Team:Brno Czech Republic/Design

Design

Design



Figure 1.: Schema of our experimental approach


Organisms used in our project


We have selected B. subtilis to be the target organism for the expression and utilization of our composite parts. We obtained the strain B. subtilis168 from Dr. Krásný who works with this bacteria regularly and with whom we have consulted various aspects of our project. Additionally, we used E. coli JM109 to replicate and store our plasmids.


We have however presumed that the gene of the repressor lacI which is necessary for IPTG induction was already integrated into the chromosome of the B. subtilis 168 as we know that IPTG induction is commonly used with this strain. That was however not the case, so in order to use IPTG induction, we would have to transform our B. subtilis 168 strains with lacl as well. We found this out too late and we were thus unable to test out the functionality of our system in B. subtilis fully. For this purpose, we also transformed expression strain E. coli BL21 (DE3) with our constructs as this strain already carries lacI in its chromosome.


Composite part A = BMC shell proteins


This composite part was designed to be used to test and characterise the production and assembly of bacterial microcompartments BMCs inBacillus subtilis. Its main components are the five shell proteins of Pdu BMC from bacteria Parageobacilus thermoglucosidasius. These genes were placed under a promoter inducible by IPTG for the purpose of verifying their production in B. subtilis.


Additionally, this construct was designed in a way that would allow for the BMC genes and their respective ribosomal binding sites to be cleaved out by restriction enzymes and subsequently ligated to the alteret Pgrac promoter in order to assemble composite part Z.


BASIC PARTS

Terminators


Terminator (BBa_B0014)

This is a bidirectional double terminator consisting of terminators BBa_B0012 and BBa_B0011.

Source: Bba_B0014


Terminator (BBa_B0015)

This is a forward double terminator, consisting of terminators BBa_B0010 and BBa_B0012.

Source: Bba_B0015


Spacers


Spacers were placed between terminators and the rest of the composite part at both ends.


Spacer_0 (BBa_K3831010)

This spacer corresponds to the spacer sp0 from a study by Guiziou et al. in 2016.

Source: Guiziou et al., 2016 [1]


Spacer_1, modified (BBa_K3831011)

We used the first 15 bp from the spacer sp1 from a study by Guiziou et al. in 2016.

Source: Guiziou et al., 2016 [1]


Promoter


Pgrac promoter (BBa_K3831035)

The Pgrac promoter was created by combining the groE promoter, gsiBSD sequence and lacO operator to allow for IPTG induction.

Source: Phan et al., 2005 [2]


Source: BBa_K3831035


Ribosomal binding sites


Synthetic Ribosome binding site RBS_a (BBa_K3831005)

Designed with RBS calculator


Synthetic Ribosome binding site RBS_b (BBa_K3831006)

Designed with RBS calculator


Ribosome binding site RBS R2 (BBa_K3831007)


Source: Guiziou et al., 2016 [1]


Ribosome binding site RBS R6 (BBa_K3831008)

Source: Guiziou et al., 2016 [1]


Synthetic Ribosome binding site RBS_c (BBa_K3831009)

Designed with RBS calculator


BMC shell proteins


We opted for the heterologous production of BMC shell proteins from the Pdu operon of the bacteria Parageobacillus thermoglucosidasius which is closely related to B. subtilis. Heterologous production and assembly of Pdu BMC in B. subtilis has been demonstrated in a study by Wade et al. (2019) [3], where they have identified five essential shell proteins. All of the Pdu shell proteins were codon-optimized for expression in B. subtilis.


