Team:Brno Czech Republic/Design
Specific project design
Design of Basic Parts
When we began designing our project, we first needed to determine the desired function of our device. Then we had to find out which DNA sequences would be able to perform the functions we had in mind. At first, we focused on the coding sequences (CDS) of our proteins of interest. Afterwards, we had to find suitable tags for the detection of our protein on Western blot, and signaling sequences that would carry our proteins through the cell membrane and cell wall of our host organism. Finally, we had to decide which promoter, ribosome binding site (RBS) and terminator we would use for the expression of our synthetic genes. Here you can find all the Basic Parts used in CYANOTRAP.
Promoter (BBa_K3590000)
The Pveg promoter is the strongest currently known constitutive promoter of Bacillus subtilis. We chose this promoter as we want our host organism to produce the target proteins in a natural environment where the use of inducers is impossible. Dr. Krásný suggested this promoter based on the information in the article by Sojka et al. in 2011 [1].
Source: Accession: MK301203, nucleotides 57-85 [2]
Ribosome binding site R0 (BBa_K3590001)
RBS R0 is the strongest of the three RBSs of B. subtilis selected from the study published by Guiziou and colleagues in 2016 [3].
Source: Guiziou et al., 2016 [3]
Ribosome binding site R1 (BBa_K3590002)
RBS R1 is the middle one in terms of strength of the three RBSs of Bacillus subtilis selected from the study published by Guiziou and colleagues in 2016 [3].
Source: Guiziou et al., 2016 [3]
Ribosome binding site R2 (BBa_K3590003)
RBS R2 is the weakest of the three Bacillus subtilis RBSs selected from the study published by Guiziou and colleagues in 2016 [3].
Source: Guiziou et al., 2016 [3]
Terminator derived from Bacillus subtilis rrnO (BBa_K3590004)
TrrnO is a terminator derived from the rrn operon of Bacillus subtilis, which it encodes rRNA. It can be used as the final component of a transcription unit. However, according to Dr. Libor Krásný from Laboratory of Microbial Genetics and Gene Expression in Prague (Czech Republic), it is not necessary to put a terminator at the end of each transcriptional unit. In his experience, it does no harm to Bacillus subtilis if the transcription of a heterologous gene is not terminated by a terminator and is left to end spontaneously anywhere downstream the stop codon.
Source: Accession: CP052842.1, nucleotides 328469-328381 [4]
SacB signal peptide of Bacillus subtilis levansucrase (BBa_K3590005, BBa_K3590006,BBa_K3590007, BBa_K3590008)
SacB is a signal peptide which ensures the transportation of tagged peptide into the extracellular space. In CYANOTRAP, we use four different versions of this gene's DNA sequence in order to avoid unwanted homologous recombination between our synthetic genes when combined into one operon.
Source: BioBrick BBa_K2273063 designed by Henri Deda from iGEM17_TU_Dresden [5]
3x His-tag (BBa_K3590009, BBa_K3590010, BBa_K3590011)
3x His-tag is a sequence coding 18 histidines in a row. It is used for the detection and purification of tagged proteins in Western blotting or affinity chromatography. In CYANOTRAP, we use three different versions of this gene's DNA sequence in order to avoid unwanted homologous recombination between our synthetic genes when combined into one operon.
Source: This sequence has no source, it was created by randomly combining histidine codons (CAC, CAT).
Myc-tag (BBa_K3590012)
Myc-tag is a short peptide that is used for the detection and purification of tagged proteins in Western blotting or affinity chromatography.
The amino acid sequence of Myc-tag (EQKLISEEDL) was rewritten into a nucleotide sequence and codon-optimized for Bacillus subtilis.
Cellulose binding module (BBa_K3590013)
CBM stands for cellulose or carbohydrate-binding module. In nature, it is usually found as a part of a fusion protein with cellulolytic activity or as a part of a scaffoldin - a non-catalytic module that serves as a central part of a cellulosome.
Source: The original sequence of CBM was taken from the Scaf19L sequence published in the article by Vazana and colleagues in 2013 [6]. The sequence was codon-optimized for expression in Bacillus subtilis.
