Notebook

In this notebook, we summarize in quite some detail each week we spent in the lab working on our iGEM project. The notebook documents the chronological progress of experiments, but does not include their individual procedures and all results - these are described in detail in the Protocols, Experiments and Results sections.

Week 1 (June 28 – July 2)

The first week in our lab is finally here! In order to even start working in it, it was necessary for all of us to complete online safety training. Our new team members were introduced to the lab and its equipment. We set aside these days for general preparation of the lab for the coming weeks. From mixing LB media and agars, diluting antibiotics to the necessary working concentration, preparing plates for our bacteria, to testing transformation of E. Coli DH5-ALPHA, E. coli JM109, and Bacillus subtilis 168 competent cells and PCR. We became familiar with the workspace to make us feel at home while making sure our competent cells and primers were working as they should. After all, as they say, fortune favours the prepared!
Figure 1: Preparing the lab for work

Week 2 (July 5 – July 9)

However, the preparations are not over yet. Some of the chemicals we had from last year, some of them were brand new, so it was necessary to test them all properly so that in the future we could rely on the correctness of the experiments and their results. One of the things that needed to be tested was the functionality of the selection systems. This time we used a different type of plasmid to transform E. coli and B. subtilis than last week, so for Bacillus we had to use a different antibiotic plate. The next day we could only conclude that although the transformation of E. coli came out with a beautifully clean negative control plate, for Bacillus the negative control was overgrown... Where was the problem? There were many possibilities but one thing was certain. In the next few days we will have to repeat the experiment and find out the cause. We decided to make colony PCR from the transformants and try a new intercalation dye, Midori Green, which is non-toxic and so we will be able to manipulate it as well as take pictures of the gels directly in our lab.

Week 3 (July 12 – July 16)

We decided to divide this year's iGEM team into three groups, which will rotate in the lab every week. Each group also has a "leader" who has the most insight into what is going on in the lab and is responsible for passing on information to the next week's leader. So in the following lines, for the sake of clarity, we will talk about Míša's, Barča's, and Quency's groups. This week, Barča's group was on "duty" in the lab. They started testing the antibiotics which did not work last week and prepared a new batch. This time, after the B. subtilis transformation, the dish with the new antibiotics with negative control was exemplary, without a single colony. So probably the original antibiotics were at the wrong working concentration. Subsequently, the colony PCR conditions for Bacillus were optimized, and since we had already run out of our construct B in the commercial pUCIDT plasmid, E. coli JM109 was transformed with it. The plasmid was isolated from the transformants, restriction digested and put on a gel to confirm the insertion. Awesome! We have plasmid with our construct B in E. coli ! All that remains is to make glycerol stocks, to serve as supply for future experiments ☺ .

Week 4 (July 19 – July 23)

The Quency’s team took over the scepter in the lab and the week started again with the transformation of E. coli JM109, as we received constructs C and D in commercial plasmids. The transformation was successful and the results on the plates the next day were more than interesting. In addition to the traditionally white colonies, some of the plates were overgrown with pink colonies. These were due to the production of the mScarlet protein, which we have included in both construct C and D. What is interesting, however, is the fact that this protein is under the promoter for B. subtilis and also that the colonies were visibly pink only on the ampicillin plates. Again, it was necessary to establish overnight cultures from several colonies, isolate the plasmid from them, restriction-digest them, put them on a gel, and analyze the products. This restriction analysis confirmed the insertion of plasmids with both C and D constructs into E. coli JM109, and we were able to make additional glycerol stocks.
Figure 2: The mystery of pink colonies

Week 5 (July 26 – July 30)

With the new week, the services in the lab have rotated again, this time a group led by Míša took up residence. We started the week by isolating plasmids from last week's validated E. coli transformants. The following days we continued subcloning. We restriction-digested the C and D constructs in IDT's commercial plasmids, visualized them using gel electrophoresis, and purified them. We ligated the digested and purified constructs C and D with the destination vector overnight. The next morning, we enthusiastically set about transforming cloning E. coli with the aforementioned ligation mixture. We were thrilled with the result. We grew potential transformants on the selection plates. Those that should contain the C construct, but also those that should have the D construct. Are we getting excited prematurely?

