iGEM 2020 — Team Brno_Czech_Republic

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# Team:Brno Czech Republic/Notebook

[![](https://static.igem.org/mediawiki/2020/a/ad/T--Brno_Czech_Republic--Logo_CYANOTRAP.svg)](https://2020.igem.org/Team%3ABrno_Czech_Republic)

* [TEAM](https://2020.igem.org/Team%3ABrno_Czech_Republic/Team)

  [Attributions](https://2020.igem.org/Team%3ABrno_Czech_Republic/Attributions)
  [Our NPO](https://2020.igem.org/Team%3ABrno_Czech_Republic/Excellence)
* PROJECT DESIGN

  [Project description](https://2020.igem.org/Team%3ABrno_Czech_Republic/Description)
  [Theory](https://2020.igem.org/Team%3ABrno_Czech_Republic/Theory)
  [Specific Project Design](https://2020.igem.org/Team%3ABrno_Czech_Republic/Design)
  [Our device](https://2020.igem.org/Team%3ABrno_Czech_Republic/Device)
  [Modeling](https://2020.igem.org/Team%3ABrno_Czech_Republic/Model)
  [Parts](https://2020.igem.org/Team%3ABrno_Czech_Republic/Parts)
* WET LAB

  [Safety](https://2020.igem.org/Team%3ABrno_Czech_Republic/Safety)
  [Experiment](https://2020.igem.org/Team%3ABrno_Czech_Republic/Experiments)
  [Engineering success](https://2020.igem.org/Team%3ABrno_Czech_Republic/Engineering)
  [Conclusion & Results](https://2020.igem.org/Team%3ABrno_Czech_Republic/Results)
  [Notebook](https://2020.igem.org/Team%3ABrno_Czech_Republic/Notebook)
* FUTURE

  [Proposed implementation](https://2020.igem.org/Team%3ABrno_Czech_Republic/Implementation)
  [Plans for the future](https://2020.igem.org/Team%3ABrno_Czech_Republic/Future)
* OUTREACH

  [Collaboration](https://2020.igem.org/Team%3ABrno_Czech_Republic/Collaborations)
  [Human Practices](https://2020.igem.org/Team%3ABrno_Czech_Republic/Human_Practices)
  [Education](https://2020.igem.org/Team%3ABrno_Czech_Republic/Education)
  [Contribution to iGEM](https://2020.igem.org/Team%3ABrno_Czech_Republic/Contribution)
* Documentation

  [Protocols](https://2020.igem.org/Team%3ABrno_Czech_Republic/Protocols)
  [Medals and awards](https://2020.igem.org/Team%3ABrno_Czech_Republic/Medals_and_Awards)

### 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.

![](https://static.igem.org/mediawiki/2020/8/8a/T--Brno_Czech_Republic--ContributionsIgem-i2.svg)

[![](https://static.igem.org/mediawiki/2020/8/8b/HQ_page_logo.jpg)](https://2020.igem.org)

# Team:Brno Czech Republic/Engineering

[![](https://static.igem.org/mediawiki/2020/a/ad/T--Brno_Czech_Republic--Logo_CYANOTRAP.svg)](https://2020.igem.org/Team%3ABrno_Czech_Republic)

* [TEAM](https://2020.igem.org/Team%3ABrno_Czech_Republic/Team)

  [Attributions](https://2020.igem.org/Team%3ABrno_Czech_Republic/Attributions)
  [Our NPO](https://2020.igem.org/Team%3ABrno_Czech_Republic/Excellence)
* PROJECT DESIGN

  [Project description](https://2020.igem.org/Team%3ABrno_Czech_Republic/Description)
  [Theory](https://2020.igem.org/Team%3ABrno_Czech_Republic/Theory)
  [Specific Project Design](https://2020.igem.org/Team%3ABrno_Czech_Republic/Design)
  [Our device](https://2020.igem.org/Team%3ABrno_Czech_Republic/Device)
  [Modeling](https://2020.igem.org/Team%3ABrno_Czech_Republic/Model)
  [Parts](https://2020.igem.org/Team%3ABrno_Czech_Republic/Parts)
* WET LAB

  [Safety](https://2020.igem.org/Team%3ABrno_Czech_Republic/Safety)
  [Experiment](https://2020.igem.org/Team%3ABrno_Czech_Republic/Experiments)
  [Engineering success](https://2020.igem.org/Team%3ABrno_Czech_Republic/Engineering)
  [Conclusion & Results](https://2020.igem.org/Team%3ABrno_Czech_Republic/Results)
  [Notebook](https://2020.igem.org/Team%3ABrno_Czech_Republic/Notebook)
* FUTURE

  [Proposed implementation](https://2020.igem.org/Team%3ABrno_Czech_Republic/Implementation)
  [Plans for the future](https://2020.igem.org/Team%3ABrno_Czech_Republic/Future)
* OUTREACH

  [Collaboration](https://2020.igem.org/Team%3ABrno_Czech_Republic/Collaborations)
  [Human Practices](https://2020.igem.org/Team%3ABrno_Czech_Republic/Human_Practices)
  [Education](https://2020.igem.org/Team%3ABrno_Czech_Republic/Education)
  [Contribution to iGEM](https://2020.igem.org/Team%3ABrno_Czech_Republic/Contribution)
* Documentation

  [Protocols](https://2020.igem.org/Team%3ABrno_Czech_Republic/Protocols)
  [Medals and awards](https://2020.igem.org/Team%3ABrno_Czech_Republic/Medals_and_Awards)

### Engineering success

**Using the tools of synthetic biology, we assembled and proved the transfer of one of our synthetic constructs - firstly into the cloning strain of *Escherichia coli* and then into the final host - *Bacillus subtilis*. This way we accomplished one of the aspects of our CYANOTRAP project and demonstrated our Engineering success.**

*Notice: This page is only a summary of our engineering success and the path to it. All the detailed information about the design, parts, experiments and results can be found at wiki pages [PROJECT DESCRIPTION](https://2020.igem.org/Team%3ABrno_Czech_Republic/Description), [THEORY](https://2020.igem.org/Team%3ABrno_Czech_Republic/Theory), [SPECIFIC PROJECT DESIGN](https://2020.igem.org/Team%3ABrno_Czech_Republic/Design), [DESIGNING OUR DEVICE](https://2020.igem.org/Team%3ABrno_Czech_Republic/Device), [MODELING](https://2020.igem.org/Team%3ABrno_Czech_Republic/Model), [PARTS](https://2020.igem.org/Team%3ABrno_Czech_Republic/Parts), [EXPERIMENT](https://2020.igem.org/Team%3ABrno_Czech_Republic/Experiments), [RESULTS](https://2020.igem.org/Team%3ABrno_Czech_Republic/Results) and [PROTOCOLS](https://2020.igem.org/Team%3ABrno_Czech_Republic/Protocols).*

### Research

We began our mission for the better world via synthetic biology in December. Our first task was choosing the topic of our iGEM project. So we started to look for issues affecting our neighborhood, city and the whole Czech republic. We studied and discussed many environmental issues of our country, such as unsustainable agriculture or deforestation. The final inspiration came when looking at the dam located near the city of Brno. This huge body of water has one problem – it is often affected by cyanobacterial overpopulation. The more we thought about it, the more we realised just how many places are facing the overabundance of these organisms. It is not just a challenge for Brno, but that is a huge problem worldwide. So we started to speculate how it is possible to solve this issue with tools of synthetic biology.

### Imagine

We asked ourselves the first fundamental question: How to stop cyanobacteria from growing? The answer was easy. Destroy them. But how? How about engineering a bacterial strain which would help us tackle this issue! So another round of research began and we started to imagine the solution. We searched for enzymes, which are able to lyse cyanobacteria and degrade their toxins. We found lysozyme and Mlr enzymes of the microcystin degradation pathway. We thought about ways to immobilize these proteins together and increase their effectiveness. We also needed a way to keep our genetically modified bacteria from escaping into nature. We found cellulosomes. We slowly came up with an idea of a device, floating on the water surface, containing engineered organisms producing these proteins. And the idea of CYANOTRAP was born. The general public and experts liked the idea and gave us essential feedback. So we continued with our design.

### Design

During the Design part, we needed to transform our ideas from theoretical concepts to a feasible project. Firstly we chose a chassis which will be suitable for our purposes. We decided for *Bacillus subtilis*. This gram-positive soil bacterium is relatively hefty and we thought that the overall robustness is a very important feature of our modified organism. Also *B. subtilis* is known for its high protein secretion, which is great as the concept of CYANOTRAP is based on extracellular display of proteins. Another argument for choosing this bacterium was that it has been engineered to carry cellulosomes, which we wanted to use in our project [1].

We thought through every part of our device. If you want to know more about their design, sequences and why we chose them, **we highly recommend** visiting the corresponding wiki pages. We spend a lot of time with their draft and we can confidently say that these parts are also ready for the wet lab. But due to epidemiological issues we did not have time to work with every construct. So we focused on just one aspect of the project.

One of many questions which we were forced to solve was, how to stop our engineered bacteria from escaping from the device. We decided to combine the Cellulose Binding Module (CBM), and the LysM domain in order to immobilize our bacteria. In nature CBM is found as a component of cellulosomes. The CBM is able to bind cellulose and thus brings the cellulolytic enzymes of the collulosome closer to their substrate [2]. The other component - LysM - is a quite common small globular module and it is involved in binding peptidoglycans or chitin. It provides a strong ionic connection to the cell wall as was demonstrated by You and colleagues in 2012 [1].

In CYANOTRAP, we wanted to connect these two proteins via a linker and create an anchor which would be able to immobilize our modified *B. subtilis* on a cellulose matrix (**Fig 1**). CBM has already been successfully used for this purpose in *E. coli* by Wang *et al.* in 2001 [3]. In our case, the cellulose matrix would be cellulose microbeads. The device would be filled with them and as the beads would provide large surface area for immobilization and, as they would be too big to pass through the filters in our device, they would stay inside of CYANOTRAP. If you want to know more about CBM or LysM, we recommend visiting the corresponding wiki pages.

![](https://static.igem.org/mediawiki/2020/c/c8/T--Brno_Czech_Republic--Engineering_Bacouch.svg)

**Figure 1: Scheme of part of the CYANOTRAP project, which we tried to achieve this year.** The *Bacillus subtilis* cells are connected to the cellulose microbead through the Immobilization module, which comprises the LysM, linker, and cellulose binding domain (CBM).

We named this protein, containing CBM connected to LysM, the Immobilization Module, or IM in short. We designed the sequence for the expression of IM. The simplified draft could be seen in Figure 2.

![](https://static.igem.org/mediawiki/2020/8/85/T--Brno_Czech_Republic--Engineering_sekvence.svg)

**Figure 2: The scheme of the Immobilization Module sequence.** The sequence is 1405 bp long. At the 5' end of the IM there is the Pveg promoter (red). The Pveg promoter is the strongest currently known constitutive promoter of *B. subtilis*. The promoter is followed by the RBS R2 (light orange), which is strong, but not too much. We did not want to burden the cell with our modifications. The coding sequence (CDS) encodes several functional modules - SacB (yellow) signal sequence for extracellular transport, Myc-tag (light green) for Western blot detection, CBM (dark green), linker (light blue) and three copies of the LysM domains (dark blue). At the 3' end of the CDS, there is a STOP codon. We also added restriction sites EcoRI (pink) and HindIII (purple) at the ends and NheI (dark orange) between the promoter and RBS sequences for following restrictions and possible modifications. Sources of all sequences can be found at wiki page.

As it is almost a rule in genetic engineering, the next step was to choose appropriate vectors, which would be able to transfer the IM sequence into the *B. subtilis*. In our project we worked with vectors, which were recommended to us by Dr. Krásný from the Laboratory of Microbial Genetics and Gene Expression in Prague. Our plasmids are synthetic vectors designed especially for ectopic integration of genes into the chromosome of *B. subtilis*. We used the plasmid pDG3661 [4] for the cloning and integration of the IM (**Fig. 3**). It is a “shuttle vector” which means that it can be used in both *E. coli* and *B. subtilis*. The origin of replication is recognized only in *E. Coli* - *B. subtilis* is not able to replicate the vectors themselves. In *B. subtilis* the circular molecule is integrated into the chromosome immediately after transformation. The integration takes place through a double recombination event.

![](https://static.igem.org/mediawiki/2020/e/ec/T--Brno_Czech_Republic--Engineering_pdg3661.svg)

**Figure 3: The scheme of the plasmid pDG3661. The vector is 10407 bp long.** Sequence *ori* (red) on the picture indicates the origin of replication in *E. Coli*. Gene *bla* (pink) encodes antibiotic resistance for ampicillin. Parts of the *amyE* gene (purple and light orange) secures the integration into the *amyE* gene in the chromosome of *B. subtilis*. Gene *spoVG-lacZ* (dark blue) is the fusion of genes *spoVG* and *lacZ* and expresses *β*-galactosidase. This could be used for the Blue-White selection. Restriction sites HindIII (light blue) and EcoRI (green) were used for the cloning of IM into the vector. Cat gene (yellow) and *spc* gene (dark orange) encodes resistance to antibiotic chloramphenicol.