Cohesin derived from Acetivibrio cellulolyticus (BBa_K3590014)
Cohesin is a binding domain that attaches proteins containing a complementary dockerin domain to a protein scaffold (called scaffoldin) which is usually anchored to the surface of the cell. The whole protein complex is called the cellulosome. Thanks to the non-covalent interactions between cohesins and dockerins, enzymes are immobilized in close proximity with each other as well as close to the surface of the cell. The cohesin-dockerin interaction is species-specific. This cohesin is derived from Acetivibrio cellulolyticus and will interact only with the dockerin from the same species (BBa_K3590015).
Source: The original sequence of CohAc was taken from the Scaf19L sequence published in the article by Vazana and colleagues in 2013 [6]. The sequence was codon-optimized for expression in Bacillus subtilis.
Dockerin derived from Acetivibrio cellulolyticus (BBa_K3590015)
Dockerin is a binding domain, typically found in enzymes with cellulolytic activity, as it serves to immobilize them on a protein scaffold (scaffoldin) thanks to its interaction with complementary cohesin domain. The whole protein complex is called the cellulosome. Thanks to the non-covalent interactions between cohesins and dockerins, enzymes are displayed in close proximity with each other as well as close to the surface of the cell. The cohesin-dockerin interaction is species-specific. This dockerin is derived from Acetivibrio cellulolyticus and will interact only with the cohesin from the same species (BBa_K3590014).
Source: The original sequence of DocAc was taken from the Scaf19L sequence published in the article by Vazana and colleagues in 2013 [6]. The sequence was codon-optimized for expression in Bacillus subtilis.
Cohesin derived from Bacteroides cellulosolvens (BBa_K3590016)
Cohesin is a binding domain that attaches proteins containing a complementary dockerin domain to a protein scaffold (called scaffoldin) which is usually anchored to the surface of the cell. The whole protein complex is called the cellulosome. Thanks to the non-covalent interactions between cohesins and dockerins, enzymes are immobilized in close proximity with each other as well as close to the surface of the cell. The cohesin-dockerin interaction is species-specific. This cohesin is derived from Bacteroides cellulosolvens and will interact only with the dockerin from the same species (BBa_K3590017).
Source: The original sequence of CohBc was taken from the Scaf19L sequence published in the article by Vazana and colleagues in 2013 [6]. The sequence was codon-optimized for expression in Bacillus subtilis.
Dockerin derived from Bacteroides cellulosolvens (BBa_K3590017)
Dockerin is a binding domain, typically found in enzymes with cellulolytic activity, as it serves to immobilize them on a protein scaffold (scaffoldin) thanks to its interaction with complementary cohesin domain. The whole protein complex is called the cellulosome. Thanks to the non-covalent interactions between cohesins and dockerins, enzymes are displayed in close proximity with each other as well as close to the surface of the cell. The cohesin-dockerin interaction is species-specific. This dockerin is derived from Bacteroides cellulosolvens and will interact only with the cohesin from the same species (BBa_K3590016).
Source: The original sequence of DocBc was taken from the Scaf19L sequence published in the article by Vazana and colleagues in 2013 [6]. The sequence was codon-optimized for expression in Bacillus subtilis.
Cohesin derived from Clostridium thermocellum (BBa_K3590018)
Cohesin is a binding domain that attaches proteins containing a complementary dockerin domain to a protein scaffold (called scaffoldin) which is usually anchored to the surface of the cell. The whole protein complex is called the cellulosome. Thanks to the non-covalent interactions between cohesins and dockerins, enzymes are immobilized in close proximity with each other as well as close to the surface of the cell. The cohesin-dockerin interaction is species-specific. This cohesin is derived from Clostridium thermocellum and will interact only with the dockerin from the same species (BBa_K3590019).
Source: The original sequence of CohCt was taken from the Scaf19L sequence published in the article by Vazana and colleagues in 2013 [6]. The sequence was codon-optimized for expression in Bacillus subtilis.
Dockerin derived from Clostridium thermocellum (BBa_K3590019)
Dockerin is a binding domain, typically found in enzymes with cellulolytic activity, as it serves to immobilize them on a protein scaffold (scaffoldin) thanks to its interaction with complementary cohesin domain. The whole protein complex is called the cellulosome. Thanks to the non-covalent interactions between cohesins and dockerins, enzymes are displayed in close proximity with each other as well as close to the surface of the cell. The cohesin-dockerin interaction is species-specific. This dockerin is derived from Clostridium thermocellum and will interact only with the cohesin from the same species (BBa_K3590018).
Source: The original sequence of DocCt was taken from the Scaf19L sequence published in the article by Vazana and colleagues in 2013 [6]. The sequence was codon-optimized for expression in Bacillus subtilis.