Pdu shell protein derived from P. thermoglucosidasius PduA (BBa_K3831000)

Source: Wade et al. (2019) [3]


Pdu shell protein derived from P. thermoglucosidasius PduB (BBa_K3831001)

Source: Wade et al. (2019) [3]


Pdu shell protein derived from P. thermoglucosidasius PduK (BBa_K3831003)

Source: Wade et al. (2019) [3]


Pdu shell protein derived from P. thermoglucosidasius PduJ (BBa_K3831002)

Source: Wade et al. (2019) [3]


Pdu shell protein derived from P. thermoglucosidasius PduN (BBa_K3831004)

Source: Wade et al. (2019) [3]


Composite part A (BBa_K3831030)


Bidirectional terminator BBa_B0014 is located at the 5' end of composite part A to prevent the activation of our genes from a native promoter in the chromosome of B. subtilis. This terminator is separated from the inducible Pgrac by Spacer_0.


Five genes encoding the shell proteins of Pdu BMC from P. thermoglucosidasius are placed downstream of the Pgrac promotor with their corresponding ribosomal binding site in the following order. Translation rate for every sequence and RBS was predicted using an online RBS calculator .


RBS_a + PduA (Translation rate = 58 368.33)

RBS_b + PduB (Translation rate = 67 646.67)

PBS_R2 + PduJ (Translation rate = 57 863.55)

RBS_R6 + PduK (Translation rate = 9 221.97)

RBS_c + PduN (Translation rate = 5 343.89)


Translation rates for PduK and PduN are significantly lower than for the rest of the BMC shell proteins. This should not be an issue as in the Pdu BMCs of S. enterica, which is highly similar to the Pdu BMCs of P. thermoglucosidasius, PduK and PduN are present less than PduA, PduB and PduJ [3].


Forward double terminator BBa_B0015 was placed at the 3' to prevent activation of genes located downstream from the composite part. It was separated from the rest of the sequence by spacer_1 BBa_K3831011.


Due to higher degree of similarity in the sequences of the shell proteins of Pdu BMC, this construct could not be synthesized as a whole. It was thus split into two constructs which were then optimised for Golden Gate cloning to allow for them to be ligated together once they have arrived. However, this has caused a bit of a delay. In the end, the synthesized sequences arrived too late and we were unable to proceed with golden gate cloning due to time constraints.
Figure 2.: Schema of composite part A.


References


  1. 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.


  1. Phan, T. T., Nguyen, H. D., & Schumann, W. (2006). Novel plasmid-based expression vectors for intra- and extracellular production of recombinant proteins in Bacillus subtilis. Protein expression and purification, 46(2), 189–195.


  1. Wade Y., Daniel R. A., Leak D. J. 2019. Heterologous Microcompartment Assembly in Bacillaceae: Establishing the Components Necessary for Scaffold Formation. ACS Synth. Biol. 8: 1642-1654.


  1. Kerfeld C. A., Sutter M. 2020. Engineered bacterial microcompartments: apps for programing metabolism. Curr. Opin. Biotechnol. 65: 225-232.



Composite part B = Encapsulation of protein into BMC


This composite part was designed to test and characterise the encapsulation of a protein into assembled bacterial microcompartments (BMC) in Bacillus subtilis. Its main components are sfGFP derived from Aequorea victoria and leading short peptide sequence (Pdu-tag), which should guide GFP into the BMC during it’s assembling. These genes were placed under a promoter inducible by IPTG for the purpose of verifying their production in B. subtilis.


The sfGFP could later on be replaced by an enzyme polyphosphate kinase (PPK), which should then behave similarly to this reporter and thus be encapsulated in the BMC. This enzyme links phosphates into polyphosphates which would then remain inside of the BMCs and thus increase phosphate accumulation by B. subtilis. In our project we have not reached this step as we have not yet been able to verify the production and formation of BMCs.


BASIC PARTS


Terminators


Terminator (BBa_B0014)

This is a bidirectional double terminator consisting of terminators BBa_B0012 and BBa_B0011.


Source: Bba_B0014


Spacers


Spacers were used to separate terminators from the rest of our composite part.


Spacer_5 (BBa_K3831012)

This spacer corresponds to the spacer sp5 from a study by Guiziou et al. in 2016.