The last missing piece was to decide how to insert the IM into the pDG3661. We choose restriction cloning, as Dr. Krásný recommended to us. The gist of this method is quite simple. The sequence of IM, which was commercially synthesized for us, was specifically designed to have restriction sites for EcoRI and HindIII at its 3' and 5' ends. These restriction sites are also present in the vector pDG3661 between the *amyE* sequence. In theory, if the IM sequence and the vector are digested with EcoRI and HindIII and then ligated together, the IM sequence should join the vector in the required direction. Our synthetic construct - the pDG3661 plasmid with our insert - was named pDG3661\_IM (**Fig. 4**).

![](https://static.igem.org/mediawiki/2020/6/64/T--Brno_Czech_Republic--Engineering_pdg3661IM.svg)

**Figure 4: The scheme of the synthetic construct pDG3661\_IM.** The construct is 11789 bp long. It contains genes and parts described in the pictures above. The IM sequence in the plasmid is marked by light green color.

### Build

#### Preparation of genetic modules in *Escherichia coli* cloning host

We can divide our path to the successful integration of the IM into the chromosome of *B. subtilis* into several steps. Firstly we needed to prepare our synthetic construct pDG3661\_IM and use it to transform a cloning host. We chose *E. coli* chemocompetent cells DH5α from NEB® for this purpose.

The restriction cloning was conducted as follows. Empty pDG3661 vectors were cleaved by already mentioned restriction enzymes - EcoRI and HindIII. During the restriction digestion, the Antarctic phosphatase was added to prevent the plasmid from re-ligating. Afterwards, we separated the samples from restriction enzymes and other impurities. Our fragment was amplified with PCR and subsequently cleaved with the same restriction enzymes under the same conditions (without Antarctic phosphatase). The restriction mix was also purified. Digested nucleotides were then mixed together with T4 ligase.

In theory, the ligation mixture should now contain vector pDG3661\_IM. and we used this mix to transform the chemocompetent *E. coli* cells. The cells were incubated and plated on agar plates with corresponding antibiotics. The colonies of transformants were tested for the presence of pDG3661\_IM via restriction analysis and sequencing (**Fig. 5**). The results confirmed our first experimental success, we assembled synthetic construct pDG3661\_IM. The complete results can be seen on wiki page [Results](https://2020.igem.org/Team%3ABrno_Czech_Republic/Results).

![](https://static.igem.org/mediawiki/2020/7/71/T--Brno_Czech_Republic--Engineering_sekvenace.png)

**Figure 5: Scheme of sequencing results.** The two sequencing results cover the whole synthetic sequence of the Immobilization module.

#### Integration of the expression cassette with IM into the chromosome of *Bacillus subtilis*

In the next phase, we focused on the transformation of *B. subtilis* with pDG3661\_IM. Working with this bacterium was complicated as no one at our university had any previous experience with it. It was not only new to us but also to our PIs. Despite this, we've managed to perform our experiments successfully. In order to share our experience with future iGEM teams, we've decided to write a handbook: **How to handle *Bacillus subtilis*.** You can find it in the [Contributions](https://2020.igem.org/Team%3ABrno_Czech_Republic/Contribution) section.

For our purposes, we used our own naturally competent cells prepared from *B. subtilis* strain 168. The protocol for preparing *B. subtilis* competent cells can be found in our Experiment section. As described above, the vector pDG3661 is designed to integrate into the chromosome of *B. subtilis*. After a routine transformation, the transformants were selected using chloramphenicol. Now we were ready to finally verify the success of the integration of the expression cassette into a specific region of *B. subtilis* chromosome - our second experimental achievement. The whole path to this point is again summarized in *Fig. 6*.

![](https://static.igem.org/mediawiki/2020/f/fa/T--Brno_Czech_Republic--Engineering_Transformace.svg)

**Figure 6: Scheme of our experimental workflow.** Orange color represents the amyE sequence. Red color represents the IM module. The experiment was conducted as follows. Firstly, the synthetic IM sequence as well as the integrative shuttle vector pDG3661 were digested by two restriction enzymes. Secondly, digested nucleotides were ligated together. Thirdly, this construct was used to transform competent *E. Coli* cells. Then, from the obtained transformants, large amounts of plasmid PDG3661 \_IM were isolated. And finally, *B. subtilis* 168 was transformed with the plasmid. Expression cassette with IM module subsequently integrated from the plasmid into a specific location of the chromosome of *B. subtilis*.

### Test

We demonstrated the presence of pDG3661\_IM in the chromosome with two methods. Firstly we performed agarose electrophoresis of PCR products, where isolated chromosomal DNA was used as a template. The primers we used were complementary to the chromosomal sequence upstream and downstream of the integration site. The PCR amplicons should thus contain a part of the *B. subtilis* chromosome, the expression cassette with the IM sequence and also *lacZ* gene and a part of the pDG3661 vector. The size of the amplicons should be 8074 bp as you can see in Table 1.

**Table 1. A comparison of length of each band, which should be seen on resulting gel.**

| Part of completed vector | Count of base pair |
| --- | --- |
| Immobilisation module | 1405 |
| *lacZ* gene | 3060 |
| Expression cassette | 7708 |
| Amplified regions of chromosome on either side of the expression cassette | 158 + 208 |
| Total length of the amplicon | 8074 |
| Negative control → Part of the chromosome without insert | 2346 |

Secondly we checked the expression of *α*-amylase. The integration of our expression cassette disrupts the gene encoding this enzyme. This is called the AmyE test. Agar with two layers is used for this method - the first layer is regular agar containing corresponding antibiotic and the second layer is thin agar with a starch supplement. After the addition of a few crystals of iodine, the starch will turn dark blue. The *α*-amylase is able to degrade starch. If the integration site is intact, the *α*-amylase is produced and a clear halo surrounding the colonies can be observed after iodine treatment. If the integration into the chromosome is successful, the enzyme is not produced, starch is not degraded and agar after iodine treatment is blue.

We also performed other tests, but due to a lack of time, caused by the worsening epidemiological situation in September and October, we were not able to optimize these methods. We have tried to confirm the presence of the insert in the chromosome via sequencing and also to verify the expression and secretion of the IM protein. We also tested if its production enhances the ability of the cells to bind cellulose.

To achieve this, we had to cultivate the cells in a medium that is favorable for extracellular protein production. During and after the cultivation we collected samples for SDS-PAGE and Western blot analysis to find out whether the target protein is expressed by the transformants. After the cultivation, we performed a set of experiments to observe the effect of IM expression on the cell's affinity for cellulose when compared to the original strain (control) without chromosomal insertion. However, the results so far are inconclusive, as this experiment still needs to be optimized. Description of all of the performed tests could be found on wiki pages [Experiment](https://2020.igem.org/Team%3ABrno_Czech_Republic/Experiments) and [Protocols](https://2020.igem.org/Team%3ABrno_Czech_Republic/Protocols).

### Learn

In **Figure 7**, you can see the agarose gel DNA electrophoresis of PCR products obtained from the first test explained above. From the result we can see that three tested strains produced amplicons of the required length.

![](https://static.igem.org/mediawiki/2020/b/b9/T--Brno_Czech_Republic--ResultsFig10.jpg)

**Figure 7: Agarose gel DNA electrophoresis of amplicons of the integration region of *B. subtilis* chromosome.** Line 1 contains the IM. The lines 2, 3 and 4 contain amplicons from the chromosomes of transformants of *Bacillus subtilis* (indicated by the blue arrow). Line number 5 contains negative control - an amplicon from a chromosome of *Bacillus subtilis* which was not transformed. M is a marker 1kb GeneRuler from ThermoFisher Scientific. The lengths of the fragments are explained in **Table 1**.

In Figures **8A;8B;8C**, you can see plates with starch, stained by iodine, obtained after the second test explained above. From the result we can see that tested strains do not create a halo effect in comparison to the non-transformed *B. subtilis* 168.

![](https://static.igem.org/mediawiki/2020/thumb/f/fd/T--Brno_Czech_Republic--Results_11A.png/607px-T--Brno_Czech_Republic--Results_11A.png)
![](https://static.igem.org/mediawiki/2020/thumb/2/29/T--Brno_Czech_Republic--Results_11B.png/607px-T--Brno_Czech_Republic--Results_11B.png)
![](https://static.igem.org/mediawiki/2020/5/5b/T--Brno_Czech_Republic--Results_11C.png)

**Figure 8: Proof of chromosomal integration in *B. subtilis* by AmyE test.** In the Fig. 7A you can see the *Bacillus subtilis* 168 which was not transformed - there is a clear halo surrounding the colonies (in detail in Fig. 7C). The plate in Fig. 7B contains bacteria which were transformed with pDG3661\_IM. The integration was successful as there was no halo visible on the plate. In 7C you can see the clear halo from 7A in detail.

Results from both experiments confirmed that we achieved successful integration of expression cassette into a specific region of *B. subtilis* chromosome. We have also optimized the protocol for the transformation of *B. subtilis* with plasmid pDG3661. We now have a functional methodology for the integration of synthetic DNA sequence in the chromosome of *B. subtilis*. In the future, we would like to transform *B. subtilis* with other constructs containing the sequences for the rest of our proteins. Detailed description of our results and other experiments we've performed can be found on the wiki page [Results](https://2020.igem.org/Team%3ABrno_Czech_Republic/Results) and [Experiment](https://2020.igem.org/Team%3ABrno_Czech_Republic/Experiments).

We have also learned other things, for example how to order synthetic sequences and what to look out for. We have found out that there was a misunderstanding when we ordered the synthesis of our sequences. We discovered this as we were submitting the synthetic sequences used in CYANOTRAP to the Parts Registry. Because the sequence was too complex to be synthetized as it was, we needed to optimize it before the final order was placed. Accidentally, the optimization software changed some of the nucleotides which unfortunately created two forbidden restriction sites in the sequence. From now on, we will definitely remember to check the optimized sequence for restriction sites before confirming it.

When we learned this fact, most of the experiments were already done and we couldn't enter the university campus due to the Covid-19 situation. However, the change did not interfere with our cloning strategy and it occurred only on the nucleotide level (the amino acid sequence was not changed) which means it could only influence the translation rate (which was not measured during our experiments) and our experiments should work the same with a slightly altered nucleotide sequence.

Luckily, as we already suggested, our experimental success does not stand on the creation of a new part. Despite inconveniences, we strongly believe that we reached the main goal of our experiments for this year, as described above. In conclusion this is the list of thing we have achieved and learned:

1. We have learned how to design synthetic sequences for a given purpose and we have designed all of the sequences needed for our project.
2. We have learned how to work with *B. subtilis*.
3. We have cloned the synthetic sequence encoding the Immobilization module into the plasmid pDG3661, creating our construct pDG3661\_IM. We have verified this using restriction digestion, long PCR and sequencing.
4. We have managed to propagate the pDG3661\_IM construct in *E. Coli* and isolate a sufficient amount for further experiments.
5. *B. subtilis* was successfully transformed and the integration of our expression cassette into a specific region of its chromosome was confirmed with longPCR and with the AmyE test.
6. We have optimized the protocol for the transformation of *B. subtilis* with plasmid pDG3661.
7. We have also done a series of experiments to confirm the expression and functionality of IM in *B. subtilis*.

### Improve

We know that the most important thing in science is to analyze our results, discuss and learn from the mistakes, suggest improvements and tirelessly continue working. These are our suggestions:

*All of the conducted experiments and their results are in detail described on wiki page Results and Experiment and Protocol. This is mainly discussion.*

In our cultivation experiment we have used the 2x Mal medium. For the next cultivation, we will use a different recipe for the 2x Mal medium, as we later discovered, that our medium was not prepared in the most common way. We will also not perform the second cultivation in 2x Mal medium but use the cells from the first cultivation in this medium when they grow to OD600 = 3. Because we found out in the literature, that there is no need to perform this second cultivation.

As for the SDS-page and Western blot transfer, we think we could optimize the concentration and the type of antibodies used. Instead of using the c-myc tag, we could use antibodies specific to our protein. However, we do not think that there is an issue in the Western blot design, because the gels and membranes do show proteins and patterns. We rather think that there is a problem with the protein expression protocol and sample processing. For expression we could optimize the type of medium and its pH during the cultivation of *B. subtilis*. We should also determine an optimal OD600 for the sample collection. We have to also select an ideal method of sample processing and optimize it, because we did not figure out which method is the best.

When redoing the cellulose binding experiment, we will work only with one dilution of the 2x Mal medium culture (OD600 = 0.1) and perform several dilutions of the samples before putting them on LB agar plates. We will also try to measure the volume of the microbeads as precisely as possible in order to determine the number of cells attached to one microbead. This will make our work more effective.

The Immobilization module (IM), a synthetic sequence which we used during our experiments, contains a region encoding the LysM domain. It contains two forbidden restriction sites by mistake, as mentioned above. Another one of our synthetic sequences (ScafD) contains a region encoding the same protein (same amino acid sequence) which is compatible with one of the iGEM required standards and could thus be used in the IM sequence as well. There are several possible strategies to achieve this goal. The simplest and most straight-forward one is to update the sequence and have it synthesized again without the forbidden restriction sites. Another option is to exchange the faulty sequence with the correct one using the Overlap extension PCR (**Fig. 9**). This method is an easy way to splice two sequences together using specific primers and two consecutive PCR reactions. We designed two primers (32 and 33, see Primers in [Specific project design](https://2020.igem.org/Team%3ABrno_Czech_Republic/Design#p-38)) which partially anneal to the IM and partially to the ScafD. This ensures that the products of the first (separate) PCR reactions anneal one to the other when mixed together.