3x LysM domain derived from Bacillus subtilis LytE (BBa_K3590020, BBa_K3590021)
The LysM domain is a small globular module made up of approximately 40 amino acid residues that got its name from the ever-present lysin motif. It can be found in a wide range of prokaryotic and eukaryotic organisms including Bacillus subtilis in proteins interacting with peptidoglycans or chitin (Letunic et Bork, 2017; [7]).
Source: Accession: U38819, nucleotides 515 to 1132 [8]. The sequence was codon-optimized for easier gene synthesis and a few codons have been changed in version 2 to avoid homologous recombination between the linkers in one construct.
Lysozyme derived from Bacillus licheniformis (BBa_K3590022)
Lysozyme is an enzyme which cleaves the peptidoglycan layer of the bacterial cell wall and thus kills the affected bacteria. This lysozyme is derived from Bacillus licheniformis. It has been shown that LysBL does not have a negative effect on B. subtilis but its activity against cyanobacteria is, to our knowledge, still unknown [9].
Source: Accession: CP034569, nucleotides 1476276 to 1477226 [10]
Lysozyme derived from Gallus gallus (BBa_K3590023)
Lysozyme is an enzyme which cleaves the peptidoglycan layer of the bacterial cell wall and thus kills the affected bacteria. This lysozyme is derived from Gallus gallus.
Source: BBa_K284001 [11]
Microvirin (BBa_K3590024)
Microvirin is a lectin that specifically binds to the lipopolysaccharides in the cyanobacterial cell wall.
Source: BBa_K1378003 [12]
MlrA (BBa_K3590025)
MlrA is a protease that linearises circular microcystins, a group of cyanobacterial toxins. MlrA is the first enzyme in the microcystin-degradation pathway.
Source: BBa_K1378001 [13]. The sequence was codon-optimized for expression in Bacillus subtilis.
MlrB (BBa_K3590026)
MlrB is a protease that further digests microcystins, a group of cyanobacterial toxins. MlrB is the second enzyme in the microcystin-degradation pathway.
Source: BBa_K2960007 [14]. The sequence was modified to get rid of undesired restriction sites.
MlrC (BBa_K3590027)
MlrC is a protease that further digests microcystins, a group of cyanobacterial toxins. MlrC is the third enzyme in the microcystin-degradation pathway.
Source: BBa_K2960008 [15]. The sequence was codon-optimized for expression in Bacillus subtilis.
Linker 1 (BBa_K3590028, BBa_K3590029, BBa_K3590030, BBa_K3590031)
Linkers are sequences connecting different modules of the same protein. The most common amino acids in likers are Pro, Ser, and Thr.
Source: The original sequence of L1 was taken from the Scaf19L sequence (where it is called Linker 2) published in the article by Vazana and colleagues in 2013 [6]. The sequence was codon-optimized for expression in Bacillus subtilis and a few codons have been changed in versions 2, 3 and 4 to avoid homologous recombination between the linkers in one construct.
Linker 2 (BBa_K3590032, BBa_K3590033)
Linkers are sequences connecting different modules of the same protein. The most common amino acids in likers are Pro, Ser, and Thr.
Source: The original sequence of L2 was taken from the Scaf19L sequence (where it is called Linker 3) published in the article by Vazana and colleagues in 2013 [6]. The sequence was codon-optimized for expression in Bacillus subtilis and a few codons have been changed in version 2 to avoid homologous recombination between the linkers in one construct.
Linker in front of a dockerin (BBa_K3590034, BBa_K3590035)
Linkers are sequences connecting different modules of the same protein. The most common amino acids in likers are Pro, Ser, and Thr.
Source: Our Secondary PI Dr. Pavel Dvořák told us the best amino acid composition and length for a linker connecting a dockerin with the protein attached to it. We chose the most suitable codons of those amino acids for Bacillus subtilis production and put the sequence together in the order our Secondary PI advised us. A few codons have been changed in the second version to avoid homologous recombination between the linkers in one operon.
Design of Composite Parts
After completing the list of our Basic Parts, we had to combine them into functional units. It was like taking single LEGO bricks of different colors and building something that makes sense. Only without the user manual.
The order of regulatory elements was clear - first comes a promoter and then an RBS. Then there is the coding sequence which ends with a STOP codon. On both ends of this sequence we added restriction sites (EcoRI at the 5' end and HindIII at the 3'end) so it can be cloned into the ectopic integration plasmids pDG3661 and pDG1664. When designing our constructs, we used codon optimization to avoid the EcoRI and HindIII sites within the coding sequence.
