Team:Hong Kong HKUST/pneumosensor/modules

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<p>The mechanism we adopted is controlled by a two-component regulatory system (TCS), which consists of the histidine kinase (HK) ComD and its  
<p>The mechanism we adopted is controlled by a two-component regulatory system (TCS), which consists of the histidine kinase (HK) ComD and its  
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cognate response regulator (RR) ComE.  When CSP binds to it, ComD autophosphorylates to become phospho-ComD, ComD~P; at the expenditure of ATP.  
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cognate response regulator (RR) ComE (<a href="http://parts.igem.org/Part:BBa_K1379051">BBa_K1379051</a>).  When CSP binds to it, ComD autophosphorylates to become phospho-ComD, ComD~P; at the expenditure of ATP.  
The cytosolic protein ComE is then phosphorylated by ComD~P through transphosphorylation reactions, producing ComE~P. Genes coding for ComD and ComE are constitutively  
The cytosolic protein ComE is then phosphorylated by ComD~P through transphosphorylation reactions, producing ComE~P. Genes coding for ComD and ComE are constitutively  
expressed in our Pneumosensor.
expressed in our Pneumosensor.
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<p class="first_letter_enhanced">In order to achieve the functionality of pneumosensor, we must have a highly specific reporting system which will only give fluorescent signal under the presence of <i>S. pneumoniae</i>. In search for the suitable gene circuit, the discovery by Prof. Morrison on the competence for genetic transformation in <i>S. pneumoniae</i> which depends on quorum-sensing system to control many competence-specific genes acting in DNA uptake, processing, and integration has provided the ideal framework for this module. (Lee and Morrison, 1999) There is a link between this quorum-sensing system and the competence-specific genes, which is an alternative &sigma;<sup>x</sup> (ComX protein) that serves as a competence-specific global transcription modulator. (Luo and Morrison, 2003) In <i>S. pneumoniae</i>, competence (a state capable of being genetic transformed) happens transiently during the log phase growth, and is regulated by a quorum sensing system utilizing the Competence Signal Peptide (CSP). Upon stimulation by CSP, &sigma;<sup>x</sup> will be expressed and associated with RNA polymerase apoenzyme. The resulting holoenzyme will then be guided by &sigma;<sup>x</sup> to initiate transcription of a set of “late” genes enabling genetic transformation and other unknown functions. Characterized genes regulated by &sigma;<sup>x</sup> were found to contain an 8 base pairs consensus sequence TACGAATA known as the Cin-Box or the Com-Box. (Piotrowski, Luo, & Morrison, 2009). Taking advantage of this competence-specific mechanism, it is now able to produce the <i>S. pneumoniae</i> sensing device of high specificity by incorporating this system into <i>E. coli</i>. <br>
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<p class="first_letter_enhanced">In order to achieve the functionality of pneumosensor, we must have a highly specific reporting system which will only give fluorescent signal under the presence of <i>S. pneumoniae</i>. In search for the suitable gene circuit, the discovery by Prof. Morrison on the competence for genetic transformation in <i>S. pneumoniae</i> which depends on quorum-sensing system to control many competence-specific genes acting in DNA uptake, processing, and integration has provided the ideal framework for this module. (Lee and Morrison, 1999) There is a link between this quorum-sensing system and the competence-specific genes, which is an alternative &sigma;<sup>x</sup> (ComX protein) that serves as a competence-specific global transcription modulator. (Luo and Morrison, 2003) In <i>S. pneumoniae</i>, competence (a state capable of being genetic transformed) happens transiently during the log phase growth, and is regulated by a quorum sensing system utilizing the competence-stimulating peptide (CSP). Upon stimulation by CSP, &sigma;<sup>x</sup> will be expressed and associated with RNA polymerase apoenzyme. The resulting holoenzyme will then be guided by &sigma;<sup>x</sup> to initiate transcription of a set of “late” genes enabling genetic transformation and other unknown functions. Characterized genes regulated by &sigma;<sup>x</sup> were found to contain an 8 base pairs consensus sequence TACGAATA known as the Cin-Box or the Com-Box. (Piotrowski, Luo, & Morrison, 2009). Taking advantage of this competence-specific mechanism, it is now able to produce the <i>S. pneumoniae</i> sensing device of high specificity by incorporating this system into <i>E. coli</i>. <br>
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<h6><b>Figure 2. &sigma;<sup>x</sup> - <i>comW</i> Interaction Diagram</h6></b><br>
<h6><b>Figure 2. &sigma;<sup>x</sup> - <i>comW</i> Interaction Diagram</h6></b><br>