Source: Guiziou et al., 2016 [1]


Spacer_7, modifies (BBa_K3831015)

We used the first 15 bp from the spacer sp7 from a study by Guiziou et al. in 2016.

Source: Guiziou et al., 2016 [1]


Promoter


Pgrac promoter (BBa_K3831035)

The Pgrac promoter was created by combining the groE promoter, gsiBSD sequence and lacO operator to allow for IPTG induction.

Source: Phan et al., 2005 [2]


Source: BBa_K3831035


Ribosomal binding sites


Ribosome binding site RBS R1 (BBa_K3831014)

This native ribosomal binding site from B. subtilis was taken from a study by Guiziou et al. from 2016.

Source: Guiziou et al., 2016 [1]


BMC guiding tag PduP


We opted for the heterologous production of BMC shell proteins from the Pdu operon of the bacteria Parageobacillus thermoglucosidasius which is closely related to B. subtilis. Heterologous production and assembly of Pdu BMC in B. subtilis has been demonstrated in a study by Wade et al. (2019) [3], where they have used 24 N-terminal amino acid residues from PduP protein as a tag to encapsulate protein inside of Pdu BMCs. PduP tag binds to C-terminal helixes of shell proteins of BMC and mediate incorporation of fused protein, in our case sfGFP, into the BMC.


Pdu BMC guiding peptide derived from N-terminal sequence of PduP of P. thermoglucosidasius (BBa_K3831017)

Source: Wade et al. (2019) [3]


Linker


Linker was used to connect the PduP tag and sfGFP protein.


GGGGS-linker (BBa_K3831013)


Source: Chen et al., 2013


sfGFP


Superfolder GFP is a basic green fluorescent protein derived from Aequorea victoria.


Superfolder green fluorescent protein(BBa_K3831016)


Source: Pédelacq et al., 2006


Composite part B (BBa_K3831031)


Bidirectional terminators BBa_B0014 are located at the 5' and 3' ends of composite part B to prevent the activation of our genes from a native promoter in the chromosome of B. subtilis and to prevent activation of genes located downstream from the composite part. These terminators are separated from the inducible Pgrac by Spacer_5 and from sfGFP by Spacer_7.


The coding sequence is placed downstream of the Pgrac promotor with corresponding ribosomal binding site. The PduP tag derived from the Pdu operon of P. thermoglucosidasius is connected to sfGFP by a GGGGS-linker. The translation rate of this fusion protein is predicted to be 9 221.97 by RBS calculator.
Figure 3.: Schema of composite part B.


Sources


CHEN, Xiaoying, Jennica L. ZARO a Wei-Chiang SHEN. Fusion protein linkers: Property, design and functionality. Advanced Drug Delivery Reviews [online]. 2013, 65(10), 1357-1369 [cit. 2021-10-7]. ISSN 0169409X. Accessible from; : doi:10.1016/j.addr.2012.09.039.


Wade Y., Daniel R. A., Leak D. J. 2019. Heterologous Microcompartment Assembly in Bacillaceae: Establishing the Components Necessary for Scaffold Formation. ACS Synth. Biol. 8: 1642-1654.


PÉDELACQ, Jean-Denis, Stéphanie CABANTOUS, Timothy TRAN, Thomas C TERWILLIGER a Geoffrey S WALDO. Engineering and characterization of a superfolder green fluorescent protein. Nature Biotechnology [online]. 2006, 24(1), 79-88 [cit. 2021-10-7]. ISSN 1087-0156. Accessible from: doi:10.1038/nbt1172



Composite part C = Testing response to phosphate concentrations


We designed this composite part to test the function of PPho promoter in Bacillus subtilis. PPho is a native promoter of B. subtilis. It is Bacillus subtilis activated by the Pho signaling pathway which is in turn activated by low phosphate concentrations. This promoter was described in a study by Hulett et. al, [1]. We used the predicted sequence from this study from the 5' end of the promoter to the start codon of the first protein in the native operon sequence.