![](https://static.igem.org/mediawiki/2020/a/a0/T--Brno_Czech_Republic--engineering-oePCR.png)

**Figure 9: Overlap Extension PCR strategy for improving the Immobilization module.** The Overlap Extension PCR method is an easy method for splicing two sequences using two consecutive PCR reactions with special overlapping primers. Thanks to this method, we will be able to exchange the faulty LysM module in the original Immobilization module sequence with a part of the LysM module from Scaffoldin D.

This way we are closing the engineering design cycle: Research → Imagine → Design → Build → Test → Learn → Improve → Research. It was quite a ride. We learned, achieved and experienced a lot this year and we are looking forward to iGEM year 2021. See you later!

#### References

1. You C., Zhang X. Z., Sathitsuksanoh N., Lynd L. R. and Zhang Y. H. P. 2012. Enhanced microbial utilization of recalcitrant cellulose by an ex vivo cellulosome-microbe complex. Appl. Environ. Microb. 78 (5): 1437–1444. DOI: 10.1128/AEM.07138-11
2. Mota T. R., de Oliveira D. M., Marchiosi R., Ferrarese-Filho O., and dos Santos W. D. 2018. Plant cell wall composition and enzymatic deconstruction. AIMS Bioeng. 5 (1): 63–77. DOI: 10.3934/bioeng.2018.1.63
3. Wang A. A., Mulchandani A. and Chen W. 2001. Whole-Cell Immobilization Using Cell Surface-Exposed Cellulose-Binding Domain. Biotechnol. Prog. 17: 407–411. DOI:10.1021/bp0100225
4. Krásný, L., and Gourse, R. L. 2004. An alternative strategy for bacterial ribosome synthesis: *Bacillus subtilis* rRNA transcription regulation. The EMBO journal 23(22): 4473–4483. DOI: 10.1038/sj.emboj.7600423

![](https://static.igem.org/mediawiki/2020/3/3d/T--Brno_Czech_Republic--Engineering_Icon_Wrench.svg)

[![](https://static.igem.org/mediawiki/2020/8/8b/HQ_page_logo.jpg)](https://2020.igem.org)

# Team:Brno Czech Republic/Experiments

[![](https://static.igem.org/mediawiki/2020/a/ad/T--Brno_Czech_Republic--Logo_CYANOTRAP.svg)](https://2020.igem.org/Team%3ABrno_Czech_Republic)

* [TEAM](https://2020.igem.org/Team%3ABrno_Czech_Republic/Team)

  [Attributions](https://2020.igem.org/Team%3ABrno_Czech_Republic/Attributions)
  [Our NPO](https://2020.igem.org/Team%3ABrno_Czech_Republic/Excellence)
* PROJECT DESIGN

  [Project description](https://2020.igem.org/Team%3ABrno_Czech_Republic/Description)
  [Theory](https://2020.igem.org/Team%3ABrno_Czech_Republic/Theory)
  [Specific Project Design](https://2020.igem.org/Team%3ABrno_Czech_Republic/Design)
  [Our device](https://2020.igem.org/Team%3ABrno_Czech_Republic/Device)
  [Modeling](https://2020.igem.org/Team%3ABrno_Czech_Republic/Model)
  [Parts](https://2020.igem.org/Team%3ABrno_Czech_Republic/Parts)
* WET LAB

  [Safety](https://2020.igem.org/Team%3ABrno_Czech_Republic/Safety)
  [Experiment](https://2020.igem.org/Team%3ABrno_Czech_Republic/Experiments)
  [Engineering success](https://2020.igem.org/Team%3ABrno_Czech_Republic/Engineering)
  [Conclusion & Results](https://2020.igem.org/Team%3ABrno_Czech_Republic/Results)
  [Notebook](https://2020.igem.org/Team%3ABrno_Czech_Republic/Notebook)
* FUTURE

  [Proposed implementation](https://2020.igem.org/Team%3ABrno_Czech_Republic/Implementation)
  [Plans for the future](https://2020.igem.org/Team%3ABrno_Czech_Republic/Future)
* OUTREACH

  [Collaboration](https://2020.igem.org/Team%3ABrno_Czech_Republic/Collaborations)
  [Human Practices](https://2020.igem.org/Team%3ABrno_Czech_Republic/Human_Practices)
  [Education](https://2020.igem.org/Team%3ABrno_Czech_Republic/Education)
  [Contribution to iGEM](https://2020.igem.org/Team%3ABrno_Czech_Republic/Contribution)
* Documentation

  [Protocols](https://2020.igem.org/Team%3ABrno_Czech_Republic/Protocols)
  [Medals and awards](https://2020.igem.org/Team%3ABrno_Czech_Republic/Medals_and_Awards)

### Experiments

Here, you can find more information about the experiments we've done. You can read about our reasons for performing the experiment, its theoretical background and a short description of our workflow. You will find the result of each experiment in the section Results & Conclusion. If you are interested in more specific information about the methodist we used, you can take a look at our Protocols [*here*](https://2020.igem.org/Team%3ABrno_Czech_Republic/Protocols) or through the hypertext link mentioned with specific experiments.

At Masaryk university, we were allowed to work in a laboratory, which is usually used for teaching practical courses during the semester. There we had everything we needed - flow boxes, cyclers, centrifuges, pipettes and so on. We also had the support of doctoral students and technical assistants of the Experimental biology department. Unfortunately, the coronavirus situation in our country worsened at the end of the summer, so we had to stop with our experiments earlier than planned.

We have however already planned out what to improve in the future. We have also already brainstormed how to characterize the rest of our synthetic constructs which we will hopefully share with you next year.

![](https://static.igem.org/mediawiki/2020/3/3a/T--Brno_Czech_Republic--Results_Parafilm.jpg)

![](https://static.igem.org/mediawiki/2020/5/58/T--Brno_Czech_Republic--Results_StandaPozuje.jpg)

#### Basic experiments

##### Preparation of growth media

The protocols for media and agar preparation can be found here: [PROTOCOL: Preparation of growth media](https://static.igem.org/mediawiki/2020/9/9b/T--Brno_Czech_Republic--prot-grow-med.pdf).

##### Preparation of synthetic constructs and primers

###### **Preparation of synthetic constructs**

**Motivation:**
For the transformation of organism *B. subtilis* we had 6 synthetic constructs prepared by company IDT within the offer mediated by iGEM contest.

These synthetic modules are IM, ScafL, LysBL-DocCt, LysGG-DocCt, ScafD, MlrB-DocBc, MlrC-DocAc and are further described in the section [Project design](https://2020.igem.org/Team%3ABrno_Czech_Republic/Design).

**Short description:**

1. Genes were delivered in a powder form and they had to be resuspended according to the instructions provided by the producer.
2. Synthetic genes were shortly centrifuged at Short spin mode and then resuspended in 50 µl nuclease free H2O in the flowbox.

* Resuspended genes are less stable, therefore it was necessary to keep them on ice or put them in a freezer at -20 °C.
* We worked with them only in Flowbox to minimize risk of DNA contamination.
* It is better to use pipette tips with filters for the gene stocks, because pipettes might contain aerosols from previous pipetting. Possible contamination could devalue the whole stock.

###### **Preparation of primers**

**Motivation:**
Within the CYANOTRAP project we worked with a lot of primers. Their detailed description is [here](https://2020.igem.org/Team%3ABrno_Czech_Republic/Design#p-38).

**Short description:**
Our primers were also synthesised by the IDT company. They were also delivered in powder form and it was necessary to resuspend them in nuclease free H2O, according to provided instructions.When working with primers, we always used 10 nM solution, which we prepared from 100 nM stock solution. The stock solution was created by resuspending delivered powder. We assigned each primer a number to be able to distinguish them and talk about them easily.

##### Long-Range PCR of synthetic constructs

**Protocol is available** **here:** [PROTOCOL: Long-Range PCR](https://static.igem.org/mediawiki/2020/1/1e/T--Brno_Czech_Republic--prot-long-range-PCR.pdf)

**Motivation:**
We routinely used long-Range PCR in our experiment for the amplification of our DNA. Another reason was our issues with colony PCR. Amplified DNA was used for many purposes, for example as gel electrophoresis, sequencing or storage.

**Short description:**
When performing Long-Range PCR, we followed a protocol that came with the GoTaq® Long PCR Master Mix by the company PROMEGA [here](https://worldwide.promega.com/-/media/files/resources/protocols/technical-manuals/101/gotaq-long-pcr-master-mix-protocol.pdf?la=en)). While working, it is necessary to keep everything sterile. Positive and negative controls are also necessary.

##### Preparation of glycerol stocks and vectors for cloning

**Protocols are available here** [(PROTOCOL: Preparation of glycerol stocks)](https://static.igem.org/mediawiki/2020/f/f1/T--Brno_Czech_Republic--prot-glyc-stocks.pdf) and here [(PROTOCOL: Isolation of plasmids)](https://static.igem.org/mediawiki/2020/9/96/T--Brno_Czech_Republic--prot-izo-plas.pdf).

**Motivation:**
We received our strain of *B. subtilis* and the vectors for our project from Dr. Libor Krásný. He is the head of the Laboratory of Microbial Genetics and Gene Expression at the Academy of Sciences of Czech Republic. After the plates with strains of *B. subtilis* and *E. coli* cells containing our vectors were successfully delivered, we needed to make glycerol stocks from them and isolate our vectors from *E. coli*.

**Short description:**
In total we got 5 plates with following strains:

|  |  |  |
| --- | --- | --- |
| Strain | Resistance | Plasmid |
| B. subtilis BSB1 wt-derivative 168 trp+ | None | None |
| E. coli Dh6α | Ampicillin | pDG1663 |
| E. coli Dh6α | Ampicillin | pDG1664 |
| E. coli Dh6α | Ampicillin | pDG1731 |
| E. coli Dh6α | Ampicillin | pDG3661 |

You can read more about our bacterial strains and vectors in [Project design](https://2020.igem.org/Team%3ABrno_Czech_Republic/Design)

In the end, we've worked mainly with vector pDG3661 and a little bit with pDG1664. Other strains carrying our vectors were stored at - 80 °C.

One colony from each plate was then streaked on a fresh plate with Lysogeny broth (LB) agar. For *E. coli* strains, we used a medium with ampicillin (concentration: 100 µg/ml). Due to initial miscommunication, we thought that the ideal temperature for the growth of *B. subtilis* is 28 °C. We used this temperature in this experiment and in the following weeks. After realising our mistake, we proceeded to use the correct temperature of 37 °C. Lower temperature caused delayed growth. The plates with *E. coli* were incubated at 37 °C.

Next day one larger colony from the plate with *B. subtilis* was chosen and it was densely spread on 6 different plates. One colony was chosen from each plate with *E. coli* and each colony was densely spread on 2 different plates. We've inoculated two plates with each colony, to have a backup in case of contamination.

Following day the plates were harvested and the bacteria were transported into 2 ml of liquid LB medium with 20% glycerol. These glycerol stocks were then stored at -80 °C.

Plasmid were isolated from a liquid overnight culture of corresponding *E. coli* strain, we used PureYield™ Plasmid Miniprep System by Promega Corporation.

![](https://static.igem.org/mediawiki/2020/0/0b/T--Brno_Czech_Republic--Results_SabcaMedia.jpg)

![](https://static.igem.org/mediawiki/2020/e/ed/T--Brno_Czech_Republic--Results_StandaPipetuje.jpg)

#### Preparation of genetic modules in *Escherichia coli* cloning host

##### Test of vector background of *Escherichia coli*

**Protocol is available here:** [PROTOCOL: Test of vector background of *Escherichia coli*](https://static.igem.org/mediawiki/2020/9/91/T--Brno_Czech_Republic--prot-vec-test.pdf)

**Motivation:**
After transforming *E. coli* with our vectors after restriction cloning, we got only a small number of transformants. Therefore, we tested the transformation background to determine if the growth we are observing is just the background. Background refers to the fact that in some cases even a vector which is cleaved with two different restriction enzymes and dephosphorylated with antarctic phosphatase, could reconnect.

**Short description:**
One batch of competent cells was transformed with supercoiled plasmid. Another batch of cells was transformed (see transformation experiment: Cloning and preparation of *E. coli* below) with a vector cleaved with two restriction enzymes - in our case HindIII and EcoRI - and also dephosphorylated with Antarctic phosphatase. Both batches were inoculated at the LB agar plate with appropriate antibiotics. Plates were incubated overnight in 37°C.

##### Preparation of genetic modules in *Escherichia coli* cloning host

**Protocol is available here:** [PROTOCOL: Preparation of genetic modules in *Escherichia coli* cloning host](https://static.igem.org/mediawiki/2020/8/86/T--Brno_Czech_Republic--prot-clon-ecoli.pdf)

**Motivation:**
We transformed our constructs into *E. coli* Dh6α NEB® 5-alpha Competent (High Efficiency) in order to multiply our vectors with inserts and to make glycerol stocks.

**Short description:**
First, the plasmid was isolated from a well grown overnight culture of *E. coli*. Then the empty vector pDG3661 was cleaved by two different restriction enzymes - EcoRI and HindIII. Antarctic phosphatase was added to keep the plasmid from self-ligating. As for our insert, we used amplified PCR products of synthetized genes by IDT. Subsequently, they were digested with the same restriction enzymes under the same conditions (with no Antarctic phosphatase added).