Our expression cassettes do not contain a terminator because Dr. Krásný advised us not to use it (based on his vast experience with gene expression in B. subtilis). We obtained one plasmid from Dr. Krásný, that already contains a terminator sequence and if the expression did not work as is, it would be possible to clone it at the 3' end of our expression cassette. It could be done through the BamHI restriction site, conveniently placed near the 3' end of the region where we want to insert our construct.
When creating the constructs, we made some mistakes at first - like putting the tags in front of the signal peptide sequence, which would have resulted in the loss of the tag. Fortunately, our PIs checked everything we prepared and made us aware of every problem. So we managed to build our own set of synthetic gene cassettes - the Composite Parts of CYANOTRAP.
Immobilization module - IM (BBa_K3590036)
The purpose of the Immobilization module (IM; Fig. 1) is to immobilize the cells of B. subtilis on a cellulose matrix. This construct was designed to ensure that the genetically modified bacteria will not escape our floating device and endanger the environment.
At the 5' end of the IM there is the Pveg promoter followed by the RBS R2. The coding sequence (CDS) encodes several functional modules - SacB signal sequence for extracellular transport, Myc-tag for Western blot detection, followed by the cellulose binding module (CBM) through which it will attach to the microbeads in our device. The CBM is connected by a linker to three copies of the LysM domains which anchor the whole system to peptidoglycans in the cell wall of B. subtilis. At the 3' end of the CDS, there is a STOP codon.
Figure 1. The Immobilization module. Synthetic DNA sequence (top) and synthetic protein (bottom).
Scaffoldin for lysis of cyanobacteria - ScafL (BBa_K3590037)
ScafL (Fig. 2) was designed to display proteins that would work together to lyse overpopulated cyanobacteria in affected water basins.
At the 5' end of ScafL there is the Pveg promoter followed by RBS R2. The CDS encodes several functional modules - the SacB signal sequence for extracellular transport, the His-tag for Western blot detection, followed by microvirin which is able to bind to the cyanobacterial cell wall and thus keep the target cells close to our host organism and to lysozyme. The microvirin module is connected by a linker to cohesin from Clostridium thermocellum which will interact with its dockerin counterpart connected to our lysozyme and thus attach this enzyme to the protein scaffold. Another linker then connects the previous parts of the fusion protein to three LysM domains that anchor the whole system to peptidoglycans in the cell wall of B. subtilis. At the 3' end of the CDS, there is a STOP codon.
Figure 2. Scaffoldin for lysis of cyanobacteria - ScafL. Synthetic DNA sequence (top) and synthetic protein (bottom).
Lysozyme derived from Bacillus licheniformis fused with dockerin derived from Clostridium thermocellum - LysBL-DocCt(BBa_K3590038)
This synthetic construct was designed to lyse cyanobacterial cell walls while being displayed on ScafL.
At the 5' end of LysBL-DocCt (Fig. 3) there the Pveg promoter followed by RBS R1. The CDS encodes several functional modules - SacB signal sequence for extracellular transport, His-tag for Western blot detection followed by lysozyme derived from Bacillus licheniformis (LysBL). It has been shown that LysBL does not have a negative effect on B. subtilis but its activity against cyanobacteria is, to our knowledge, still unknown. The LysBL module is followed by a linker connecting it to the dockerin domain of Clostridium thermocellum. This dockerin interacts exclusively with a cohesin domain originating from the same species so it ensures the attachment of this whole fusion protein to ScafL. At the 3' end of the CDS, there is a STOP codon.
Figure 3. Lysozyme derived from Bacillus licheniformis fused with dockerin derived from Clostridium thermocellum - LysBL-DocCt. Synthetic DNA sequence (top) and synthetic protein (bottom).
Lysozyme derived from Gallus gallus fused with a dockerin derived from Clostridium thermocellum - LysGG-DocCt(BBa_K3590039)
This synthetic construct was designed to lyse the cyanobacterial cell wall while being connected to the ScafL.