Reporter protein mScarlet-I has been added downstream of this promoter to monitor its function. The cI repressor, which is tested by construct D (BBa_K3831033), has also been placed under the control of PPho promotor.


Additionally, this construct was designed in a way that would allow for the Pho promotor to be cleaved out by restriction enzymes and subsequently ligated with other sequences to assemble composite part Z.


Basic parts


Terminator (Bba_B0014)


This is a bidirectional double terminator consisting of terminators BBa_B0012 and BBa_B0011. In the C composite part, this terminator is located on both sides of the construct.


Source: BBa_B0014


Spacers


Spacers were placed between terminators and the rest of the composite part at both ends.


Spacer_0 (BBa_K3831010)

In our project design, we used spacers according to the study by Guiziou et al. in 2016 [2]

This space corresponds to the sp0 from the mentioned study.


Spacer_3, modified (BBa_K3831023)

In our project design, we used spacers from the study by Guiziou et al. in2016 [2]

This spacer corresponds to the sp3 from the mentioned study. In this case, we used the first 15 bp of the spacer.


Promoter


PPho promoter region (BBa_K3831022)

This promoter is the most important part of construct C. The original sequence was described by Hulett et. al, [1]. We used the predicted sequence from this study from the 5' end of the promoter to the start codon of the first protein in the native operon sequence. This promoter is activated by the Pho pathway and is thus able to respond to the concentration of phosphates in the media. The promoter is activated in low phosphate levels. To demonstrate its functionality and ability to respond to phosphate concentrations, we designed the reporter system with mScarlet-I fluorescent protein.


Ribosomal binding sites


RBS R0 (BBa_K3831018)

Native RBS R0 from this study: Guiziou et al., 2016 [1]


RBS R3 (BBa_K3831020)

Artificial RBS R3 from this study: Guiziou et al., 2016 [1]


Coding sequences


mScarlet-I (BBa_K3831021)

This red fluorescent protein serves as a reporter of the activity of PPho  promoter. We found its protein sequence on FPbase, we translated it back to DNA and codon-optimized it for expression in B. subtilis.

Source: FPbase [3]


cI repressor (BBa_K3831024)

Repressor cI is natively produced by phage lambda and controls the decision between initiating lytic or lysogenic cycle. This repressor binds to the O1, O2 and O3 operators, forms multimers and bends the DNA molecule, preventing transcription initiation from the pR promoter. We added one of the operator sequences to the Pgrac promoter to create Pgrac-OcI which should thus be repressed by the cI repressor.

We codon-optimized the cI sequence to be expressed in B. subtilis.

Source: NC_049948


Degradation tag SVN (BBa_K3831019)

The degradation tag ensures quick degradation of the tagged protein.

Source: Guiziou et al., 2016 [1]


Composite part C (BBa_K3831032)


Two genes have been placed under the control of the PPho promoter-reporter protein mScarlet-I to visualize the activity of this promoter and cI repressor (BBa_K3831024) which is a component of our switch system. Repressor cI is able to repress Pgrac-OcI promoter by binding to the sequence of its operator OcI, which was artificially modified. The translation of these coding sequences is then controlled by these RBSs. Their translation rates were predicted using RBS calculator:


RBS_R0 + cI repressor (Translation rate = 38 689)

RBS_R3 + mScarlet-I (Translation rate = 19 000)


Bidirectional terminator BBa_B0014 is located at the 5' and 3' ends of composite part C to prevent the activation of our genes from a native promoter in the chromosome of B. subtilis as well as the activation of B. subtilis genes located downstream from the Pgrac-OcI  promoter.
Figure 4.: Schema of composite part C.


References


  1. Hulett, F. M., Lee, J., Shi, L., Sun, G., Chesnut, R., Sharkova, E., Duggan, M. F., & Kapp, N. (1994). Sequential action of two-component genetic switches regulates the PHO regulon in Bacillus subtilis. Journal of bacteriology, 176(5), 1348–1358.