Week 6 (August 2 – August 6)

The enthusiasm and joy of the successful transformation late last week was only half broken by Bára's announcement that construct C had been transformed back into E. coli in a commercial vector from IDT. The good news, however, is that construct D is already present in the transformants in our destination vector. Therefore, we did not have to wait for anything and immediately started transforming B. subtilis. Then, we saw something has grown on the selection plates again, which might have suggested that we should not get our hopes up prematurely. Leaving nothing to chance, we are going in hard to test our potential success. We have set up the AmyE assay and mixed up the mix for the LongPCR reaction. Both experiments were successful and we can finally celebrate. After a month of hard work, we managed to integrate our first construct into the B. subtilis chromosome!
Figure 3: We were very pleased with the results of the AmyE test

Week 7 ( August 9 – August 13)

Quency is in charge this week. After the most successful week we have had so far in this year's iGEM competition, on Monday we started again from the beginning. The goal now was to continue subcloning the B and C constructs that are in pUCIDT plasmids. Hence, we set about doing a restriction digestion of these constructs and inserting them into our destination vectors. Unfortunately, restriction digestion of these constructs did not listen to us this week. The gels from the electrophoresis were uneasy to read and we had a hard time guessing whether the constructs were correctly digested or not. Simply put, restriction digestion was playing a provocative game with us. It was only on Friday that we were able to transform E. coli with a ligated destination vector and the C construct. On Saturday, Quency, full of anticipation, went on campus to evaluate the transformation. The colonies are on the plates, but will the correct plasmid with construct C be in the transformants?

Week 8 (August 16 – August 20)

A new week started and with it another group came to work again. Now under the leadership of Míša. On Monday morning, construct B in the pUCIDT plasmid was restriction digested and at the same time the presence of construct C in plasmid 3661 in E. coli was tested by cPCR. Unfortunately, both experiments came out negative. The next day we repeated the cPCR, again unsuccessfully = construct C probably failed to ligate into pDG3661 and transform E. coli. Nothing left to do but restriction-digest again, verify products on electrophoretic gels, isolate them from gel, then ligate and transform. On Wednesday, we isolated the plasmids needed for restriction cloning from overnight cultures of E. coli. This time we were let down by the equipment, the thermostat set at 37°C only warmed up to 28°C overnight for some unknown reason. Therefore, the concentrations of the isolated plasmids were miserably low. In addition, on this day we transformed competent E. coli JM109 with hopefully successfully ligated products from yesterday. The following morning we evaluated the transformation. One of the ligated products (pDG3661 + construct C) yielded many transformants on the plates. However, we rejoiced prematurely. After performing cPCR of more than 30 randomly selected colonies and gel electrophoresis, we are sad to say that our ligated product is not present in any colony. This week, perhaps, everything that was planned went wrong. We were exhausted and unmotivated. We hoped that Barča's group will do much better next week!
Figure 4: Even if you work hard and accurately, experiments can just fail.

Week 9 (August 23 – August 27)

On Monday, Barča's group started lightly. The functionality of the new polymerases was tested and overnight cultures were established. On Tuesday, it was possible to confirm with 100% certaintibility that the putative ligated products from last week are actually still our initial pUCIDT+C. Restriction cloning of this construct from the pUCIDT vector is probably not the way to go, so primers for PCR cloning will be designed. In addition, restriction digestion of plasmid 1664 and plasmid pUCIDT with the B construct was successful and will be ligated and subsequently used to transform E. coli. The following day, work was done on transforming cloning E. coli with the ligation mixture from the previous day. Work was also done to test the functionality of the AmyE assay. On Thursday, the transformation was verified by cPCR. However, this did not turn out the best, apparently we inadvertently used the wrong primers. Therefore, on Friday we decided to verify the transformants one more time, but now by using restriction analysis. It turned out that the transformation was successful and construct B was located in the destination vector pDG1664. Finally, we can transform B. subtilis!

Week 10 (August 30 – September 3)

Quency's group had a big thing coming up on Monday. After a long time, we again set about transforming B. subtilis, but this time with plasmid pDG1664 containing construct B. In addition, the functionality of the polymerases for LongPCR was verified. The latter is important to amplify the transition between the B. subtilis chromosome and our integrated constructs, which could be sent for sequencing. In the morning, we spotted B. subtilis colonies on agar plates. At that moment, we were all a little breathless, hoping that the transformation had been successful and we would be able to excitedly announce to our PIs that we had managed to integrate a second construct into the chromosome. However, we were unable to verify it by PCR. We were trying to optimize the conditions of the PCR reaction, trying to use different primers, even annealing temperatures. It was only on Friday that we were able to successfully verify the presence of the B construct in the B. subtilis chromosome for the first time.
Figure 5: Proof of successful integration of B construct into B. subtilis´; chromosome

Week 11 (September 6 – September 10)

Míša's group decided to leave nothing to coincidence and try again to verify the transformation of B. subtilis by construct B. After all, those verification PCR reactions last week did not quite come out the way they were supposed to. So we tried to repeat the success of the Quency group using their optimization tweaks. Unfortunately, our first verification did not turn out positive. After visualization by electrophoresis, we saw only narrow faint bands on the gel. The next day we repeated the PCR reaction again, again unsuccessfully. We are beginning to worry if the positive result from last week was significant. It was not until the third day and the third attempt that we were also able to successfully verify that the transformation was successful. We sent the purified PCR amplicons for sequencing on Thursday. On the same day, we visited the Loschmidt lab to measure the spectra absorbed by B. subtilis samples with construct B and D after IPTG induction. On the last day of this week, we finally started working on PCR cloning of construct C. We were able to successfully amplify and purify it. We also prepared glycerol stocks of B. subtilis with construct B and stored them in a -80°C freezer.