<h7>&sigma;<sup>x</sup> and ComW protein are both produced by a constitutive promoter <a href= "http://parts.igem.org/wiki/index.php?title=Part:BBa_J23100">BBa_J23100</a>, which continuously expresses &sigma;<sup>x</sup> required for P<i><sub>celA</sub></i> and P<i><sub>comFA</sub></i> promoters induction, and ComW protein is required for &sigma;<sup>x</sup> stabilization. ComW protein acts as a barrier that protects &sigma;<sup>x</sup> from being degraded by ClpXP degradation enzyme, hence it increases the production of &sigma;<sup>x</sup>. The increase in &sigma;<sup>x</sup> production will increase the expression of green fluorescence protein by P<i><sub>celA</sub></i> and P<i><sub>comFA</sub></i> promoters.</h7>
<h7>&sigma;<sup>x</sup> and ComW protein are both produced by a constitutive promoter <a href= "http://parts.igem.org/wiki/index.php?title=Part:BBa_J23100">BBa_J23100</a>, which continuously expresses &sigma;<sup>x</sup> required for P<i><sub>celA</sub></i> and P<i><sub>comFA</sub></i> promoters induction, and ComW protein is required for &sigma;<sup>x</sup> stabilization. ComW protein acts as a barrier that protects &sigma;<sup>x</sup> from being degraded by ClpXP degradation enzyme, hence it increases the production of &sigma;<sup>x</sup>. The increase in &sigma;<sup>x</sup> production will increase the expression of green fluorescence protein by P<i><sub>celA</sub></i> and P<i><sub>comFA</sub></i> promoters.</h7>
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<p class="first_letter_enhanced">To complete the story of competence regulation mechanism from <i>S. Pneumoniae</i>, we would also like to integrate another positive factor involved in competence regulation which was later found out to be ComW. Prof. Morrison's lab released another research paper on the identification of a new component in the regulation of genetic transformation in <i>S. Pneumoniae</i>. The gene <i>comW</i> (SP0018) was found to be  
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<p class="first_letter_enhanced">To complete the story of competence regulation mechanism from <i>S. pneumoniae</i>, we would also like to integrate another positive factor involved in competence regulation which was later found out to be ComW. Prof. Morrison's lab released another research paper on the identification of a new component in the regulation of genetic transformation in <i>S. pneumoniae</i>. The gene <i>comW</i> (SP0018) was found to be  
regulated by the quorum-sensing system and is required for a high-level of competence (Luo, Li, and Morrison, 2004). Coexpression of ComW with &sigma;<sup>x</sup> restores the accumulation of &sigma;<sup>x</sup> and the expression of late genes as ComW contributes to the stabilization of the alternative sigma factor X against proteolysis by ClpXP and is required for full activity of &sigma;<sup>x</sup> in directing transcription of late competence genes (Piotrowski, Luo, and Morrison, 2009).  
regulated by the quorum-sensing system and is required for a high-level of competence (Luo, Li, and Morrison, 2004). Coexpression of ComW with &sigma;<sup>x</sup> restores the accumulation of &sigma;<sup>x</sup> and the expression of late genes as ComW contributes to the stabilization of the alternative sigma factor X against proteolysis by ClpXP and is required for full activity of &sigma;<sup>x</sup> in directing transcription of late competence genes (Piotrowski, Luo, and Morrison, 2009).  
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Based on these findings, we tried to integrate this ComW into the mechanism to see whether and how the presence of ComW affects &sigma;<sup>x</sup>. We firstly cloned out the <i>comX</i> gene expressing &sigma;<sup>x</sup>, and <i>comW</i> genes from the genomic DNA of <i>S. pneumoniae</i> NCTC 7465 strain. We then used <a href= "http://parts.igem.org/Part:BBa_K880005">BBa_K880005</a> (consisting of constitutive promoter <a href= "http://parts.igem.org/wiki/index.php?title=Part:BBa_J23100">BBa_J23100</a> and strong RBS <a href= "http://parts.igem.org/wiki/index.php?title=Part:BBa_B0034">BBa_B0034</a>) from the BioBricks to express those genes.<br><br>
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Based on these findings, we tried to integrate this ComW into the mechanism to see whether and how the presence of ComW affects &sigma;<sup>x</sup>. First, we cloned out the <i>comX</i> gene expressing &sigma;<sup>x</sup> and <i>comW</i> genes from the genomic DNA of <i>S. pneumoniae</i> NCTC 7465 strain. We then used <a href= "http://parts.igem.org/Part:BBa_K880005">BBa_K880005</a> (consisting of constitutive promoter <a href= "http://parts.igem.org/wiki/index.php?title=Part:BBa_J23100">BBa_J23100</a> and strong RBS <a href= "http://parts.igem.org/wiki/index.php?title=Part:BBa_B0034">BBa_B0034</a>) from the BioBricks to express those genes.<br><br>
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Latest revision as of 16:05, 1 September 2015