We used different ligation strategies during preparation of each of these plates. T4 ligase from Promega was used for the first ligation reaction. This reaction ran overnight at room temperature. The cells transformed with this reaction mix were inoculated on plate 2 (see [Results](https://2020.igem.org/Team%3ABrno_Czech_Republic/Results)). The ligation reaction, with which we transformed the cells plated on plate 3, ran for 15 min at room temperature. This reaction mix contained T4 ligase from NEB. Our last ligation reaction was used to transform cells plated on plate 4. Unlike in previous reactions, the products of restriction digestion were first separated on agarose gel and then extracted using the kit mentioned above. The ligation reaction ran for 15 min at room temperature. Ligation mixes were added to *E. coli* cells, which then underwent heat-shock and were incubated for 75 min before being inoculated on a LB plate containing ampicillin. In this experiment we also used positive control. Our positive control of competence ability of competent cells contained empty supercoiled plasmid pUC19. The cells grew overnight at 37 °C. Next day we evaluated the results.

##### Proof of transformation of *Escherichia coli*

**Protocol is available here:** [PROTOCOL: Proof of transformation of *Escherichia coli*](https://static.igem.org/mediawiki/2020/5/52/T--Brno_Czech_Republic--prot-tran-ecoli.pdf)

**Motivation:**
We needed to determine the success of our transformation and the presence of our vector. We've had some issues with colonyPCR, so we've had to use different methods of proof - restriction digestion and sequencing.

**Short description:**
Firstly, we purified our plasmid from overnight culture. Isolated plasmid was then cleaved by two different restriction enzymes - EcoRI and HindIII. The restriction reaction lasted 2 hours at 37°C. Afterwards, we used agarose electrophoresis to show the size of the fragments.

##### Preparation of vector pDG3661 for sequencing

**Motivation:**
The aim of this experiment was to verify the sequence of isolated plasmid pDG3661, specifically the sequence of our insert - the Immobilization module (IM). This experiment further demonstrates the success of the cloning and *E. coli* Dh6α transformation.

**Short description:**
We followed the protocol for sequencing of plasmid templates, provided by the SEQme company. We used isolated plasmid pDG3661\_IM (See [PROTOCOL: Isolation of plasmids](https://static.igem.org/mediawiki/2020/9/96/T--Brno_Czech_Republic--prot-izo-plas.pdf)). We added forward and reverse primers to the samples of isolated plasmid. Both primers were added separately, so there were two sequencing mixes for each sample and the sequence was ready to be read from both strands. We send these samples to the company SEQme based in Czech republic.

#### Integration of the exprssion casette with IM into the chromosome of *Bacillus subtilis*

##### Preparation of competent cells of *Bacillus subtilis*

**Protocol is available here:** [PROTOCOL: Preparation of competent cells of *Bacillus subtilis*](https://static.igem.org/mediawiki/2020/7/71/T--Brno_Czech_Republic--prot-test-com.pdf)

**Original publications:**

**Spizizen J. (1958)**. TRANSFORMATION OF BIOCHEMICALLY DEFICIENT STRAINS OF BACILLUS SUBTILIS BY DEOXYRIBONUCLEATE. Proceedings of the National Academy of Sciences of the United States of America, 44(10), 1072–1078. <https://doi.org/10.1073/pnas.44.10.1072>

* modified by dr. Krásný - Laboratory of Microbial Genetics and Gene Expression

**Dubnau, D., & Davidoff-Abelson, R. (1971)**. Fate of transforming DNA following uptake by competent *Bacillus subtilis*. I. Formation and properties of the donor-recipient complex. Journal of molecular biology, 56(2), 209–221. https://doi.org/10.1016/0022-2836(71)90460-8

* modified by dr. Krásný - Laboratory of Microbial Genetics and Gene Expression

**Motivation:**
We needed to prepare natural competent cells of *B. subtilis*. The Laboratory of Microbial Genetics and Gene Expression and dr. Krásný from Prague really helped us at the beginning of our experiment. He also recommended this type of competence indication - as he has very good experience with this protocol.

**Theory:**
The preparation of naturally competent cells is necessary for successful transformation of *B. subtilis*. It is also possible to prepare electrocompetent cells from *B. subtilis*, but that wasn't necessary in our case. This method can be done only in certain media and in the post-exponential phase of bacterial culture growth. These competent cells are able to efficiently take in exogenous high-molecular-weight DNA. In the competent culture, not every cell is in its competent state. It is however enough to generate a sufficient amount of transformants. Their preparation includes a two-step process using glucose minimal salt-based media with Spizizen salts.

**Short description:**
This experiment requires the preparation of two different media - SPI & SPII and an overnight culture of *B. subtilis.* The next day, overnight culture has to be inoculated into the SPI medium in duplicate - one for measurement and one for later use. After inoculation, the OD600 of the culture must be measured. The cultivation must continue until the values indicate the departure from the exponential rate (this is very important). This usually takes around 5 hours and 30 min. As the values deviate from the exponential curve, culture is inoculated into the SPII medium and cultivated under the same conditions for 90 minutes. Afterwards, the culture is placed on ice to stop its growth and centrifuged. Pellet is resuspended in a fraction of the supernatant and glycerol. Aliquots can then be made and stored at -80°C.

![](https://static.igem.org/mediawiki/2020/c/c5/T--Brno_Czech_Republic--Results_NasPlazmid.jpg)

##### Testing competent cells of *Bacillus subtilis*

**Protocol is available here:** [PROTOCOL: Transformation of *Bacillus subtilis*](https://static.igem.org/mediawiki/2020/6/6b/T--Brno_Czech_Republic--PROTOCOL-Test_of_Bacillus_subtilis_competent_cells.pdf)

**Motivation:**
The ability of our natural competent cells to accept plasmid DNA needed to be verified. We performed this experiment many times before the synthetic genes arrived to optimize the process. In the end we determined the best conditions for our transformation.

**Theory:**
This experiment primarily verifies the competency of the cells and the efficiency of their transformation. The use of controls is essential for this experiment. *B. subtilis* cells without plasmid pDG3661 carrying a resistance to chloramphenicol were used as a negative control. These bacteria should not be able to grow in the presence of this antibiotic. It is also necessary to confirm that our competent cells are alive by growing them in LB agar with no antibiotics, where colonies should appear regardless of the success of our transformation.

**Short description:**
Our natural competent cells were transformed with plasmids pDG3661\_IM. We then followed this protocol (time of shaking was 60 min). Afterwards, we inoculated the bacteria onto LB plates with chloramphenicol. Cells transformed with empty plasmid pDG3661 were used as positive control. The cells were cultivated overnight at 37 °C.

##### Transformation of *Bacillus subtilis*

**Protocol is available here:** [PROTOCOL: Transformation of *Bacillus subtilis*](https://static.igem.org/mediawiki/2020/0/07/T--Brno_Czech_Republic--prot-tran-bac.pdf)

**Motivation:**
We transformed naturally competent *B. subtilis* 168 in order to produce our proteins - specifically the IM.

**Short experimental description:**
We first had to isolate pDG3661\_IM from *E. coli* Dh6αfrom previous experiments. The naturally competent *B. subtilis* cells were preserved in the form of glycerol stocks. We added 1 µg of plasmid to 100 µl of natural competent cells. We also prepared positive and negative controls. The positive control contained empty supercoiled plasmid pDG3661 carrying antibiotic resistance. The competent cells without plasmid were used as the negative control. These controls confirmed that the transformation was performed well and that the antibiotic was working on non-resistant cells. We incubated the tubes containing competent cells and plasmids on a shaker at 200 rpm and 37 °C for 1 hour. Afterwards, we planted the competent cells on agar plates with selection medium containing chloramphenicol. The cells grew overnight at 37 °C. Next day we evaluated the results.

##### TOPO cloning

**Protocol is available here:** [PROTOCOL: TOPO cloning](https://static.igem.org/mediawiki/2020/2/28/T--Brno_Czech_Republic--prot-topo-clon.pdf)

**Motivation:**
Beside restriction cloning we also performed TOPO cloning, in case the restriction cloning wouldn't be successful.

**Theory:**
TOPO cloning is a tool of synthetic biology, which enables cloning of an insert into a vector without using a ligase. It utilizes the ability of Taq polymerase to add a single deoxyadenosine to the 3'-end of the PCR products. Taq polymerase thus creates a sticky end of the insert, which can then be used to ligate it to our vector. Important role also plays DNA topoisomerase I., which recognises a specific DNA sequence and catalyzes its digestion and the subsequent re-ligation. Under optimal conditions, the insert can be ligated to the specific vector thanks to the activity of topoisomerase I.

**Short description:**
First we amplified our synthetic gene using a Long-Range PCR. We've then added this insert to a plasmid specifically designed for topo cloning - Topoisomerase I-activated pCR™4-TOPO® vector

To transform the competent *E. coli* cells with this mix, we followed the variant 2 in Protocol. Transformants were then streaked on Xgal plates with ampicillin. The plates were incubated overnight at 37 °C in the dark.

As we were trying out the TOPO cloning, we've decided to redo restriction cloning as well. In the end, we've gotten colonies of transformed cells in both cases. We've opted to continue with the transformants from restriction cloning because in that case, our insert was already in the correct plasmid for the chromosomal integration in *B. subtilis.*

![](https://static.igem.org/mediawiki/2020/f/f6/T--Brno_Czech_Republic--Results_SelfieDavid.jpg)

##### Proof of the successful transformation of *Bacillus subtilis*

**Protocol is available here:** [PROTOCOL: Proof of the successful transformation of Bacillus subtilis](https://static.igem.org/mediawiki/2020/3/30/T--Brno_Czech_Republic--prot-succ-tran.pdf)

**Motivation:**
After transforming *B. subtilis* with our vectors, we needed to confirm that the integration of our IM gene and the *lacZ* gene into the chromosome of *B. subtilis* was successful. As we had some issues with colony PCR, we had to use different methods of proof. Therefore we decided to isolate genomic DNA of *B. subtilis* and use standard long-Range PCR for its amplification. We then used sequencing and also the AmyE test to confirm the integration of our gene.

**Theory:**
We transformed *B. subtilis* with the vector pDG3661\_IM containing also the *lacZ* gene. The sequence is designed for integration into the *amyE* region of the chromosome of our bacteria*.* Our primers were designed to be complementary to the chromosomal sequence in the proximity of the integration site. PCR amplicons should thus contain a part of the *B. subtilis* chromosome, our synthetic, the *lacZ* gene and a component of vector pDG3661 for ectopic integration. The size of these amplicons should be around 8074 bp.

There are many different methods of isolating the genomic DNA of the *B. subtilis*. We tried two and tested their effectiveness. The first method - we called it the physical method - used extreme temperature changes. The second - chemical - method utilized a lysis buffer. We also tested the impact of the age of our cultures, as *B. subtilis* forms endospores which are not suitable for DNA isolation. As we expected, fresh overnight culture gave better results than a culture which was stored for a couple of days.

**Short description:**
We used fresh overnight cultures and older cultures grown on plates. Both methods of chromosomal isolation are described in detail in the protocol. The physical method resulted in higher yield of DNA (about 150 ng/µl). As for purity, there was no difference between the two methods.

We used the isolated DNA as a template for long PCR. The amplicons were then visualised on agarose gel.

##### AmyE test (Starch hydrolysis test)

**Protocol is available here:** [PROTOCOL: AmyE test (Starch hydrolysis test)](https://static.igem.org/mediawiki/2020/b/b6/T--Brno_Czech_Republic--prot-amy-test.pdf)

**Original publication:**
Guérout-Fleury, A. M., Frandsen, N., & Stragier, P. (1996). Plasmids for ectopic integration in *Bacillus subtilis*. *Gene*, *180*(1-2), 57–61. [https://doi.org/10.1016/s0378-1119(96)00404-0](https://doi.org/10.1016/s0378-1119%2896%2900404-0)

**Motivation:**
AmyE test is a simple method of demonstrating ectopic integration into the chromosome of *B. subtilis*. The integration site in the chromosome encodes 𝛼-amylase, which is able to degrade starch in the media. If the integration site is intact, the 𝛼-amylase is produced and a clear halo around the colonies can be observed after iodine treatment as the starch gets digested. If the integration into chromosome is successful and the sequence encoding 𝛼-amylase is split by the inset, the enzyme is not produced and starch is not degraded. In our experiment we tried using an AmyE test to confirm the transformation of our *B. subtilis* with oue vectors*.* We compared our samples with *B. subtilis* 168, which was not transformed with any DNA.

**Short description:**
Two layer agar is used for this method - the first layer is a regular agar containing corresponding antibiotic and the second layer is a thin agar with starch supplement. After the addition of a few crystals of iodine, the starch will turn dark blue. Do not forget to work in the laboratory hood as iodine fumes are harmful.

##### Modified Blue-White selection

**Protocol is available here:** [PROTOCOL: Modified Blue-White selection](https://static.igem.org/mediawiki/2020/1/17/T--Brno_Czech_Republic--prot-mod-sel.pdf)

**Motivation:**
We wanted to also use a modified Blue-White selection in order to verify chromosomal integration. Unfortunately, after a few confusing results, we realized that our construct, which was cloned upstream of the *spoVG-lacZ* gene, has a STOP codon at the end and *spoVG-lacZ* does not have its own RBS site. Due to this flaw in our design, we could not use this selection method. If we decided to perform this test in the future, we would also place an RBS at the end of our synthetic construct. We realized that iGEM is also (and maybe mainly) about learning from our mistakes.