  1. 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.


  1. https://www.fpbase.org/protein/mscarlet-i/



Composite part D = Testing the response of the newly created Pgrac OcI to the cI repressor


We designed this composite part with the aim to test the function of the newly created promoter Pgrac OcI in Bacillus subtilis and its response to the presence of the cI repressor originating from phage lambda.


BASIC PARTS


Terminators


Terminator (BBa_B0010)

This is a transcriptional terminator originating from the E. coli rrnB gene.

Source: BBa_B0010


Terminator (BBa_K3831044)

This is a transcriptional terminator originating from the B. subtilis gyrase gene.

Derived from:BBa_K780000


Terminator (BBa_B0014)

This is a bidirectional double terminator consisting of terminators BBa_B0012 and BBa_B0011.

Source: BBa_B0014


Spacers


Spacers were placed between terminators and the rest of the composite part at 3' and 5' end and also at both sides of the bidirectional terminator located in the middle of the composite part.


Spacer_1SP (BBa_K3831038)

Source: Guiziou et al., 2016 [1], modified


Spacer_2 (BBa_K3831026)

Source: Guiziou et al., 2016 [1], modified


Spacer_4 (BBa_K3831028)

Source: Guiziou et al., 2016 [1], modified


Spacer_6 (BBa_K3831027)

Source: Guiziou et al., 2016 [1], modified


Promoters


Pgrac OcI promoter (BBa_K3831025)

The original Pgrac promoter was created by combining the groE promoter, gsiBSD sequence and lacO operator to allow for IPTG induction. We took out the lacO sequence and replaced it by the cI operator (OcI) sequence. This design should allow the strong promoter to constitutively initiate expression until the cI repressor from Phage lambda binds to OcI and the expression is stopped.

Source:BBa_K1074012, Atsumi et Little, 2004 [2]


Phyperspank (BBa_K3831039)

Phyperspank is an IPTG-inducible promoter. In order for this promoter to truly be inducible, the cell must constitutively express lacI transcription regulator, which binds to the operator of the promoter and prevents the initiation of transcription from happening. When IPTG is added to the medium, it removes lacI from the operator and thus allows for the initiation of transcription.

Source: BBa_K143015


Ribosomal binding sites


RBS_R0 (BBa_K3831018)

RBS_R0 is a strong natural ribosome binding site which we had already used in 2020.

Source: Guiziou et al., 2016 [1]


RBS_R2 (BBa_K3831051)

RBS_R2 is a natural ribosome binding site of medium strength.

Source: Guiziou et al., 2016 [1]


RBS_R3 (BBa_K3831020)

RBS_R3 is an artificial ribosome binding site of medium strength.

Source: Guiziou et al., 2016 [1]


Coding sequences


cI repressor (BBa_K3831024)

Repressor cI is natively produced by phage lambda and controls the decision between initiating lytic or lysogenic cycle. This repressor binds to the O1, O2 and O3 operators, forms multimers and bends the DNA molecule, preventing transcription initiation from the pR promoter. We added one of the operator sequences to the Pgrac promoter to create Pgrac-OcI which should thus be repressed by the cI repressor.

We codon-optimized the cI sequence to be expressed in B. subtilis.

Source: NC_049948


m-Scarlet-I (BBa_K3831021)

This red fluorescent protein serves as a reporter of Pgrac-OcI activation. We found its protein sequence on FPbase, we translated it back to DNA and codon-optimized it for expression in B. subtilis.

Source: FPbase [3]


sfGFP (BBa_K3831029)

The Green Fluorescent protein serves as a reporter of cI repressor function. We found its protein sequence on FPbase, we rendered it back to DNA and codon-optimized it to be expressed in B. subtilis.

Source: FPbase [4]


SVN degradation tags (BBa_K3831040,BBa_K3831045,BBa_K3831041)

The degradation tags ensure quick degradation of the tagged protein.