At the 5' end of LysGG-DocCt (Fig. 4) there is the Pveg promoter followed by RBS R1. The CDS encodes several functional modules - SacB signal sequence for extracellular transport, His-tag for Western blot detection followed by lysozyme derived from Gallus gallus (LysGG). It has been shown that LysGG lyses the cyanobacterial cell wall. The LysGG module is followed by a linker that connects it to the dockerin domain of Clostridium thermocellum. This dockerin interacts exclusively with a cohesin domain originating from the same species, ensuring the attachment of this fusion protein to ScafL. At the 3' end of the CDS, there is a STOP codon.
Figure 4.Lysozyme derived from Gallus gallus fused with a dockerin derived from Clostridium thermocellum - LysGG-DocCt. Synthetic DNA sequence (top) and synthetic protein (bottom).
Scaffoldin for degradation of microcystin MC-LR - ScafD (BBa_K3590040)
ScafD (Fig. 5) was designed to display enzymes MlrA, B and C from the microcystin degradation pathway. Thanks to the close proximity of those enzymes, displayed on this scaffoldin, microcystin degradation will be more efficient compared to freely released enzymes.
At the 5' end of ScafD there is the Pveg promoter followed by RBS R2. The CDS encodes several functional modules - SacB signal sequence for extracellular transport, His-tag for Western blot detection followed by the MlrA enzyme that linearizes the circular molecule of MC-LR. The MlrA module is connected by a linker to a cohesin molecule that originates from Bacteroides cellulosolvens which is followed by another linker and cohesin from Acetivibrio cellulolyticus. Those cohesin domains ensure the attachment of MlrB and MlrC enzymes to the ScafD, respectively. The attachment of these enzymes is achieved through a species-specific interaction of cohesins and their counterparts - dockerins, which are fused with said enzymes. Another linker then connects the previous parts of the fusion protein to three LysM domains that anchor the whole system to peptidoglycans in the cell wall of B. subtilis. At the 3' end of the CDS, there is a STOP codon.
Figure 5. Scaffoldin for degradation of microcystin MC-LR - ScafD. Synthetic DNA sequence (top) and synthetic protein (bottom).
Enzyme MlrB derived from Sphyngopyxis sp. fused with a dockerin derived from Bacteroides cellulosolvens - MlrB-DocBc (BBa_K3590041)
MlrB is the second enzyme in the microcystin degradation pathway. The purpose of the MlrB-DocBc (Fig. 6) is to degrade the product of the reaction catalyzed by MlrA into even shorter linear peptides and thus contributing to microcystin degradation. This enzyme should fulfill its task while being displayed on ScafD.
At the 5' end of MlrB-DocBc there is the Pveg promoter followed by the RBS R1. The CDS encodes several functional modules - SacB signal sequence for extracellular transport, His-tag for Western blot detection followed by the MlrB module, the function of which is described above. MlrB is connected by a short linker to a dockerin of Bacteroides cellulosolvens. This dockerin interacts exclusively with a cohesin domain originating from the same species ensuring the attachment of this fusion protein to the specific position on ScafD. At the 3' end of the CDS, there is a STOP codon.
Figure 6. Enzyme MlrB derived from Sphyngopyxis sp. fused with a dockerin derived from Bacteroides cellulosolvens - MlrB-DocBc. Synthetic DNA sequence (top) and synthetic protein (bottom).
Enzyme MlrC derived from Sphyngopyxis sp. fused with a dockerin derived from Acetivibrio cellulolyticus - MlrC-DocAc (BBa_K3590042)
MlrC is the third enzyme of the microcystin degradation pathway. The purpose of MlrC-DocAc (Fig. 7) is to further degrade the products of MlrA and MlrB enzymatic activity into even shorter linear peptides and amino acids (one of its products is ADDA). It thus contributes to the reduction of microcystin induced toxicity. The enzyme should fulfill its task while being displayed on the ScafD.
At the 5' end of MlrC-DocAc there is the Pveg promoter followed by the RBS R0. The CDS encodes several functional modules - SacB signal sequence for extracellular transport, His-tag for Western blot detection followed by the MlrC module, the function of which is described above. MlrC is connected by a short linker to the dockerin from Acetivibrio cellulolyticus. This dockerin interacts exclusively with a cohesin domain originating from the same species so it ensures the attachment of this fusion protein to the specific position on ScafD. At the 3' end of the CDS, there is a STOP codon.
Figure 7. Enzyme MlrC derived from Sphyngopyxis sp. fused with a dockerin derived from Acetivibrio cellulolyticus - MlrC-DocAc.Synthetic DNA sequence (top) and synthetic protein (bottom).