Pneumosensor Module Description


Overview

Pneumosensor primarily adopts the quorum sensing pathway components in Streptococcus pneumoniae to detect populations of S. pneumoniae. The main advantage of this system is its detection specificity- the Gram-positive quorum sensing mechanism is incorporated into the Gram-negative bacteria E. coli to eliminate possible cross-talk of the autoinducer molecule, competence-stimulating peptide (CSP) with native E. coli molecules.

There are two modules to our Pneumosensor- the Detection Module and the S. pneumoniae σx Promoters Module. The Detection Module comprises of the CSP receptor ComD, its response regulator ComE and the promoter PcomCDE which is induced by phosphorylated ComE. The σx Promoters Module involves a highly specific reporting system, whereby σx is associated with RNA polymerase and binds to promoters PcelA and PcomFA which are specific to σx for activation. These promoters then drive the expression of GFP as a reporting system. Another protein, ComW, is expressed alongside the σx as its stabilizer against proteolysis.



Detection Module Description



Figure 1. Detection Module Diagram

CSP released by S. pneumoniae is detected by its receptor ComD, which autophosphorylates at the expenditure of ATP. ComD~P then phosphorylates ComE. ComE~P then induces the promoter PcomCDE, which drives the expression of GFP.

Transformation in Streptococcus pneumoniae, like many other species, depends on specialized state called competence. Competence is achieved during the exponential growth stage of pneumococcal culture by the secretion of competence-stimulating peptide (CSP), which is a 17- residue long pheromone that is species-specific. Our team aims to adopt the specific CSP sensing mechanism of S. pneumoniae to detect its populations.


The mechanism we adopted is controlled by a two-component regulatory system (TCS), which consists of the histidine kinase (HK) ComD and its cognate response regulator (RR) ComE (BBa_K1379051). When CSP binds to it, ComD autophosphorylates to become phospho-ComD, ComD~P; at the expenditure of ATP. The cytosolic protein ComE is then phosphorylated by ComD~P through transphosphorylation reactions, producing ComE~P. Genes coding for ComD and ComE are constitutively expressed in our Pneumosensor.