Source: Guiziou et al., 2016 [1]


Composite part D (BBa_K3831033)


This composite part consists of two expression units. One expression unit contains the IPTG-inducible Phyperspank (BBa_K3831039) which controls the production of the cI repressor (BBa_K3831024) and the reporter protein m-Scarlet-I (BBa_K3831021). The other expression unit contains the newly designed Pgrac OcI (BBa_K3831025) which controls the expression of the reporter protein sfGFP (BBa_K3831029).


This system as a whole should work like a switch. Without the presence of IPTG, B. subtilis cells should produce GFP. When induced, the cells should start producing the cI repressor and m-Scarlet-I. m-Scarlet-I fluorescence would serve to confirm the expression of the two proteins. The cI repressor should bind itself to its operator located in Pgrac OcI and stop the expression of GFP. The fall of GFP fluorescence would thus serve as a reporter of the cI mediated repression. To ensure the reversibility of the switch as well as a quick reaction of the system, all proteins contain a SNV degradation tag.


The whole composite part is flanked by two terminators (BBa_B0010 and BBa_K3831044) to prevent reciprocal influence on the transcription of both the composite part and the plasmid backbone (and later the B. subtilis chromosome).


This composite part is designed in a way which allows its further modification in order to create the composite part Z.
Figure 5.: Schema of composite part D.


References


  1. 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

  2. Atsumi S. and Little J. W. A synthetic phage λ regulatory circuit. 2006. PNAS 103 (50): 19045–19050. DOI: 10.1073/pnas.0603052103 DOI: 10.1073/pnas.0603052103

  3. https://www.fpbase.org/protein/mscarlet-i/

  4. https://www.fpbase.org/protein/superfolder-gfp/



Composite part Z = Testing the production of BMCs controlled by changes in phosphate concentrations


The composite part Z is composed of certain components of composite parts A (BBa_K3831030), C (BBa_K3831032), and D (BBa_K3831033).


Composite part Z will be assembled by modifying composite part D. Promotor Phyperspank would be replaced by the PPho promoter from composite part C which responds to the phosphate concentration in the growth medium. This modification would be done using restriction sites HindIII and NcoI.


Additionally, the GFP sequence would be replaced by the sequences of the Pdu BMC shell proteins from composite part A. This modification would be done using restriction sites NdeI and XhoI.


By using this clever design, we were able to avoid having to pay for the synthesis of another large construct while making very efficient use of the other constructs.


Basic parts


Terminators


Terminator, modified (BBa_K3831044)

This is a transcriptional terminator originating from the B. subtilis gyrase gene.

Derived from:BBa_K780000


Terminator (Bba_B0014)

This is a bidirectional double terminator consisting of terminators BBa_B0012 and BBa_B0011. In the Z composite part, this terminator is located on both ends of the construct.

Source: BBa_B0014


Spacers


In our project design, we used spacers from the study by Guiziou et al. in 2016 [1]


Spacer_0 (BBa_K3831010)

This space corresponds to the sp0 from the mentioned study.

Source: Guiziou et al., 2016 [1]


Spacer_2 (BBa_K3831026)

Source: Guiziou et al., 2016 [1], modified


Spacer_4 (BBa_K3831028)

Source: Guiziou et al., 2016 [1], modified


Spacer_6 (BBa_K3831027)

Source: Guiziou et al., 2016 [1], modified


Promotors


PPho promoter region (BBa_K3831022)

The original sequence was described by Hulett et. al, 1994 [2]. We used the predicted sequence from this study from the 5' end of the promoter to the start codon of the first protein in the native operon sequence. This promoter is activated by the Pho pathway and is thus able to respond to the concentration of phosphates in the media. The promoter is activated in low phosphate levels.