Vectors used in CYANOTRAP
In our project we worked with two vectors, which were recommended by the Laboratory of Microbial Genetics and Gene Expression and Krásný Ph.D from Prague. Our plasmids are synthetic vectors designed especially for ectopic integration of genes into the genome of Bacillus subtilis. We used vectors pDG1664 (Fig. 8) and pDG3661 (Fig. 9). They are “shuttle vectors” which means that they can be used in both Escherichia coli and Bacillus subtilis, although in each of them there will be a different system for selecting transformed cells. The origin of replication is recognized only in Escherichia coli and the plasmids cannot be replicated in Bacillus subtilis. As a consequence, these plasmids can be propagated as episomes in E. coli, but must be integrated into the Bacillus subtilis chromosome immediately after transformation to confer antibiotics resistance. The integration takes place through a double recombination event.
In our experiments we used Escherichia coli strain DH5α commercial competent cells and Bacillus subtilis 168.
Every method mentioned here is also described in the Experiment section in greater detail.
pDG1664
Figure 8. Vector pDG1664.
Published: Guérout-Fleury et al.,1996 [16]
GenBank identificator: U46201.1 [17]
We decided to use pDG1664 for the majority of our project. We planned to clone 6 of our 7 synthetic genes into this vector. This year we tried inserting the gene sequence into our vectors using Restriction cloning. After a few unsuccessful experiments we decided to prioritize the successful transformation of Bacillus subtilis and our main focus shifted to vector pDG3661 described below.
This plasmid contains 3 genes coding antibiotic resistance - gene erm (resistance to erythromycin), gene spc (resistance to spectinomycin) and gene bla (resistance to ampicillin). The gene of interest is cloned into restriction sites for EcoRI and HindIII in the middle of the thrC gene, which is also responsible for chromosomal integration in Bacillus subtilis. Transformants of Escherichia coli are easily detectable by their resistance to ampicillin. We are able to select the successfully transformed cells of Bacillus subtilis using a combination of antibiotics erythromycin and lincomycin. This is known as MLS selection. There are several methods we used to validate that the expression cassette was successfully integrated into chromosome colonyPCR, restriction cleavage or sequencing.
pDG3661
Figure 9. Vector pDG3661.
Published: Krásný et Gourse, 2004 [18]
GenBank identificator: AY618310.1 [19]
The vector pDG3661 is 10407 bp long and we used it for the cloning of the Immobilization module, which was the construct we decided to focus on in the end. This plasmid also contains the spoVG-lacZ gene, which came to be by fusing Bacillus subtilis genes spoVG and lacZ. This sequence can be replaced by different recombinant DNA using the BamHI restriction site. This gene can also remain in the plasmid and serve as an indicator gene for modified Blue-White selection. Our construct gets integrated into the amyE gene. The selection of transformed Escherichia coli is done using the bla gene which codes ampicillin resistance. Spc gene and cat gene, which encode resistance to antibiotic chloramphenicol, are used for the selection of transformed Bacillus subtilis cells. The sequence of the Immobilization module was inserted in front of the spoVG-lacZ gene. Integration into the chromosome will happen in amyE sequence so the cat gene and 3060 bp long spoVG-lacZ gene are inserted as well as our synthetic Immobilization module.
Modified Blue-White selection is in our case a very easy way of determining the success of chromosomal integration. When Xgal is added to LB agar on agar plates, the Bacillus subtilis colonies with spoVG-lacZ gene turn blue and the colonies without our insert stay colourless. As the Immobilization module is integrated together with the spoVG-lacZ gene, which encodes β-galactosidase, transformants are able to turn a medium containing Xgal blue. Escherichia coli with pDG3661 will also be able to change the colour of the media to blue, but in our case we would not be able to distinguish colonies with empty plasmids from the ones containing our inserts. In order to determine the success of cloning our insert into this plasmid we used restriction cleavage. Unfortunately, there is a STOP codon between our gene and the lacZ gene, so the enzyme crucial for Blue-white selection is currently not being produced even in the case of successful integration We will thus need to add a RBS before the lacZ gene to make this selection method work.
We also used the AmyE test to prove that the integration into the Bacillus subtilis chromosome took place. Our construct is inserted into the amyE sequence, which encodes α-amylase. In the case of unsuccessful integration, the starch in agar medium gets degraded by this enzyme and the addition of a few crystals of iodine leads to the surrounding medium getting brighter, creating a clear halo. Medium in the proximity of colonies where the chromosomal integration did not take place is much brighter then in the proximity of colonies where our gene got integrated successfully.