Week 12 (September 13 – September 17)

This week has started off challenging. On Monday the semester at the university started and so in the next few weeks we will not be able to be in the lab with the full line-up. The older members of the team already have to work hard on their diploma theses and the younger ones have lectures almost every day. Nevertheless, we have decided that we still want to gather the last remnants of strength and try to get the C construct into the shuttle vector pDG3661 and then transform B. subtilis with it. So on Monday, the amplicon of product C and pDG3661 were restriction-digested, the products on gel were purified and ligated overnight. The next morning, the ligation mixture was used to transform competent E. coli JM109 cells. This was the last chance. Either it works or it does not, and we will have to resign ourselves to the fact that the C construct will forever remain only in the pUCIDT plasmid. On Wednesday morning, we arrived at the lab with anticipation, took the plates out of the incubator... and there were the transformants! But we knew we had not won yet. We have had several instances where construct C has, for reasons we do not understand, ligated again with the pUCIDT plasmid, which carries the same antibiotic resistance as our shuttle vectors. So we rushed to verify the colonies by colony PCR. This time, for each colony, we verified not only the presence of construct C in pDG3661, but also the presence of the pUCIDT plasmid. In the first batch of colonies that we tested, all had a positive product signal corresponding to construct C in the pUCIDT plasmid. All but one - the colony with the assigned number 1. The faint band corresponding to the product size of construct C in pDG3661 gave us hope. And so the next day we tested another batch, including colony #1, for confirmation. And there it was! Even in the other two colonies. And so, after two months of trying, our dream became a reality. We have construct C in the shuttle vector pDG3661! The week was capped off by transforming the E. coli BL21(DE3) expression strain with constructs B and D in pUCIDT plasmids.
Figure 6: And so, after two months of trying, our dream became a reality. We have construct C in the shuttle vector pDG3661!

Week 13 (September 20 – September 24)

After isolating plasmid pDG3661 with construct C from an ON culture of E. coli, we finally attempted to transform our B. subtilis. However, due to the low concentration of plasmid DNA and a minor transformation error, we were unsuccessful the first time. So we established another ON culture of E. coli and the next day we tried to isolate the plasmids again. This time we had beautiful concentration and purity. The transformation process went smoothly, the plates were put in the incubator and were looked at the next day. At the same time, we were able to successfully verify the transformed E. coli BL21(DE3) with constructs B and D in the pUCIDT plasmids using colony PCR. And not to forget, we also received a shipment from Dr. Krásný from Prague with various strains of B. subtilis and E. coli that we will use as controls in the following experiments. So at the end of the week, we made glycerol stocks of these strains and the AmyE test with the B. subtilis transformants was set up. And sure enough, it turned out great!

Week 14 (September 27 – October 1)

The AmyE test came out above all our expectations, suggesting that B. subtilis had a broken AmyE site in its chromosome and our C construct integrated into it. However, verification is not yet complete. On Sunday evening, ON cultures were established from B. subtilis transformants and on Monday we set about isolating the chromosome. Subsequently, long PCR was performed. One of the primers was complementary with the interface of the chromosome and part of the plasmid pDG3661, which was integrated. The other one was complementary on some part of the construct. The PCR came out positive and the products of the expected sizes shone on the gel! We then additionally purified the PCR mixtures and sent the samples for sequencing. The latter will have the last word. Thursday was scheduled to be undoubtedly the most logistically complex experiment of the season. Everyone who could was involved in thinking up and executing it. And every hand was more than needed. On this day we were scheduled to measure at Tecan in Loschmidt's labs, and in the afternoon we had arranged to go finally see our beloved bacteria under the fluorescence microscope at CEITEC. Starting next week there will be lectures in our iGEM lab so we will have limited access there.
Figure 7: Bacterial culture of E. coli producing mScarlet protein

Week 15 (October 4 – October 8)

This week was reserved for writing the wiki. The wiki freeze is inexorably approaching, so it is time to write down everything we have been working on for the last few months. There is currently blood and coffee flowing through our veins :).