Team:Hong Kong HKUST/pneumosensor/module two

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<div class='content_1'><h3><i>S. pneumoniae</i> σ<sup>x</sup> promoters module </h3>
<div class='content_1'><h3><i>S. pneumoniae</i> σ<sup>x</sup> promoters module </h3>
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<h6>Figure 1. &sigma;<sup>x</sup>-Com-Box promoter mechanism</h6><br>
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<h6><b>Figure 1. &sigma;<sup>x</sup>-Com-Box promoter mechanism</h6></b><br>
<h7> The reporter system contains 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 Com-Box promoter induction. &sigma;<sup>x</sup> will then bind to Com-Box promoter and express green fluorescence protein. The whole construct was built in <i>E. coli</i> DH10B strain. </h7>
<h7> The reporter system contains 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 Com-Box promoter induction. &sigma;<sup>x</sup> will then bind to Com-Box promoter and express green fluorescence protein. The whole construct was built in <i>E. coli</i> DH10B strain. </h7>
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<p class="first_letter_enhanced">Prof. Donald A. Morrison’s research lab in University of Illinois at Chicago published several papers on the competence for genetic transformation in <i>Streptococcus pneumoniae</i> which depends on quorum-sensing system to control many competence-specific genes acting in DNA uptake, processing, and integration. There is a link between this quorum-sensing system and the competence-specific genes, which is an alternative &sigma;<sup>x</sup>. &sigma;<sup>x</sup> (ComX protein) serves as a competence-specific global transcription modulator. 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 a 8 base pairs consensus sequence TACGAATA known as the Cin-Box or the Com-Box. (Piotrowski, Luo, & Morrison, 2009)  
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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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iGEM 2014 Hong_Kong_HKUST Team has cloned &sigma;<sup>X</sup> from <i>S. pneumoniae</i> strain NCTC7465 and characterized its ability to initiate transcription of two downstream promoters: P<sub>celA</sub> (<a href= "http://parts.igem.org/Part:BBa_K1379000">BBa_K1379000</a>) and P<sub>comFA</sub> (<a href= "http://parts.igem.org/Part:BBa_K1379001">BBa_K1379001</a>).
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P<sub>celA</sub> and P<sub>comFA</sub> promoters can be found on many different regions within the genomic DNA of <i>Streptococcus Pneumoniae</i> strains. These promoters have different lengths and consensus sequences. Though much information about the 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.<br><br> 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.
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iGEM 2014 Hong_Kong_HKUST Team has cloned &sigma;<sup>x</sup> from <i>S. pneumoniae</i> strain NCTC7465 and characterized its ability to initiate transcription of two downstream promoters with different lengths: P<sub>celA</sub> (<a href= "http://parts.igem.org/Part:BBa_K1379000">BBa_K1379000</a>) and P<sub>comFA</sub> (<a href= "http://parts.igem.org/Part:BBa_K1379001">BBa_K1379001</a>), 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. <br>
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<h6>Figure 2. &sigma;<sup>x</sup> - <i>comW</i> Interaction Diagram</h6><br>
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<h6><b>Figure 2. &sigma;<sup>x</sup> - <i>comW</i> Interaction Diagram</h6></b><br>
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<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<sub>celA</sub> and P<sub>comFA</sub> 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<sub>celA</sub> and P<sub>comFA</sub> promoters.</h7>
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<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">Besides &sigma;<sup>x</sup>, another positive factor involved in competence regulation was later found out to be ComW. The gene <i>comW</i> (SP0018) is
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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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regulated by the quorum-sensing system and is required for a high-level of competence. 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.  
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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 integrated this alternative sigma factor system from Gram-positive <i>Streptococcus pneumoniae</i> into Gram-negative <i>Escherichia coli</i>. 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>. 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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Lastly, we combined these constructs with P<sub>celA</sub> and P<sub>comFA</sub> promoters and GFP generator to check the functionality of the system, and calculate the Relative Promoter Unit (R.P.U) of the promoters.
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M. S. Lee & D. A . Morrison. (1999). Identification of a New Regulator in <i>Streptococcus pneumoniae</i> Linking Quorum Sensing to Competence for Genetic Transformation. <i>Journal of Bacteriology</i>, 181(16), 5004-5016.
M. S. Lee & D. A . Morrison. (1999). Identification of a New Regulator in <i>Streptococcus pneumoniae</i> Linking Quorum Sensing to Competence for Genetic Transformation. <i>Journal of Bacteriology</i>, 181(16), 5004-5016.
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Piotrowski Andrew, Luo Ping, & Morrison Donald. (2009).<i> Competence for genetic transformation in Streptococcus pneumoniae: termination of activity of the alternative sigma factor ComX is independent of proteolysis of ComX and ComW.</i> Journal of Bacteriology. doi:10.1128/JB.01750-08
 
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Latest revision as of 17:51, 14 October 2014



S. pneumoniae σx promoters module

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 Signal 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 and 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. We firstly 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

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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