**Theory:**
The vectors which we chose for our project include *spoVG-lacZ* gene. This gene was created by fuzing the *spoVG* gene from *B. subtilis* and *lacZ* gene which encodes ß-galactosidase. This enzyme is able to degrade the Xgal substrate to gain a colorful product. Colonies with active ß-galactosidase turn blue and colonies without it stay colorless. In our case, colonies containing our insert would thus turn blue, which is not how the Blue-white method is usually done. In our project the transcription of the *spoVG-lacZ* gene is controlled by constitutive promoter *Pveg*, so we did not use IPTG to induce the protein expression.

**Short description:**
In this experiment LB agar with added Xgal is used. We prepared this agar and then we inoculated the bacteria onto the plates containing this medium.

![](https://static.igem.org/mediawiki/2020/b/b9/T--Brno_Czech_Republic--Results_Miska.jpg)

##### Preparation of sample for sequenation - *Bacillus subtilis*

**Motivation:**
We needed to verify the sequence which was integrated into the chromosome of *B. subtilis*. This would demonstrate the success of our transformation and chromosomal integration. We tried to get a good sequencing result but unfortunately, we were not able to prepare the sample properly.

**Short description:**
We used the services of the company SEQme, based in Czech republic. The sequencing was performed three times. In the first attempt we used the whole chromosomal DNA of *B. subtilis* 168, which was transformed with vector pDG3661\_IM. We used primer 1 and 29, which allowed us to obtain a sequence of the main part of our synthetic gene – the immobilisation module. More information can be found in the section [Primers](https://2020.igem.org/Team%3ABrno_Czech_Republic/Design#p-38). However, the results weren't optimal, so we performed this experiment once again with optimised sample preparation.

When repeating the sequencing, we prepared chromosomal DNA isolated with the same method. We then amplified desired DNA sequence, using the same primers as were being used for sequencing, specifically primer 1 and 13. The PCR product was then separated from chromosomal DNA on 0,6 % agarose gel. Afterwards, we performed gel extraction and prepared the samples for sequencing according to this protocol and sent them for sequencing. In the third attempt, we used the same method. Despite our efforts, the results were not optimal.

#### Characterization of immobilization module

Calibration of the device with iGEM Measurement kit should precede this experiment. We however did not receive it this year and, due to a lack of time and the coronavirus situation, we also weren’t able to handover this material to another iGEM team. We have preformed the experiment in the final days of our lab work and we consider its result as unclear and inconclusive. Next year we will try to get the iGEM Measurement kit for calibration of the device in order to measure OD600 in time.

##### Cultivation experiment

**Protocol is available here:** [PROTOCOL: Cultivation experiment](https://static.igem.org/mediawiki/2020/6/6e/T--Brno_Czech_Republic--prot-cul-exp.pdf)

**Original publication:**
You C., Zhang X. Z., Sathitsuksanoh N., Lynd L. R. and Zhang Y. H. P. 2012. Enhanced microbial utilization of recalcitrant cellulose by an ex vivo cellulosome-microbe complex. Appl. Environ. Microb. 78 (5): 1437–1444. DOI: 10.1128/AEM.07138-11

**Motivation:**
For optimal extracellular production of heterologous proteins, *B. subtilis* should be cultivated in 2x Mal medium. This experiment was inspired by the work of You *et al*. in 2012.

**Short description:**
Expression cultures after an overnight cultivation and the control strains were inoculated into 10 ml of liquid LB medium with and without chloramphenicol, respectively. This selection strategy was kept the same for the whole experiment. After 16 hours of growth at 37 °C and 200 rpm, 20 ml of fresh LB medium were inoculated with the cultures so that the starting OD600 would be 0.05. OD600 was then measured every 30 minutes. When the culture reached OD600 of 1.2, 50 ml of modified 2x Mal medium were inoculated with these cultures to get the starting OD600 of 0.0048. OD600 was then measured every 30 minutes. After the culture reached OD600 of 1.5, fresh 50 ml of modified 2x Mal medium were inoculated with these cultures to get the starting OD600 of 0.0025. The culture was cultivated for 14 hours and then used for further experiments. The protocol contains the preparation of the modified 2x Mal medium as well as the cultivation procedure.

![](https://static.igem.org/mediawiki/2020/4/44/T--Brno_Czech_Republic--Results_MisaMisky.jpg)

##### Collection of samples and their SDS-PAGE and Western blot analysis

**Protocol is available here:** [PROTOCOL: Bradford protein assay , SDS-PAGE, Coomassie staining, Western blot,](https://static.igem.org/mediawiki/2020/6/67/T--Brno_Czech_Republic--prot-brad-prot.pdf) [Proccesing of samples for SDS-PAGE](https://static.igem.org/mediawiki/2020/a/a6/T--Brno_Czech_Republic--prot-proc-samples.pdf)

**Motivation:**
We used SDS-page and Western blotting to prove the expression of the IM in *B. subtilis*. We tried to gather the samples from the cultures with different OD600 and several different methods of sample processing to obtain the best results.

**Theory:**
SDS-page is a method used to separate proteins based on their size, using a highly linked gel matrix and direct electrical current. The effect of different charges is suppressed as the proteins are denatured and encapsulated by negatively charged SDS molecules.

Western blot is used to verify the presence of a protein through its interaction with specific antibodies, which are then detected by chemiluminescence. From the SDS gel, the proteins are transferred to the PVDF membrane. Primary antibodies can then interact and bind to the Tag on our protein. Afterwards, secondary antibodies carrying chemiluminescent enzymes bind to primary antibodies.

**Short description:**
The first round of sample collection was performed thusly. At first the samples were collected from an overnight culture in LB, then from 2 x Mal medium at the OD600 of 1.2 and finally after 24 h cultivation in LB. These samples were processed with B-PER reagent and 2 M urea. SDS-page and Western blot was carried out using c-Myc antibody. We tried antibodies from mouse and rabbit. Primary mouse antibody was monoclonal c-Myc (9E10) from Franz Klein lab. Secondary was anti-mouse IgG + IgM, (H+L), conjugated to HRP, from Pierce # 31444. Primary rabbit antibody was polyclonal c-Myc (A-14) from Santa Cruz Biotechnology, INC. Secondary rabbit antibody was anti-rabbit IgG, (H+L), conjugated to HRP, from Pierce # 31460. The first concentrations of antibodies were 1:1000 of the primary antibodies and 1:15000 of the secondary antibodies.

Unfortunately, the results yielded from negative control were rather similar to the expression strains and there is no clear proof of the presence of the IM. The gel was also stained with coomassie blue, but also there was no visible difference between the control and expression samples.

For our second attempt, we tried to make some adjustments. The first incubation was accidentally done at 30 °C. We used the correct temperature of 37 °C for the second run. This time we also gathered whole cell lysates and whole cells. We also adjusted the concentrations of primary and secondary antibodies to 1:500. The first samples were collected from overnight cultures growing in LB medium, another batch of samples was obtained from 2x Mal medium at the OD600 of 1.5. Final samples were collected after the cultures were incubated in 2x Mal medium for 24 hours.

![](https://static.igem.org/mediawiki/2020/d/de/T--Brno_Czech_Republic--TomPipetuje.jpg)

##### Preparation of cellulose microbeads

**Protocol is available here:** [PROTOCOL: Preparation of cellulose microbeads](https://static.igem.org/mediawiki/2020/b/b6/T--Brno_Czech_Republic--prot-prep-cel.pdf)

**Motivation:**
We needed to clean the cellulose microbeads from the 20% ethanol in which they were stored.

**Short description:**
Approximately 0,5 ml of microbeads were transferred from the storage container to Eppendorf tubes and left to sediment. The ethanol was then removed and the rest of it was left to evaporate in a thermoblock at 50 °C. After that, the microbeads were resuspended in 0,5 ml of PBS buffer. This suspension was left to sediment and the PBS buffer was discarded. The washing was repeated three times and at the end the microbeads were resuspended in a small amount of PBS. You can find the detailed protocols here.

##### Cellulose binding experiment

**Protocol is available here:** [PROTOCOL: Cellulose binding experiment](https://static.igem.org/mediawiki/2020/2/26/T--Brno_Czech_Republic--prot-cel-bin.pdf)

**Motivation:**
We wanted to find out if the IM enhances the ability of *B. subtilis* to bind to cellulose and to simulate the conditions in the CYANOTRAP device.

**Short description:**
The experiment was done using two expression strains and a control strain of unmodified *B. subtilis* 168 (C). The cellulose microbeads mixed only with the PBS buffer were also used to test the sterility of our microbeads (M). The cells were cultivated as described in the [Cultivation experiment](#cul-exp). After reaching the OD600 of 1.6 (approximately), two sets of samples were collected. In the first set, 0.75 ml of each strain in the PBS buffer at the OD600 of 1; 0.1 and 0.01 were prepared. In the second set, 5 ml of each strain in the PBS buffer at the OD600 of 1 were prepared.

The first set of samples was mixed with approximately 0,75 ml of cellulose microbeads that were prepared following this protocol. The second set was mixed with approximately 5 ml of the same material. The cells were then incubated with the microbeads for 1 hour at room temperature with rotation.

After that, the microbeads were left to sediment for 15 minutes. The supernatant was separated from the pellet and 100 µl of the supernatant of each sample (11.1 of the IM strain; 11.5 of the IM strain; control, M) from the first set of samples were applied onto LB agar plates. The pellets of the same set were washed twice with the PBS buffer to wash away any unbound cells and then the 100 µl of each type of sample were applied onto LB agar plates as well. These plates were incubated overnight at 37 °C and photographed the next day.

The pellet of the second set of samples was also washed twice with PBS for the same reason and then 2 ml of the pellet,resuspended in a small amount of the PBS buffer, were transferred to a plastic gravity column with the cutoff limit of 20 µm. This cutoff ensures that the cells bound to cellulose microbeads will stay above the filter while the unbound cells will appear in the flowthrough. 20 ml of fresh PBS was added to the microbeads with (presumably) bound cells and it was left to drop to a collection tube. After the entirety of the PBS buffer passed through the column, 100 µl of each sample was applied to LB agar plates. The plates were then incubated overnight at 37 °C.

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# Team:Brno Czech Republic/Results

[![](https://static.igem.org/mediawiki/2020/a/ad/T--Brno_Czech_Republic--Logo_CYANOTRAP.svg)](https://2020.igem.org/Team%3ABrno_Czech_Republic)

* [TEAM](https://2020.igem.org/Team%3ABrno_Czech_Republic/Team)

  [Attributions](https://2020.igem.org/Team%3ABrno_Czech_Republic/Attributions)
  [Our NPO](https://2020.igem.org/Team%3ABrno_Czech_Republic/Excellence)
* PROJECT DESIGN

  [Project description](https://2020.igem.org/Team%3ABrno_Czech_Republic/Description)
  [Theory](https://2020.igem.org/Team%3ABrno_Czech_Republic/Theory)
  [Specific Project Design](https://2020.igem.org/Team%3ABrno_Czech_Republic/Design)
  [Our device](https://2020.igem.org/Team%3ABrno_Czech_Republic/Device)
  [Modeling](https://2020.igem.org/Team%3ABrno_Czech_Republic/Model)
  [Parts](https://2020.igem.org/Team%3ABrno_Czech_Republic/Parts)
* WET LAB

  [Safety](https://2020.igem.org/Team%3ABrno_Czech_Republic/Safety)
  [Experiment](https://2020.igem.org/Team%3ABrno_Czech_Republic/Experiments)
  [Engineering success](https://2020.igem.org/Team%3ABrno_Czech_Republic/Engineering)
  [Conclusion & Results](https://2020.igem.org/Team%3ABrno_Czech_Republic/Results)
  [Notebook](https://2020.igem.org/Team%3ABrno_Czech_Republic/Notebook)
* FUTURE

  [Proposed implementation](https://2020.igem.org/Team%3ABrno_Czech_Republic/Implementation)
  [Plans for the future](https://2020.igem.org/Team%3ABrno_Czech_Republic/Future)
* OUTREACH

  [Collaboration](https://2020.igem.org/Team%3ABrno_Czech_Republic/Collaborations)
  [Human Practices](https://2020.igem.org/Team%3ABrno_Czech_Republic/Human_Practices)
  [Education](https://2020.igem.org/Team%3ABrno_Czech_Republic/Education)
  [Contribution to iGEM](https://2020.igem.org/Team%3ABrno_Czech_Republic/Contribution)
* Documentation

  [Protocols](https://2020.igem.org/Team%3ABrno_Czech_Republic/Protocols)
  [Medals and awards](https://2020.igem.org/Team%3ABrno_Czech_Republic/Medals_and_Awards)

### Results

Despite the adverse conditions in our country due to the coronavirus situation, we were able to demonstrate the functionality of some aspects of our CYANOTRAP project. Using tools of synthetic biology, we proved transfer of one of our synthetic constructs - firstly into the cloning strain of Escherichia coli and then also into the final host - *Bacillus subtilis*.