Source: Hulett et. al, 1994 [2]


Pgrac OcI promoter (BBa_K3831025)

The Pgrac promoter was created by combining the groE promoter, gsiBSD sequence and lacO operator to allow for IPTG induction. We took out the lacO sequence and replaced it by the cI operator into the promoter sequence. This design should allow the strong promoter to constitutively activate the translation of the corresponding genes until the expression is stopped by the cI repressor originating from phage lambda.

Source: BBa_K1074012, Atsumi et Little, 2004 [3]


RBS


RBS R0 (BBa_K3831018)

Native RBS R0 from the study by Guiziou et al. in 2016 [1]


RBS R3 (BBa_K3831020)

Artificial RBS R3 from the study by Guiziou et al. in 2016 [1]


RBS_a (BBa_K3831055)

Synthetic RBS designed with RBS calculator


RBS_b (BBa_K3831053)

Synthetic RBS designed with RBS calculator


RBS R2 (BBa_K3831051)

Native RBS R2 from the study by Guiziou et al. in 2016 [1]

Source: Guiziou et al., 2016 [1]


RBS R6 (BBa_K3831049)

Artificial RBS R6 from the study by Guiziou et al. in 2016 [1]

Source: Guiziou et al., 2016 [1]


RBS_c (BBa_K3831047)

Synthetic RBS designed with RBS calculator


Coding sequences


mScarlet-I (BBa_K3831021)

This red fluorescent protein serves as a reporter of the activity of PPho  promoter. We found its protein sequence on FPbase, we translated it back to DNA and codon-optimized it for expression in B. subtilis.

Source: FPbase [4]


cI repressor (BBa_K3831024)

Repressor cI is natively produced by phage lambda and controls the decision between initiating lytic or lysogenic cycle. This repressor binds to the O1, O2 and O3 operators, forms multimers and bends the DNA molecule, preventing transcription initiation from the pR promoter. We added one of the operator sequences to the Pgrac promoter to create Pgrac-OcI which should thus be repressed by the cI repressor.

We codon-optimized the cI sequence to be expressed in B. subtilis.

Source: NC_049948


SVN Degradation tags (BBa_K3831019,BBa_K3831040,BBa_K3831045,BBa_K3831041)

The degradation tag ensures quick degradation of the tagged protein.

Source: Guiziou et al., 2016 [1]


Pdu shell protein derived from P. thermoglucosidasius PduA (BBa_K3831054)

Source: Wade et al. (2019) [5]


Pdu shell protein derived from P. thermoglucosidasius PduB (BBa_K3831052)

Source: Wade et al. (2019) [5]


Pdu shell protein derived from P. thermoglucosidasius PduK (BBa_K3831048)

Source: Wade et al. (2019) [5]


Pdu shell protein derived from P. thermoglucosidasius PduJ (BBa_K3831050)

Source: Wade et al. (2019) [5]


Pdu shell protein derived from P. thermoglucosidasius PduN (BBa_K3831046)

Source: Wade et al. (2019) [5]


Composite part Z (BBa_K3831034)


This part has been combined from previously mentioned parts A, C, and D. Using this concept, we are able to combine our composite parts and test the entire system.


Using this part, we will test whether BMC shall proteins are expressed and correctly assembled when under the control of the modified Pgrac Oc  promoter. We can also verify the production of the cI repressor via mScarlet-I. At low phosphate concentrations, PPho promoter should activate the transcription of the cI sequence and lead to the production of the cI repressor. This repressor inhibits transcription initiation from the Pgrac Oc  promoter and thus prevents the formation of more BMCs. This system ensures that BMCs are produced only when the bacteria encounter high phosphate concentrations.
Figure 6.: Schema of composite part Z.


References


  1. 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.

  2. Hulett, F. M., Lee, J., Shi, L., Sun, G., Chesnut, R., Sharkova, E., Duggan, M. F., & Kapp, N. (1994). Sequential action of two-component genetic switches regulates the PHO regulon in Bacillus subtilis. Journal of bacteriology, 176(5), 1348–1358.

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    Igem Team Brno, Czech Republic 2021