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>
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<div class='content_1'><h3> Module Two what'sthename Description </h3>
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<h6><b>Figure 1. &sigma;<sup>x</sup>-Com-Box promoter mechanism</h6></b><br>
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<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>Prof. Donald A. Morrison&#39;s research lab in University of Illinois at Chicago published several papers on the competence for genetic transformation  
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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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in <i>Streptococcus pneumoniae</i> which depends on a quorum-sensing system to control many competence-specific genes which play a role in DNA uptake,  
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processing, and integration. There is a link between this quorum-sensing system and the competence-specific genes, which is an alternative sigma  
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factor ComX. Two identical copies of gene (<i>comX1</i> and <i>comX2</i>) encode a competence-specific alternative sigma factor, &sigma;<sup>x</sup>.  
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Expression of ComX allows the transcription of many genes that are involved in transformation and specifically expressed during competence. These late
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genes share a conserved 8-bp sequence in their promoter regions, TACGAATA (combox) which is specifically induced by &sigma;<sup>x</sup>-containing
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RNA polymerase.  
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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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P<sub>celA</sub> and P<sub>comFA</sub> promoters have high specificity to &sigma;<sup>x</sup> for activation, so genes downstream the promoters will be translated only if &sigma;<sup>x</sup> is present. Hence, by using fluorescence protein as a reporting mechanism, this &sigma;<sup>x</sup>, P<sub>celA</sub> and P<sub>comFA</sub> promoters system could be further utilized as a specific reporter device in <i>E. coli</i> DH10B strain that could be used by iGEM communities.
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<p>The combox promoter is a promoter associated with initiating transcription of a class of genes (commonly referred to as late competence genes)
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coding for bacterial competence proteins and bacterial transformation in <i>Streptococcus Pneumoniae</i> strains. As described before, it contains
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a general consensus sequence of TACGAATA for recognition by its promoter-specific sigma factor X, which is a protein expressed from the early
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competence gene <i>comX</i>.
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<p>In fact, the combox promoter can be found on many different regions within the genomic DNA of <i>Streptococcus
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Pneumoniae</i> strains. Researches and investigations have also reported variants of the combox promoter having different lengths and consensus
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sequences, but generally the range of variety has been kept small.
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<div class='content_1'><h3>σ<sup>x</sup> and ComW mechanism</h3>
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<p>Though much information about the combox promoter is readily available nowadays, its full characterization including promoter activity, specificity,
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sequence, as well as the biomolecular mechanism can be greatly enhanced with further investigations and experiments. As part of module II, we were
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interested in reproducing this gene circuit with all the associated genes and promoters to be combined into a single transcriptional unit.  
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To the best of our knowledge, the genes and promoters of this gene circuit for bacterial transformation are found at different locations of the
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<h6><b>Figure 2. &sigma;<sup>x</sup> - <i>comW</i> Interaction Diagram</h6></b><br>
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long genomic DNA of many <i>Streptococcus Pneumoniae</i> strains. Despite the suggested susceptibility to leakage and other factors that may
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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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hinder or interrupt the mechanism, researches have reported that the pathway was highly specific to certain environmental conditions and stress,
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suggesting minimal or no leakage in the entire process. With part I of module to focusing on ComX, part II of module II focused mainly on isolating
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the combox promoter, and also identifying the most probable length of the promoter. Characterization of the combox promoter was carried out
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together with the characterization of <i>comX</i> gene. Because combox promoter is highly specific to &sigma;<sup>x</sup> for activation,
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genes downstream of the combox promoter will be translated only if &sigma;<sup>x</sup> are present. Hence, by using fluorescence protein as a  
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reporting mechanism, this <i>comX</i>-combox system could be further utilized as a specific reporter device that could be used by the iGEM
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community.  
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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> Besides ComX, another positive factor involved in competence regulation was later found out to be ComW. The <i>comW</i> gene (SP0018) is regulated
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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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by the quorum-sensing system and is required for a high-level of competence. Coexpression of <i>comW</i> with <i>comX </i>restores the  
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accumulation of &sigma;<sup>x</sup> and the expression of late genes as ComW contributes to the stabilization of the alternative sigma  
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factor &sigma;<sup>x</sup>against proteolysis and is required for full activity of &sigma;<sup>x</sup> in directing transcription of late  
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competence genes. ComW functions to act as a barrier which covers comX protein from being degraded by the ClpXP degradation enzyme. Hence, ComW
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will be degraded instead of ComX, and the production of ComX protein will be increased.
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<p>Based on these findings, we would like to integrate this alternative sigma factor system from Gram-positive <i>Streptococcus pneumoniae</i>
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into Gram-negative <i>Escherichia coli</i>. Firstly, we cloned out the <i>comX</i> and <i>comW</i> genes from the genomic DNA of <i>S. pneumoniae</i>
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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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NCTC 7465 strain. We then used BioBrick BBa_K880005 (consisting of constitutive promoter J23100 and strong RBS B0034) to drive the expression of those
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genes. Lastly, we combined these constructs with the combox promoter and GFP generator to check the functionality of the system, and to calculate the
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relative promoter unit of combox promoter.
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<u>References</u>
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Andrew Piotrowski et al. (2009) &quot;Competence for Genetic Transformation in <i>Streptococcus pneumoninae</i>: Termination of Activity of the Alternative Sigma Factor ComX Is Independent of Proteolysis of ComX and ComW&quot; Journal of Bacteriology.
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A. Piotrowski, P. Luo, & D. A. Morrison. (2009). Competence for Genetic Transformation in <i>Streptococcus pneumoniae</i>: Termination of Activity of the Alternative Sigma Factor ComX Is Independent of Proteolysis of ComX and ComW. <i>Journal of Bacteriology</i>, 191(10), 3359-3366. doi:10.1128/JB.01750-08
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Ping Luo et al. (2003) &quot;Transient Association of an Alternative Sigma Factor, ComX, with RNA Polymerase during the Period of Competence for Genetic Transformation in <i>Streptococcus pneumonia</i>&quot; Journal of Bacteriology.
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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 <i>Streptococcus pneumoniae</i>. <i>Journal of Bacteriology</i>, 185(1), 349-358. doi: 10.1128/JB.185.1.349-358.2003
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Sung CK et al. (2005) &quot;Two distinct functions of ComW in stabilization and activation of the alternative sigma factor ComX in <i>Streptococcus pneumoniae</i>. &quot; Journal of Bacteriology.
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C. K. Sung & D. A. Morrison. (2005). Two Distinct Functions of ComW in Stabilization and Activation of the Alternative Sigma Factor ComX in <i>Streptococcus pneumoniae</i>. <i>Journal of Bacteriology</i>, 185(9), 3052-3061. doi: 10.1128/JB.187.9.3052-3061.2005
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Haiying Li et al. (2004) &quot;Identification of ComW as a new component in regulation of genetic transformation in <i>Streptococcus Pneumoniae</i>&quot; Molecular Biology.
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P. Luo, H. Li, & D. A. Morrison. (2004). Identification of ComW as a new component in the regulation of genetic transformation in <i>Streptococcus pneumoniae</i>. <i>Molecular Microbiology</i>, 54(1), 172-183. doi: 10.1111/j.1365-2958.2004.04254.x
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Myeong S.Lee et al. (1999) &quot;Identification of a New Regulator in <i>Streptococcus Pneumoniae</i> Linking Quorum Sensing to Competence for Genetic Transformation&quot;
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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.
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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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