ComE~P binds to repeat sites adjacent to the comCDE and comAB operons (Ween et al., 1999), creating a positive feedback loop, producing both pre-CSP and its required machinery for maturation and transport. The signal is thus amplified and competence is coordinated throughout the population.


As part of our detection circuit design, we make use of the inducible promoter from the comCDE operon, PcomCDE. We obtain the sequence by oligos, and will characterize it using green fluorescence protein (GFP) that we constructed the downstream of the promoter by BioBrick RFC10. Rather than using ComE~P that has to be phosphorylated by ComD~P, which involves a chain of reactions, we use a phosphorylmimetic ComE mutant, ComED58E, kindly shared with us by Bernard Martin et al. After characterization of PcomCDE, we hope to put PcomCDE together with the S. pneumoniae σx promoters module, for the ultimate goal of creating a tightly regulated automatic detection of S. pneumoniae.

S. pneumoniae σx promoters Module Description

σx - Com-Box mechanism

Figure 1. σx-Com-Box promoter mechanism

The reporter system contains a constitutive promoter BBa_J23100, which continuously expresses σx required for Com-Box promoter induction. σx will then bind to Com-Box promoter and express green fluorescence protein. The whole construct was built in E. coli DH10B strain.

In order to achieve the functionality of pneumosensor, we must have a highly specific reporting system which will only give fluorescent signal under the presence of S. pneumoniae. In search for the suitable gene circuit, the discovery by Prof. Morrison on the competence for genetic transformation in S. pneumoniae which depends on quorum-sensing system to control many competence-specific genes acting in DNA uptake, processing, and integration has provided the ideal framework for this module. (Lee and Morrison, 1999) There is a link between this quorum-sensing system and the competence-specific genes, which is an alternative σx (ComX protein) that serves as a competence-specific global transcription modulator. (Luo and Morrison, 2003) In S. pneumoniae, competence (a state capable of being genetic transformed) happens transiently during the log phase growth, and is regulated by a quorum sensing system utilizing the competence-stimulating peptide (CSP). Upon stimulation by CSP, σx will be expressed and associated with RNA polymerase apoenzyme. The resulting holoenzyme will then be guided by σx to initiate transcription of a set of “late” genes enabling genetic transformation and other unknown functions. Characterized genes regulated by σx were found to contain an 8 base pairs consensus sequence TACGAATA known as the Cin-Box or the Com-Box. (Piotrowski, Luo, & Morrison, 2009). Taking advantage of this competence-specific mechanism, it is now able to produce the S. pneumoniae sensing device of high specificity by incorporating this system into E. coli.


iGEM 2014 Hong_Kong_HKUST Team has cloned σx from S. pneumoniae strain NCTC7465 and characterized its ability to initiate transcription of two downstream promoters with different lengths: PcelA (BBa_K1379000) and PcomFA (BBa_K1379001), which have the consensus Com-Box sequence. Though much information about the promoters is readily available nowadays, its characterization of promoter activity, specificity, sequence, as well as the biomolecular mechanism can be greatly enhanced with further investigations and experiments. Hence, we were interested in reproducing this gene circuit with all the associated genes and promoters to be combined into a single transcriptional unit. Despite the suggested susceptibility to leakage and other factors that may hinder or interrupt the mechanism, researches have reported that the pathway was highly specific to certain environmental conditions and stress, suggesting minimal or no leakage in the entire process.

PcelA and PcomFA promoters have high specificity to σx for activation, so genes downstream the promoters will be translated only if σx is present. Hence, by using fluorescence protein as a reporting mechanism, this σx, PcelA and PcomFA promoters system could be further utilized as a specific reporter device in E. coli DH10B strain that could be used by iGEM communities.