Due to time constraints, we were not able to clone all of our synthetic genes and transform B. *subtilis* with the resulting plasmid constructs. We decided to focus on one synthetic gene and optimize the method of B. *subtilis* transformation for this fragment. Our chosen gene - the Immobilization module (IM) - is needed to immobilize modified B. *subtilis* on the surface of cellulose beads (**Fig. 1**). If we want the CYANOTRAP to function in natural waters, we need to prevent genetically modified B. *subtilis* from escaping into the environment, while allowing cyanobacteria to flow inside the machine and be lyzed. Immobilizing B. *subtilis* on cellulose beads, which will be larger than the pores in machine’s filters, will allow us to do so. A large area for the attachment of B. *subtilis* will be created in this way as well. This will significantly increase the number of immobilized bacteria. More details about our experimental design can be found in the section [Project Design](https://2020.igem.org/Team%3ABrno_Czech_Republic/Description).

Unfortunately, the epidemiological situation in our country in October was very bad, so we were not able to work in the laboratory anymore and thus, we could not proceed with some of the important experiments as planned.

The next text is divided into 3 main sections:

* Preparation of genetic modules in *Escherichia coli* cloning host

* Integration of the expression cassette into the chromosome of *Bacillus subtilis*

* Characterization of the Immobilization module

**The section Conclusions at the end of this document, summarizes the most important information about our Experimental success (**Fig. 2**).**

### Preparation of genetic modules in *Escherichia coli* cloning host

#### Experimental design

The synthetic sequence encoding the Immobilization module (IM, BBa\_K3590036) with some regulation regions was designed using the Benchling software. It contains a strong constitutive Pveg promoter, strong RBS R2 and a STOP codon at the end of the coding sequence (CDS). The CDS contains a cellulose binding module connected by a linker to three LysM domains. The whole synthetic sequence contains the EcoRI and HindIII restriction sites at 5' and 3’ termini, respectively. Through these restriction enzymes it was inserted into the plasmid pDG3661 from which it can be delivered into the *amyE* locus of *B. subtilis* 168 chromosome.

Synthetic sequences (Composite [Parts](https://2020.igem.org/Team%3ABrno_Czech_Republic/Parts)) received from the company IDT were synthesized as linear gBlocks® Gene Fragments. That meant that they had to be quickly cloned into a vector. We inserted this synthetic sequence in front of the *lacZ* gene of our vector. At the end of this experiment, we acquired a large amount of pDG3661 construct with the cloned Immobilization module. The success of the cloning and subsequent transformation of *E. coli* was proven by plasmid isolation, control restriction cleavage, and sequencing.

#### Material and Methods

##### *Bacterial strains*

For this experiment, we used the commercial strain of chemocompetent cells of *E. coli* DH5α NEB® 5-alpha Competent *E. coli* (High Efficiency) which we store at -80°C before use.

##### *Chemicals and media*

Our transformed bacteria were grown in the Lysogeny Broth (LB) medium containing ampicillin (100 µg/ml). For plasmid isolation, we used PureYield Plasmid Miniprep System (Promega). For purification of PCR products and DNA extraction from agarose gels, we used Wizard® Genomic DNA Purification Kit (Promega). For PCR, we used Promega's Long-Range PCR: GoTaq® Long PCR Master Mix. In our experiments we used two different restriction enzymes - EcoRI and HindIII from New England Biolabs (NEB) which are both compatible with CutSmart® Buffer from the same producer. We treated the ends of our digested vector with Antarctic Phosphatase also from NEB. For ligation reaction, we used T4 DNA Ligase from the same producer with a corresponding buffer. For the visualization of electrophoresis, we used ethidium bromide or SYBR™ Safe DNA Gel Stain from ThermoFisher Scientific.

##### *Cloning experiment*

Detailed information about our Cloning experiment can be found in the section [Experiment](https://2020.igem.org/Team%3ABrno_Czech_Republic/Experiments).

1. Preparation of vector
    The empty vector pDG3661 was cleaved by two different restriction enzymes - EcoRI and HindIII. The restriction reaction lasted 2 h at 37°C and after one hour the Antarctic phosphatase was added to prevent the plasmid from self-ligating. Afterwards, we separated the samples from restriction enzymes and other impurities.
2. Preparation of insert
    Our fragment was amplified with PCR and subsequently cleaved with the same restriction enzymes under the same conditions (without Antarctic phosphatase addition). Then we had to purify it with the kit described above.
3. Ligation
    The ligation reaction ran for 15 minutes at room temperature.
4. Transformation
    We transformed chemocompetent cells with the ligation mixture. The cells were then incubated for 1 h at 37°C and 200 rpm and then were plated on agar plates with corresponding antibiotic and left to grow overnight at 37°C. The results are shown in **Fig. 4** in the section below.
5. Isolation of plasmid DNA from transformants
    To demonstrate the presence of our vector in the transformed cells, we first needed to purify the plasmid. Twelve colonies of transformants were further tested. We used the kit from Promega which is mentioned above for the isolation. The concentration of isolated plasmid was sufficient for the following experiments.
6. Restriction cleavage of vectors
    We used the same restriction enzymes to cleave the plasmids isolated from the 12 colonies of transformed cells. The cleavage was conducted under the same conditions as previously: 2 hours and 37°C.
7. Electrophoresis
    Afterwards, we used agarose electrophoresis to show the size of cleaved fragments. We used 0,6% agarose gel and SYBR™ Safe DNA Gel Stain for visualization. Electrophoresis ran for 45 min at appropriate voltage in the 1x TAE buffer.

#### Results

We've cloned our synthetic sequence coding the IM into pDG3661 plasmid. ORI used in this vector ensures that the plasmid is high-copy and this ORI is only recognized in *E. coli*. Our synthetic construct - the pDG3661 plasmid with our insert - was named pDG3661\_IM. By inserting the synthetic sequence into the pDG3661 plasmid, we will obtain a complete expression cassette containing the IM sequence. The design of the pDG3661 plasmid allows for this expression cassette to be subsequently integrated into a specific location the chromosome of *B. subtilis*.

We had to first amplify our synthetic sequence with PCR. Our synthetic sequence as well as the plasmid were digested by two restriction enzymes - HindIII and EcoRI. Ligation reaction was then performed and the reaction mix was used to transform competent *E. coli* cells. The transformants were selected on LB agar medium with ampicillin. Due to high-copy ORI, we obtained a large amount of our construct pDG3661\_IM. We then confirmed the presence of our insert by sequencing. We also performed restriction cleavage and subsequent electrophoresis with isolated constructs in order to visualize the insert and the backbone of the vector.

This experiment was extremely important. The figures show that we were able to insert our pDG3661\_IM construct into *E. coli* and that the vector was propagated in this species.

##### Testing our competent cells

At the beginning, we tested if the available chemocompetent cells were able to accept the empty vector pDG3661. We also wanted to demonstrate that the selection process works. We recognized that the competent cells which we had from one of our PI's laboratories are quite old and we decided to use the newer ones from the producer mentioned above. With these cells we were able to get many more transformants. So it seems that the time of storage of competent cells can affect the rate of transformation.

##### Testing the vector background

After a few unsuccessful attempts to transform our competent cells, we decided to prove how vector background influences the number of transformants we got. Background refers to the fact that in some cases even a vector which is cleaved with two different restriction enzymes and dephosphorylated with antarctic phosphatase, could reconnect. One batch of competent cells was transformed with supercoiled plasmid. Another batch of cells was transformed with a vector cleaved with two restriction enzymes - in our case HindIII and EcoRI - and also dephosphorylated with Antarctic phosphatase. The plate, where the second batch of cells was plated, was clean. The first plate with cells transformed with non-digested plasmid contained thousands of colonies. This demonstrates that the digested plasmid is likely not re-ligating, as we would observe some growth in the antibiotic-containing medium otherwise. While a small number of cells can be transformed with linearized plasmid, this vector can't replicate and does not allow for extensive bacterial growth. The first batch was also used as a positive control in the experiment described in **Fig. 3**.

##### Proof of transformation of *Escherichia coli*

The presence of the construct pDG3661\_IM in *E. coli* was proved by growing the cells on LB agar plates with ampicillin. Cloning of synthetic sequence into the vector was proven by sequencing. We also performed restriction cleavage and electrophoresis with isolated vectors from colonies of transformants to check if the sizes of the fragments were correct. As a positive transformation control, we transformed the competent cells with a smaller testing vector pUC19. The control plate had many colonies, proving that the cells were capable of competence. In Fig. 3 you can see plates 2 and 3 which are plates containing transformed cells.

For cloning, we used different ligation strategies. T4 ligase from Promega was used for the first ligation reaction. This reaction ran overnight at room temperature. The cells transformed with this reaction mix were inoculated on plate 2. The ligation reaction, with which we transformed the cells plated on plate 3, ran for 15 min at room temperature. This reaction mix contained T4 ligase from NEB. Both of these transformations seemed to have been successful as colonies grew on plates 2 and 3. There were 73 colonies at plate number 2 and 45 colonies at plate number 3. However, only the transformation of colonies on plate 3 was confirmed by restriction digestion and subsequent electrophoresis (**Fig. 4**)

Our last ligation reaction was used to transform cells plated on plate 4. Unlike in previous reactions, the products of restriction digestion were first separated on agarose gel and then extracted using the kit mentioned above. The ligation reaction ran for 15 min at room temperature. This transformation was unsuccessful and no colonies grew on the plate. This was probably caused by a very low DNA concentration after gel extraction.

##### Proof by restriction cleavage and electrophoresis

As our colony PCR did not work properly, we decided to demonstrate the presence of our construct by restriction cleavage. We isolated the plasmids from transformants and then digested them with the same restriction enzymes which we used for cloning. Then we performed agarose electrophoresis (**Fig. 5**). For this, we used 12 colonies grown from *E.coli* transformants. Only the ligation reaction used for transformation of the cells spread on plate 3 was actually working.

![](https://static.igem.org/mediawiki/2020/f/f7/T--Brno_Czech_Republic--ResultsFig5nezvetseny.jpg)
**Figure 5**

**Agarose gel DNA electrophoresis showing the results of the restriction cleavage of plasmids isolated from 12** ***E. coli*** **colonies****from the plate 3*****.*** Cells in colonies 2, 6, 8, 11, and 12 contained plasmid pDG3661\_IM. The first line marked M is gene marker GeneRuler 1 kb DNA Ladder. Using the restriction reaction, we excised the 1401 bp long fragment of the IM, which is located in the lower part of the gel and indicated by the blue arrow. These positive colonies were used for the subsequent transformation of competent cells of *B. subtilis*. Positive control - an empty plasmid pDG3661 - is located in line 13.

The following transformation of *B. subtilis* was successful only with the plasmids isolated from *E. coli* colonies 11 and 12 (experiment is described below), so we proceed with only these two *B. subtilis* transformants. Additionally, we also ran a gel where the isolated pDG3661\_IM were digested with only one restriction enzyme in order to demonstrate the exact size of the construct. The length of an empty pDG3661 can also be compared with the length of pDG3661\_IM (**Fig. 6**).

**Table 1. The length of each bend on the gel in Fig. 6**

|  |  |
| --- | --- |
| **Part of completed vector** | **Number of base pair** |
| Empty vector pDG3661 (Line 1 - **Fig. 6**) | 10407 |
| Immobilisation module (Line 1 - **Fig. 10**) | 1405 |
| pDG3661\_IM (Line 2 and 3 - **Fig. 6**) | 11789 |

##### Proof by sequencing

The presence of our synthetic sequence in the pDG3661 vector was also demonstrated by sequencing (**Fig. 7**). We used the commercial service of the company SEQme based in the Czech Republic. We then aligned the results with our synthetic sequence using the Benchling platform with MAFFT algorithm. You can find the alignment *[here](https://static.igem.org/mediawiki/2020/d/d9/T--Brno_Czech_Republic--PDF_results.pdf)*

The sequencing showed that the plasmids from colony 12 had a gap of about 1 kbp in the sequence of our synthetic gene. This was however not visible on the gel we ran before sending the results (**Fig. 6**). The samples we've sent for sequencing were newly isolated form the *E. coli* colonies 11 and 12. It is possible that the gab shown in sequencing was not present in the plasmids we isolated initially and used for the agarose gel. We have however decided to only use the colony 11 from now on.

![](https://static.igem.org/mediawiki/2020/7/79/T--Brno_Czech_Republic--ResultsFig7.jpg)
**Figure 7**

**Scheme of sequencing results.** The two sequencing results cover the whole synthetic sequence of the Immobilization module.

### Integration of the expression cassette with IM into the chromosome of *Bacillus subtilis*

#### Experimental design

In the next phase of wet lab experiments, we focused on the transformation of *B. subtilis* with pDG3661\_IM. Working with this bacterium was complicated as no one at our university had any previous experience with using it. It was not only new to us but also to our PIs. Despite this, we've managed to perform our experiments successfully. In order to share our experience with future iGEM teams, we've decided to write a handbook: **How to handle** ***Bacillus subtilis*****.** You can find it in the Contributions section.

The greatest thanks goes to the Laboratory of Microbial Genetics and Gene Expression of Dr. Krásný from Prague, who really helped us at the beginning.

The vector pDG3661 is designed to integrate into the chromosome of *B. subtilis*. After a routine transformation the presence of pDG3661\_IM in the chromosome of *B. subtilis* was demonstrated by selection of cells on solid medium with chloramphenicol and by amplifying a chromosome region with inserted expression cassette by PCR. For the purpose of the latter, chromosomal DNA was isolated from *B. subtilis* transformants. AmyE test is also an elegant method showing chromosomal integration. We also sent the PCR products for sequencing to verify the DNA sequence.

