Team:Brno Czech Republic/Notebook

Notebook

Week 1 (June 6 - June 12)

We can finally start working in the lab. Baru and Standa travelled to Prague to professor Krásný’s lab. He provided us with the Bacillus subtilis strain 168 and cultures of Escherichia coli Dh5α transformed with plasmids pDG1664, pDG3661, pDG1731 and pDG1663. Bacillus subtilis culture was transferred to a new LB plate without antibiotics.

Week 2 (June 13 - June 19)

We had lab and safety training in the lab. We prepared glycerol stocks of Bacillus subtilis and Escherichia coli strains. Buffers and media for future experiments were prepared.

Week 3 (June 20 - June 26)

All of our plasmids were isolated from E. coli overnight cultures. They were subsequently digested with restriction enzymes. We got high concentrations of 200 ng/μl. Size and linear state of the plasmids were confirmed by electrophoresis. Competent cells of Bacillus subtilis were prepared.

This week, Johann Gregor Mendel’s birthday was celebrated in Brno. Two members of our team collaborated with Bioskop educational center and showed entertaining experiments to children.

Week 4 (June 27 - August 2)

We verified the competency of our Bacillus subtilis cells. We transformed them with our isolated plasmids. However, preparation of competent cells of Bacillus subtilis was not successful. There were no cells on the vital control plates. New competent cells were prepared, with some adjustments and improvements in the protocol.

Apart from working on our project in the lab. We organised a (Non)scientific pub quiz in Brno. It was a great opportunity to entertain and inform people about iGEM and our project.

Week 5 (August 3 - August 9)

We transformed the new competent cells with empty plasmids and confirmed its success with the AmyE test. We also tried to transform new superchemocompetent E. coli cells we were given from our secondary PI Pavel Dvořák.

Week 6 (August 10 - August 16)

We were still optimizing the transformation of Bacillus subtilis. We verified the functionality of our competent cells, but our negative control was also growing in the presence of the antibiotic we used for selection. We repeated the transformation many times. In the end, a technician from professor Krásný’s lab helped us with troubleshooting. There was a problem with antibiotics we were selecting transformants on. No more selecting Bacillus on ampicillin!

Week 7 (August 17 - August 23)

Our synthetic genes and primers were delivered! We made stocks and started with restriction cloning. We were following these steps - restriction digestion, electrophoresis, gel isolation, ligation. We also tested our kit for Plasmid isolation from Promega which was also delivered this week. It functions without problems. We tested our primers and Promega mastermix for colony PCR. The reaction itself seems to work - negative and positive controls are working as they are supposed to. Our constructs isolated from E. coli transformants however seem to have the wrong size.

Week 8 (August 24 - August 30)

Restriction cloning! We tried to ligate our synthetic sequences with plasmids pDG1664 and pDG3661 again. We performed restriction, ligation, transformation and colony PCR. Many attempts were made with no positive results.

This week, the majority of our experimentalists had to self-isolate by in-home quarantine as we came in contact with a person positive for Covid-19. Only two of us could come to the lab. Rest of us worked hard on text and graphics for our wiki.

Week 9 (August 31 - September 6)

We amplified our synthetic sequences using Long PCR. Again, we were trying to get our insert into plasmids using restriction cloning.

For this week, another lecture about GMOs for highschool students was planned. We had to cancel it because of the evolving pandemic situation.

We experimentalists split up into two groups and from this point, we went to the lab in shifts. If someone gets Covid-19, only the people from one group will have to self-isolate and the second half can still carry on working.

Week 10 (September 7 - September 13)

We decided to try different methods to confirm the cloning and transformation into E. coli. We also decided to focus on the immobilisation module since it is the most crucial part of our project and, when produced in B. subtilis, its functionality can be confirmed easily. We got our first successful transformants - E. coli cells were transformed with plasmids carrying the genes of chicken lysozyme, Scaffoldin B and enzyme Mlr B. Their presence was confirmed with restriction digestion. Glycerol stock of E. coli containing these plasmids was made.

Week 11 (September 14 - September 20)

Plasmids containing our genes were sent for sequencing. We tried the TOPO cloning with our immobilisation module and it was successful. Finally, we have E. coli transformant with the TOPO vector containing the gene encoding the Immobilization module. Working with plasmid might be easier than with a linear strand of DNA. Subsequently, we amplified the sequence of the Immobilisation module using long PCR. And! We isolated plasmids from the final trial of restriction cloning and transformation from the previous week and it seems like they contain our insert. Restriction cleavage of the plasmids shows that we have our Immobilisation module also in pDG3661 - in 5 of 24 tested E. coli colonies. We transformed Bacillus subtilis with these 5 variants.

After a long time of mishaps, this was a really successful week!

Week 12 (September 21 - September 27)

To prove integration of the sequence containing the Immobilization module into the chromosome of Bacillus subtilis we carried out colony PCR. This method, however, showed no results at all, so we isolated the chromosomal DNA of B. subtilis transformants and carried out long PCR and successfully proved the integration! We prepared media and samples for the characterisation of protein expression and functionality. We carried out a full-day cultivation of our genetically modified Bacillus and collected samples for SDS-PAGE and Western blot.

Week 13 (September 28 - October 4)

We send chromosomal DNA for sequencing. However, it is challenging to isolate chromosomal DNA of sufficient purity for sequencing. We also carried out the Blue-white test. We carried out the first run of the immobilisation experiment.

Week 14 (October 5 - October 11)

Our final week in the lab. We repeated the full-day cultivation and collected samples for SDS-PAGE and Western blot. We carried out these experiments as well.

Schools and universities in the Czech Republic fully converted to online classes this week and we were not allowed to come to the lab anymore. We analysed our results and prepared the presentation video, poster and finalized texts for our wiki.