Team:Brno Czech Republic/Results
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.
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) 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.
- 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. - 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. - Ligation
The ligation reaction ran for 15 minutes at room temperature. - 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. - 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. - 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. - 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.
Figure 5
Agarose gel DNA electrophoresis showing the results of the restriction cleavage of plasmids isolated from 12 E. coli coloniesfrom 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. 6Proof 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
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.
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).
Transformation experiment
Transformation experiment is also described in the experiment section in more detail.
- 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. - Transformation of Bacillus subtilis
We performed the transformation in LB medium for 1 h at 200 rpm and 37°C. - 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. - 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. - 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. - 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).
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.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).
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. 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.
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 (OD600 values) of the two clones of B. subtilis IM strain and control (C) strain without chromosomal insertion in LB medium.
Figure 12
Growth (OD600 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 (OD600 values) of the two clones of B. subtilis IM strain and control (C) strain without chromosomal insertion in modified 2x Mal medium.
Figure 13
Growth (OD600 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.
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.
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.
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.
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.
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
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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.
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We have managed to propagate the pDG3661_IM construct in E. coli and isolate a sufficient amount for further experiments.
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We have optimized the protocol for the transformation of B. subtilis with plasmid pDG3661.
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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.
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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.
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We will continue working on our project in 2021. See you later!
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).
Figure 2
Scheme of our experimental workflow.
Figure 3
Testing the plasmid background. On a plate number 1 are colonies of cells of Bacillussubtilis 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.
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.
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.
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.
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.
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.