Now we have a functional methodology for the integration of synthetic DNA sequence in the chromosome of *B. subtilis*. In the future, we would like to continue and transform *B. subtilis* with other constructs containing the sequences for the rest of our proteins.

#### Material and methods

##### *Bacterial strains:*

We used our own naturally competent cells prepared from *B. subtilis* 168. The protocol for preparing *B. subtilis* competent cells can be found in our Experiment section.

##### *Kits, media and chemicals:*

We used the same kits and chemicals as mentioned above. For the AmyE test we used two layer agar plates, the first layer is LB agar with chloramphenicol and the second layer is special agar with supplement of starch (more information in the section [Experiment](https://2020.igem.org/Team%3ABrno_Czech_Republic/Experiments)).

##### *Transformation experiment*

Transformation experiment is also described in the experiment section in more detail.

1. Isolation of plasmid from positive colonies of *Escherichia coli*
   For transformation we used plasmids isolated from *E. coli,* which were also used for the restriction reaction proving the presence of our insert. The concentration was optimal.
2. Transformation of *Bacillus subtilis*
   We performed the transformation in LB medium for 1 h at 200 rpm and 37°C.
3. Isolation of chromosomal DNA from *Bacillus subtilis*
   Initially, we used 2 different methods, from which only one worked well. We used a physical method of breaking the cell wall with high and low temperatures.
4. Proof of integration of expression cassette with IM into *Bacillus subtilis* chromosome by PCR
   We did PCR reaction with primers complementary to the parts of the chromosome near the site of integration. The result is visible from the size of fragments separated by agarose gel electrophoresis.
5. Proof of integration of the expression cassette into *B. subtilis* chromosome by AmyE test
   We also proved the integration of construct by this simple and elegant visualization.
6. Sample preparation for sequencing
   We tried to sequence the PCR product of chromosomal DNA.

##### *AmyE test*

For the integration test we had to prepare a special two layer agar. The second layer contained a supplement of starch - more can be found in the section Experiment.

#### Results

In this section we would like to demonstrate the success of the integration of pDG3661\_IM into the chromosome of *B. subtilis* and the creation of a strain of *B. subtilis* producing the IM - the IM strain*.* We have verified the integration with two methods - results from agarose gel electrophoresis and AmyE test.

##### Testing competent cells

Initially, we worked with empty vectors to prove that they can be used to transform B. subtilis cells. We tried to produce and test our competent cells. We transformed them with empty vectors pDG3661 and pDG1664 and verified the selection of transformants. After several failed experiments, we optimized the condition of transformation and edited our protocols for the best results. For example, we confirmed that the best time of incubation after transformation is 60 min.

##### Transformation of *Bacillus subtilis*

We used plasmid isolated from the verified *E. coli* transformants from previous experiments (Fig. 4 - colonies 2, 6, 8, 11 and 12) to transform competent cells of *B. subtilis*. Cells were plated on plates with chloramphenicol and incubated overnight at 37°C. The next day we observed many colonies (**Fig. 8**). Now we needed to verify whether the cells in these colonies contain the integrated expression cassette. We chose 10 colonies from each plate for further work and we restreaked them on a fresh agar plate. For our positive control, we transformed the cells with empty plasmid pDG3661. We also plated cells which were not transformed as a negative control (**Fig.9**).

![](https://static.igem.org/mediawiki/2020/f/f3/T--Brno_Czech_Republic--ResultsFig8.jpg)
**Figure 8**

**Plates with** ***B. subtilis*** **transformants.** Plates were incubated at 37°C overnight. Cells, which were streaked on these plates, were transformed with plasmids pDG3661\_IM isolated from *E. coli* colonies 2, 6, 8, 11 and 12.

##### Isolation of chromosomal DNA

From 10 colonies on each plate with transformants, three were chosen for isolation of chromosomal DNA. We tried two different methods. At the end, the physical method, using the effect of very high and very low temperatures, worked better. After isolation, we measured the DNA concentration with NanoDrop. The concentrations of these samples were all around 150 ng/µl. We used this DNA as a template for PCR reaction.

##### Proof by PCR and agarose gel DNA electrophoresis

We performed a PCR reaction using isolated chromosomal DNA as a template. The primers we used were complementary to the chromosomal sequence in the proximity of the integration site. The PCR amplicons should thus contain a part of *B. subtilis* chromosome, the expression cassette with the synthetic gene of the IM and also *lacZ* gene and a part of vector pDG3661 for integration. The size of the amplicon should be 8074 bp as you can see in **Table 2**.

**Table 2. A comparison of length of each band on the gel shown in Fig. 10.**

|  |  |
| --- | --- |
| **Part of completed vector** | **Count of base pair** |
| Immobilisation module (Line 1) | 1405 |
| lacZ gene | 3060 |
| Expression cassette | 7708 |
| Amplified regions of chromosome on either side of the expression cassette | 158 + 208 |
| Total length of the amplicon (Line 2, 3, 4) | 8074 |
| Negative control → Part of the chromosome without insert (Line 5) | 2346 |

##### Proof by AmyE test

AmyE test is a very easy method of demonstrating integration into the chromosome of *B. subtilis*. Two layers agar is used for this method - the first layer is regular agar containing corresponding antibiotic and the second layer is thin agar with starch supplement. After the addition of a few crystals of iodine, the starch will turn dark blue. The integration site in the chromosome encodes 𝛼-amylase, which is able to degrade starch in the media. If the integration site is intact, the 𝛼-amylase is produced and a clear halo around the colonies can be observed after iodine treatment. If the integration into chromosome is successful and the sequence encoding 𝛼-amylase is disrupted by the insert, the enzyme is not produced and starch is not degraded (agar after iodine treatment is blue). In our experiment we used an AmyE test to confirm the transformation of our colonies of *B. subtilis* (**Fig. 11B**)*.* We compared our samples with *B. subtilis* 168, which was not transformed with any DNA (**Fig. 11A, 11C**).

![](https://static.igem.org/mediawiki/2020/thumb/f/fd/T--Brno_Czech_Republic--Results_11A.png/607px-T--Brno_Czech_Republic--Results_11A.png)
![](https://static.igem.org/mediawiki/2020/thumb/2/29/T--Brno_Czech_Republic--Results_11B.png/607px-T--Brno_Czech_Republic--Results_11B.png)

![](https://static.igem.org/mediawiki/2020/5/5b/T--Brno_Czech_Republic--Results_11C.png)

**Figure 11**

**Proof of chromosomal integration in** ***B. subtilis*** **by AmyE test.** In the Fig. 11A you can see the *Bacillus subtilis* 168 which was not transformed - you can see clear halo surrounding the colonies (in detail in Fig. 11C). The plate in the Fig. 11B contains bacteria which were transformed with verified pDG3661\_IM. The integration was successful as there was no halo visible on the plate. At the picture 11C you can see a clear halo from 11A in detail.

##### Proof by sequencing

First we isolated chromosomal DNA of the IM strain of *B. subtilis.* We used two different methods - physical and chemical. Only the physical method however was successful. This method relies on alternating high and low temperatures to break the cell wall. More information can be found in a corresponding [Protocol](https://2020.igem.org/Team%3ABrno_Czech_Republic/Protocols). Subsequently we amplified the part of the chromosome, which we wanted to send for sequencing. The PCR product was then separated from chromosomal DNA with agarose gel DNA electrophoresis. We performed gel extraction and afterwards we diluted the samples, added primers and sent the samples for the sequencing. Unfortunately, we did not get a clear read. We've brainstormed different ways to improve our sample preparation for the iGEM 2021.

### Characterization of Immobilization module

##### Experimental design

In the last few weeks of our wet lab work we tried to verify whether our IM strain produces and secretes the target protein - the Immobilization module - and if its production enhances the ability of the cells to bind cellulose.

To achieve this, we had to cultivate the cells in a medium that is favorable for extracellular protein production. During and after the cultivation we collected samples for SDS-PAGE and Western blot analysis to find out whether the target protein is produced in the transformed cells or not.

After the cultivation, we performed a set of experiments where we wanted to prove that the presence of IM would increase the cell's affinity for cellulose when compared to the original strain (control) without chromosomal insertion. Unfortunately we were not able to obtain any presentable results. We will continue with this experiment in iGEM 2021.

Calibration of the device with iGEM Measurement kit should precede this experiment. We however did not receive it this year and, due to a lack of time and the coronavirus situation, we also weren’t able to handover this material to another iGEM team. We have preformed the experiment in the final days of our lab work and we consider its result as unclear and inconclusive. Next year we will try to get the iGEM Measurement kit for calibration of the device in order to measure OD600 in time.

##### Material and methods

###### ***Bacterial strains:***

The engineered *B. subtilis* IM strain was tested. The original strain *B. subtilis* 168 served as a negative control.

###### ***Kits, media and chemicals:***

Two types of liquid media were used for cultivation, LB and 2x Mal medium (beneficial for extracellular protein production; for details see section Experiments). LB agar Petri dishes (with and without chloramphenicol) were used for detection of the number of colony forming units after the cellulose binding experiment. The B-PER reagent from ThermoFisher Scientific was used for preparation of cell free extracts. 8 M urea was used for an acquirement of the remaining insoluble protein. A Lysis buffer with 100 μM of PMSF (phenylmethylsulfonyl fluoride) was used for preparation of whole cell lysates. Coomassie Brilliant Blue R-250 was used for staining of the gel after SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis). PVDF membrane was used for the transfer. As a primary antibody rabbit polyclonal c-Myc (A-14) from Santa Cruz Biotechnology, INC. was used. As a secondary antibody anti-rabbit IgG, (H+L), conjugated to HRP, from Pierce # 31460 was used. For the visualization SuperSignal™ West Femto Maximum Sensitivity Substrate was used. Cellulose microbeads IONTOSORB MT 100 (50-80 µm) by Iontosorb were used to test the adherence of cells to cellulose. PBS buffer was used for washing the microbeads and to incubate the cells with microbeads. Plastic XL Columns from Agarose Bead Technologies were used to stabilize the connection between the cells and cellulose. For the complete list of solutions used in SDS-PAGE and Western blot analysis and their exact composition, see relevant protocol.

###### ***Cultivation experiment***

Overnight cultures (ON) of IM strain and control strain were prepared in liquid LB medium. From the ON culture we inoculated fresh LB medium and after it had grown into desired OD600, modified 2x Mal medium was inoculated with these cells. After several hours of cultivation we transferred cells into fresh modified 2x Mal medium. The culture was growing for 14 hours and then used for further experiments.

###### ***Sample collection for SDS-PAGE and Western blot***

We've tried a few different time intervals for the collection of samples. At first the samples were collected from overnight cultures growing in LB medium, secondly when the OD600 of cells in 2x Mal medium was 1.5 and finally after the cultures were incubated in 2x Mal medium for 24 hours We've also tried to collect different types of samples. Firstly cells were only centrifuged and washed with water. Secondly whole cell lysates were prepared with the PMSF lysis buffer and with the B-PER reagent. Cell pellets obtained after the usage of the B-PER reagent, where the insoluble proteins could be present, were resuspended in 8 M urea.

###### ***SDS-PAGE***

Standard SDS-PAGE of the collected samples was performed. Before the actual electrophoresis, Bradford protein assay was conducted to measure the amount of protein in collected samples. Some of the gels were stained with Coomassie blue. For more details and conditions of the experiment, check the [Experiment](https://2020.igem.org/Team%3ABrno_Czech_Republic/Experiments).

###### ***Western blot***

Proteins were transferred from the SDS-gel to the PVDF membrane. The quality of the transfer was checked by Ponceau S staining solution. Subsequently, the membrane was incubated with the first antibody, washed and incubated again with the second antibody. Signals were visualized afterwards. More details can be found in the Experiment section.

###### ***Preparation of cellulose microbeads***

The microbeads were washed three times and resuspended in the PBS buffer.

###### ***Cellulose binding experiment***

The cells growing in the second modified 2x Mal medium were mixed with cellulose microbeads and incubated for 1 hour while being mixed slowly. By sedimentation, microbeads were separated from the liquid and then the obtained fractions were separately inoculated onto LB agar plates and incubated overnight at 37°C. Another fraction of the pellet was applied on a plastic column and washed with PBS. The flowthrough was also inoculated on LB agar plates and incubated overnight.

#### Results

##### Cultivation experiment

For this experiment, we've used 2 different colonie of the IM strain. Both of these colonies were transformed with the pDG3661\_IM construct isolated from *E. coli* colony 11. We labeled them as clone 11.1 and 11.5. The original strain *B. subtilis* 168 without integration served as a negative control (C).

The OD600 of second cultivation in LB (**Table 3** and **Fig. 12**) and the first cultivation in modified 2x Mal medium (**Table 4** and **Fig. 13**) was measured every 30 minutes.

When cultivated in LB, the culture of clone 11.5 of the IM strain grew at a lower rate than the other two cultures. The cause of the slower growth rate of this culture is not known.

However, in the modified 2x Mal medium, the culture containing the control grew faster than cultures of the IM strain. This could be caused by higher metabolic demands of heterologous protein expression and might thus suggest successful production of the IM. This is however only an indication and cannot be used to draw any conclusions.