σx-ComW mechanism

Figure 2. σx - comW Interaction Diagram

σx and ComW protein are both produced by a constitutive promoter BBa_J23100, which continuously expresses σx required for PcelA and PcomFA promoters induction, and ComW protein is required for σx stabilization. ComW protein acts as a barrier that protects σx from being degraded by ClpXP degradation enzyme, hence it increases the production of σx. The increase in σx production will increase the expression of green fluorescence protein by PcelA and PcomFA promoters.

To complete the story of competence regulation mechanism from S. pneumoniae, we would also like to integrate another positive factor involved in competence regulation which was later found out to be ComW. Prof. Morrison's lab released another research paper on the identification of a new component in the regulation of genetic transformation in S. pneumoniae. The gene comW (SP0018) was found to be regulated by the quorum-sensing system and is required for a high-level of competence (Luo, Li, and Morrison, 2004). Coexpression of ComW with σx restores the accumulation of σx and the expression of late genes as ComW contributes to the stabilization of the alternative sigma factor X against proteolysis by ClpXP and is required for full activity of σx in directing transcription of late competence genes (Piotrowski, Luo, and Morrison, 2009).


Based on these findings, we tried to integrate this ComW into the mechanism to see whether and how the presence of ComW affects σx. First, we cloned out the comX gene expressing σx and comW genes from the genomic DNA of S. pneumoniae NCTC 7465 strain. We then used BBa_K880005 (consisting of constitutive promoter BBa_J23100 and strong RBS BBa_B0034) from the BioBricks to express those genes.



References

B. Martin et al "ComE/ComE~P interplay dictates activation or extinction status of pneumococcal X-state (competence) " 2012

B. Martin et al "Cross-regulation of competence pheromone production and export in the early control of transformation in Streptococcus pneumoniae " 2000

B. Martin et al "Expression and maintenance of ComD–ComE, the two-component signal-transduction system that controls competence of Streptococcus pneumoniae mmi_7071 1513..1528 "

Cheng, Q., Campbell, E.A., Naughton, A.M., Johnson, S., and Masure, H.R. (1997) The com locus controls genetic transformation in Streptococcus pneumoniae. Mol Micro-biol 23: 683±692.

Pestova, E.V., and Morrison, D.A. (1998) Isolation and characterization of three Streptococcus pneumoniae transformation-specific loci by use of a lacZ reporter insertion vector. J Bacteriol 180: 2701±2710.

Ween, O., Gaustad, P., and Havarstein, L.S. (1999) Identification of DNA binding sites for ComE, a key regulator of natural competence in Streptococcus pneumoniae. Mol Microbiol 33: 817±827.

A. Piotrowski, P. Luo, & D. A. Morrison. (2009). Competence for Genetic Transformation in Streptococcus pneumoniae: Termination of Activity of the Alternative Sigma Factor ComX Is Independent of Proteolysis of ComX and ComW. Journal of Bacteriology, 191(10), 3359-3366. doi:10.1128/JB.01750-08

P. Luo & D. A. Morisson. (2003). Transient Association of an Alternative Sigma Factor, ComX, with RNA Polymerase during the Period of Competence for Genetic Transformation in Streptococcus pneumoniae. Journal of Bacteriology, 185(1), 349-358. doi: 10.1128/JB.185.1.349-358.2003

C. K. Sung & D. A. Morrison. (2005). Two Distinct Functions of ComW in Stabilization and Activation of the Alternative Sigma Factor ComX in Streptococcus pneumoniae. Journal of Bacteriology, 185(9), 3052-3061. doi: 10.1128/JB.187.9.3052-3061.2005

P. Luo, H. Li, & D. A. Morrison. (2004). Identification of ComW as a new component in the regulation of genetic transformation in Streptococcus pneumoniae. Molecular Microbiology, 54(1), 172-183. doi: 10.1111/j.1365-2958.2004.04254.x

M. S. Lee & D. A . Morrison. (1999). Identification of a New Regulator in Streptococcus pneumoniae Linking Quorum Sensing to Competence for Genetic Transformation. Journal of Bacteriology, 181(16), 5004-5016.


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