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, THEORY, SPECIFIC PROJECT DESIGN, DESIGNING OUR DEVICE, MODELING, PARTS, EXPERIMENT, RESULTS and PROTOCOLS.
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.
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.
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 .
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 . 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 .
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 . 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.
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.
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  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.
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).
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.
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.
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 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.
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.
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|
|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 and Protocols.
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.
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.
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 and Experiment.
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:
- We have learned how to design synthetic sequences for a given purpose and we have designed all of the sequences needed for our project.
- We have learned how to work with B. subtilis.
- 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.
- We have managed to propagate the pDG3661_IM construct in E. Coli and isolate a sufficient amount for further experiments.
- 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.
- We have optimized the protocol for the transformation of B. subtilis with plasmid pDG3661.
- We have also done a series of experiments to confirm the expression and functionality of IM in B. subtilis.
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) 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.
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!
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