****Table 3. Growth (OD****600** **values) of the two clones of B. subtilis IM strain and control (C) strain without chromosomal insertion in LB medium.****

|  |  |  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- | --- | --- |
| **Sample** | **Time (min)** | | | | | | | |
| 0 | 30 | 60 | 90 | 120 | 150 | 180 | 210 |
| 11.1 | 0.05 | 0.057 | 0.066 | 0.098 | 0.161 | 0.337 | 0.581 | 1.390 |
| 11.5 | 0.05 | 0.065 | 0.067 | 0.081 | 0.114 | 0.196 | 0.345 | 0.878 |
| C | 0.05 | 0.053 | 0.060 | 0.086 | 0.135 | 0.284 | 0.544 | 1.238 |

![](https://static.igem.org/mediawiki/2020/2/2b/T--Brno_Czech_Republic--ResultsFig12.jpg)
**Figure 12**

**Growth (OD****600** **values) of the two clones of B. subtilis IM strain and control (C) strain without chromosomal insertion in LB medium.** The control strain grew almost as fast as the culture of the clone 11.1. The culture of the clone 11.5 grew significantly slower and after 210 minutes it reached the OD600 = 0.878, while the culture of clone 11.1 and the C strains reached the OD600 = 1.390 and 1.238, respectively.

**Table 4. Growth (OD****600** **values) of the two clones of B. subtilis IM strain and control (C) strain without chromosomal insertion in modified 2x Mal medium.**

|  |  |  |  |  |  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| **Sample** | **Time (min)** | | | | | | | | | | |
| 0 | 30 | 60 | 90 | 120 | 150 | 180 | 210 | 240 | 270 | 285 |
| 11.1 | 0.0048 | 0.006 | 0.013 | 0.021 | 0.043 | 0.083 | 0.197 | 0.481 | 0.960 | 1.350 | 1.610 |
| 11.5 | 0.0048 | 0.009 | 0.015 | 0.024 | 0.046 | 0.087 | 0.203 | 0.487 | 1.200 | 1.290 | 1.470 |
| C | 0.0048 | 0.008 | 0.017 | 0.034 | 0.081 | 0.171 | 0.393 | 0.801 | 1.570 | 1.630 | 1.850 |

![](https://static.igem.org/mediawiki/2020/d/dd/T--Brno_Czech_Republic--ResultsFig13.jpg)
**Figure 13**

**Growth (OD**600** **values) of the two clones of B. subtilis IM strain and control (C) strain without chromosomal insertion in modified 2x Mal medium.** This graph shows faster growth of the C strain compared to the IM strain. After 285 minutes, the control strain reached the OD600 = 1.850 while the cultures of clones 11.1 and 11.5 reached the OD600 = 1.610 and 1.470, respectively.**

##### SDS-page and Western blot

To demonstrate the expression of our gene coding the IM in *B. subtilis,* we decided to use SDS-page and Western blot. We tried optimizing our Western blot, using a different method of sample preparation, changing the antibody concentration and time of exposure and altering the Western blot design (see more at experiment section). But unfortunately we did not have enough time to completely optimise this method and, as shown in **Fig. 14**, the results are inconclusive.

The best results have been achieved using whole cell lysates obtained from the lysis with PMSF. They can be observed in columns 3 and 5 (**Fig. 13**). The position of the signal in columns 3 and 5, would approximately correlate to the size of the IM (46 kDa). However, we can also observe the signal in column 4, which represents the negative control.

##### Preparation of cellulose microbeads

To test the purity of the cellulose microbeads, we put them in the PBS buffer and inoculated this suspension onto the LB agar plates. After cultivation, two of the plates were empty and one was covered with colonies (**Fig. 15**). We think the third got contaminated during the manipulation or a mistake was made and another sample was put on that plate.

![](https://static.igem.org/mediawiki/2020/1/12/T--Brno_Czech_Republic--ResultsFig15.jpg)
**Figure 15**

**Plates with pelleted cellulosic microbeads in the PBS buffer.** Plates number 1 and 2 are clear while number 3 contains many colonies. It probably got contaminated during manipulation or a wrong sample was accidentally put on that plate.

##### Cellulose binding experiment

Before allowing the cells to bind to the cellulose microbeads, we prepared three dilutions of the second culture in the modified 2x Mal medium. We worked with cell suspensions of OD600 = 1, OD600 = 0.1 and OD600 = 0.01, strains 11.1 and 11.5, control strain (C) and microbeads (M).

The cells were mixed with the same volume of microbeads, incubated for 1 hour and left to sediment and form a pellet. Both the supernatant and the pellet were spread onto LB agar plates and incubated overnight.

The result obtained by plating the cellulose microbeads, on which *B. subtilis* cells should be immobilized, were inconclusive. There was a lawn of colonies on plates in **Fig. 16**, **Fig. 17** and some colonies also grew on one of the plates that was supposed to contain only the microbeads (**Fig. 15**). As we cannot see distinct colonies and calculate the number of colony forming units, these results are inconclusive. We would need to repeat this experiment with multiple different dilutions in order to get colonies which can be counted. However, we didn't get the opportunity to do this, due to covid 19.

![](https://static.igem.org/mediawiki/2020/c/c7/T--Brno_Czech_Republic--ResultsFig16.jpg)
**Figure 16**

**Plates counting cellulose microbeads with (presumably) attached cells of the IM strain.** Plates 1-3 contain the cultures of the clone 11.1 of OD600 = 0.01; 0.1; and 1, respectively. Plates 4-6 contain the cultures of the clone 11.5 of OD600 = 0.01; 0.1; and 1, respectively.

![](https://static.igem.org/mediawiki/2020/e/ed/T--Brno_Czech_Republic--ResultsFig17.jpg)
**Figure 17**

**Plates with pellets containing cellulose microbeads and cells of the control strain.** Plates 1-3 contain the control strain of OD600 = 0.01; 0.1; and 1, respectively.

Supernatant, which should only contain the cells that were not able to bind to cellulose, was also plated. **Fig. 18** shows the plates with the supernatant from cultures of the IM strain. In **Fig. 19**, there are plates streaked with the supernatant from the culture of the control strain and the supernatant with no cells (obtained from the incubation of microbeads with sterile PBS). As in the previous experiment, a lawn of colonies has formed on these plates and thus we cannot calculate the colony forming units. These results are thus inconclusive and the experiment should be repeated with multiple dilutions.

![](https://static.igem.org/mediawiki/2020/e/e6/T--Brno_Czech_Republic--ResultsFig18.jpg)
**Figure 18**

**Plates with supernatants left after connection of strains 11.1 and 11.5 to cellulose microbeads.** Plates 1-3 contain the cultures of the clone 11.1 of OD600 = 1; 0.1; and 0.01, respectively. Plates 4-6 contain the cultures of the clone 11.5 of OD600 = 1; 0.1; and 0.01, respectively.

![](https://static.igem.org/mediawiki/2020/0/0b/T--Brno_Czech_Republic--ResultsFig19.jpg)
**Figure 19**

**Plates with supernatants left after incubation of the control strain and sterile PBS with cellulose microbeads.** Plates 1-3 contain the C strain of OD600 = 1; 0.1; and 0.01, respectively. Plates 4-6 contain the PBS without any cells. There is some contamination on plate 6.

##### *What could be improved*

We performed these experiments in the last stage of our wet lab work and we ended up being severely impacted by the epidemiological situation in the Czech Republic. We were not able to replicate the cultivation experiment and any of the experiments that follow. However, we have planned some ways in which we can improve when we get to repeat them.

For the next cultivation, we will use another recipe for the 2x Mal medium, as we later discovered, that our medium was not prepared in the most common way. We will also not perform the second cultivation in 2x Mal medium but use the cells from the first cultivation in this medium when they grow to OD600 = 3.

As for the SDS-page and Western blot transfer, we could improve the concentration and the type of antibodies used. Instead of using the c-myc tag, we could use antibodies specific to our protein. In any case, we do not think that there is an issue in the Western blot design, rather we might not have optimized our protein expression protocol and sample processing. For expression we could optimize the type of medium and its pH during the cultivation of *B. subtilis*. We should also determine an optimal OD600 for the collection of samples. We should also select an ideal method of sample processing and optimize it. To summarize, the experiments of this part still need to be optimized and the results are inconclusive.

When redoing the cellulose binding experiment, we will work only with one dilution of the 2x Mal medium culture (OD600 = 0.1) and perform several dilutions of the samples before putting them on LB agar plates. We will also try to measure the volume of the microbeads as precisely as possible in order to determine the number of cells attached to one microbead.

### Conclusion

1. We have successfully cloned the synthetic sequence containing the Immobilization module into the plasmid pDG3661, creating our construct pDG3661\_IM. We have verified this using restriction digestion, long PCR and sequencing.
2. We have managed to propagate the pDG3661\_IM construct in *E. coli* and isolate a sufficient amount for further experiments.
3. We have optimized the protocol for the transformation of *B. subtilis* with plasmid pDG3661.
4. *B. subtilis* was successfully transformed and the integration of our expression cassette into a specific region of its chromosome was confirmed with longPCR and with the AmyE test.
5. We have also done a series of experiments to confirm the expression and functionality of IM in *B. subtilis.* However, we did not have enough time to optimize and repeat these experiments and get conclusive results due to covid 19.
6. We will continue working on our project in 2021. See you later!

![](https://static.igem.org/mediawiki/2020/b/b0/T--Brno_Czech_Republic--ResultsFig1.jpg)
**Figure 1**

**Scheme of part of the CYANOTRAP project, which we tried to achieve this year.** The *Bacillus subtilis* cells are connected to the cellulose microbead through the Immobilization module, which comprises the LysM, linker, and cellulose binding domain (CBM).

![](https://static.igem.org/mediawiki/2020/c/c4/T--Brno_Czech_Republic--ResultsFig2.jpg)
**Figure 2**

Scheme of our experimental workflow.

![](https://static.igem.org/mediawiki/2020/d/da/T--Brno_Czech_Republic--Results_Icon_Plate.svg)

![](https://static.igem.org/mediawiki/2020/e/e5/T--Brno_Czech_Republic--ResultsFig3.jpg)
**Figure 3**

**Testing the plasmid background.** On a plate number 1 are colonies of cells of *Bacillus**subtilis* which were transformed with circular vector pDG3661. On plate 2 are cells of *Bacillus subtilis* transformed with the same vector digested by 2 different restriction enzymes EcoRI and HindIII.

![](https://static.igem.org/mediawiki/2020/2/25/T--Brno_Czech_Republic--ResultsFig4.jpg)
**Figure 4**

**Result of the transformation experiment with** ***Escherichia coli*****.** The plate number 1 contains the competence control transformed with vector pUC19. Plates number 2 and 3 contain competent cells transformed with pDG3661\_IM. Plate number 4 shows the results from another ligation strategy which was completely unsuccessful.

![](https://static.igem.org/mediawiki/2020/thumb/9/97/T--Brno_Czech_Republic--ResultsFig6.jpg/289px-T--Brno_Czech_Republic--ResultsFig6.jpg.png)

**Figure 6**

**Agarose gel DNA electrophoresis of isolated pDG3661\_IM constructs.** The first line contains a marker (1kb GeneRuler by ThermoFisher scientific), line number 1 is the isolated vector without insert. Line 2 shows plasmid isolated from colony 11 and line 3 shows plasmid isolated from colony 12. Line 4 is the negative control.

![](https://static.igem.org/mediawiki/2020/0/0d/T--Brno_Czech_Republic--ResultsFig9.jpg)
**Figure 9**

**Positive and negative control for the experiment Transformation of** ***Bacillus subtilis*****.** Plate 1 is negative control as cells that were not transformed with the plasmid were plated there. Plate 2 is positive control made by transforming our cells with empty pDG3661.

![](https://static.igem.org/mediawiki/2020/b/b9/T--Brno_Czech_Republic--ResultsFig10.jpg)
**Figure 10**

**Agarose gel DNA electrophoresis of amplikons of the integration region of** ***B. subtilis*** **chromosome.** Line 1 contains the IM. The lines 2, 3 and 4 contain amplicons from the chromosomes of transformants of *Bacillus subtilis* (indicated by the blue arrow). Line number 5 contains negative control - an amplicon from a chromosome of *Bacillus subtilis* which was not transformed. M is a marker 1kb GeneRuler from ThermoFisher Scientific. The lengths of the fragments are explained in **Table 2**.

![](https://static.igem.org/mediawiki/2020/1/1e/T--Brno_Czech_Republic--ResultsFig14.jpg)
**Figure 14**

**Western blot analysis of expression of gene for IM module.** Line M represents a marker (PageRuler™ Prestained Protein Ladder 10 – 170 kDa). The marker is unfortunately shifted, due to wrongly polymerised gel. But from the observation of the shift, three visible bands should be localized around 46 kDa. Lines 1, 2 and 3 are PMSF treated whole cell lysates from the cultivation after 24 h. Line 1 is the negative control (B. subtilis without the gene for IM module), line 2 is clone 11.1 and line 3 is clone 11.5. Lines 4, 5 and 6 are PMSF treated whole cell lysates obtained from cells collected at the OD600 = 1.5 of the culture. Line 4 is the negative control, line 5 is the clone 11.1 and line 6 is the clone 11.5. Lines 7, 8 and 9 are cells, which were not lysed with lysis buffer, but only centrifuged and washed with water. Line 7 is the negative control, line 8 is clone 11.1 and line 9 is clone 11.5.