Figure 10. Positive result of AmyE test.
Figure 11. Negative result of AmyE test.
Primers
A primer is a short single stranded DNA molecule, usually containing somewhere between 15 and 30 base pairs. It contains a sequence complementary to 3’ or 5’ terminus of the sequence that is being amplified. It can also contain some additional features based on the purpose for which this primer is used.
When designing our primers, we visualised our constructs in Benchling and used almost every tool this software offers for this task. The region of the template to which each primer should anneal to was different, depending on the purpose of the primer, but the workflow was always the same. We chose the target region and then searched for a sequence that would have a GC clamp at the 5’ end and that would be as long as possible while still keeping the melting point between 55 and 60 ºC. After we selected the sequence, we checked the energy values of possible secondary structures. If the ΔG was lower than -10 kcal (or really close to it) we tried to design a different primer, but in some cases it was not possible. Afterwards, we checked the secondary structures of primer pairs that would be used together in one reaction. The results evaluation was the same as with a single primer. After the primers were designed, we analyzed their sequences in the NEB Tm Calculator and used the Tm values calculated by this software. We strived to design the primer pairs used in the same reaction to have the Tm as close to each other as possible.
We ordered the primers from IDT. When the primers arrived, we diluted them in nuclease free water according to the manufacturer’s instructions. This way, we created 100 µM stock samples and from those we then prepared 10 µM samples that were ready to be used.
In our project, we needed four different sets of primers for different purposes. They are listed in Table 1. The name of each primer reflects its purpose as well as the plasmid (either pDG3661 or pDG1664) and the construct it anneals to. In the case of ColonyPCR primers it also indicates the bacterial species that this primer works for.
Table 1. Primers designed for CYANOTRAP
References
- Sojka L., Kouba T., Barvík I., Šanderová H., Maderová Z., Jonák J., and Krásný L. Rapid changes in gene expression: DNA determinants of promoter regulation by the concentration of the transcription initiating NTP in Bacillus subtilis. Nucleic Acids Res. 39 (11): 4598-4611. DOI: 10.1093/nar/gkr032
- https://www.ncbi.nlm.nih.gov/nuccore/MK301203
- Guiziou S., Sauveplane V., Chang H. J., Clerté C., Declerck N., Jules M. and Bonnet J. 2016. A part toolbox to tune genetic expression in Bacillus subtilis. Nucleic Acid Res. 44 (15): 7495–7508. DOI: 10.1093/nar/gkw624
- https://www.ncbi.nlm.nih.gov/nuccore/CP052842
- http://parts.igem.org/Part:BBa_K2273063
- Vazana Y., Barak Y., Unger T., Peleg Y., Shamshoum M., Ben-Yehezkel T., Mozor Y., Shapiro E., Lamed R. and Bayer E. A. 2013. A synthetic biology approach for evaluating the functional contribution of designer cellulosome components to deconstruction of cellulosic substrates. Biotechnol. Biofuels 6: 182. DOI: 10.1186/1754-6834-6-182
- Letunic I. and Bork P. 2017. 20 years of the SMART protein domain annotation resource. Nucleid Acids Res. 46: D493–D496. DOI: 10.1093/nar/gkx922
- https://www.ncbi.nlm.nih.gov/nuccore/U38819
- Zhang H., Fu G. and Zhang D. 2014. Cloning, characterization, and production of a novel lysozyme by different expression hosts. J Microbiol Biotechnol. 24(10): 1405–1412. DOI:10.4014/jmb.1404.04039
- https://www.ncbi.nlm.nih.gov/nuccore/NZ_CP034569.1
- http://parts.igem.org/Part:BBa_K284001
- http://parts.igem.org/Part:BBa_K1378003
- http://parts.igem.org/Part:BBa_K1378001
- http://parts.igem.org/Part:BBa_K2960007
- http://parts.igem.org/Part:BBa_K2960008
- Guérout-Fleury A. M., Frandsen, N., and Stragier, P. 1996. Plasmids for ectopic integration in Bacillus subtilis. Gene 180(1-2): 57–61. DOI: 10.1016/s0378-1119(96)00404-0
- https://www.ncbi.nlm.nih.gov/nuccore/1185593
- 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://www.ncbi.nlm.nih.gov/nuccore/AY618310.1
