomorrow. I also checked the pHs of the multiformis cultures and they are both in the basic range still, so that is a good sign, they still have plenty of NaCO₃ to continue their growth. (JB)

Dispersin B: I selected six colonies and labeled them A1-F1 and created a segment on a plate as well as an overnight for each colony. This should enable me to perform colony PCR on Wednesday as well as give me growth to work with in the future. Dispersin B should be ready to begin assaying by Friday.(JM)