Team:Hong Kong HKUST/pneumosensor/module two

From 2014.igem.org

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Combox promoters can be found on many different regions within the genomic DNA of Streptococcus Pneumoniae strains. The promoters were reported to have variants with different lengths and consensus sequences, but generally the range of variety has been kept small. Though much information about the combox promoter 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>
Combox promoters can be found on many different regions within the genomic DNA of Streptococcus Pneumoniae strains. The promoters were reported to have variants with different lengths and consensus sequences, but generally the range of variety has been kept small. Though much information about the combox promoter 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>
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<div class='content_1'><h3> Reporting Module Description </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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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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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>Besides comX, another positive factor involved in competence regulation was later found out to be ComW. The gene comW (SP0018) is regulated by the quorum-sensing system and is required for a high-level of competence. Coexpression of comW with comX restores the accumulation of σx and the expression of late genes as ComW contributes to the stabilization of the alternative sigma factor σx against proteolysis by ClpXP and is required for full activity of σx in directing transcription of late competence genes.  
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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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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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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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Based on these findings, we integrated this alternative sigma factor system from Gram-positive Streptococcus pneumoniae into Gram-negative Escherichia coli. We firstly cloned out the comX and comW genes from the genomic DNA of S. pneumoniae NCTC 7465 strain. We then used BBa_K880005 (consisting of constitutive promoter J23100 and strong RBS B0034) from the BioBricks to express those genes.<br><br>
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Lastly, we combined these constructs with combox promoter and GFP generator to check the functionality of the system, and calculate the relative promoter unit of combox promoter.
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Revision as of 14:47, 3 October 2014


Reporting Module Description

Prof. Donald A. Morrison’s research lab in University of Illinois at Chicago published several papers on the competence for genetic transformation in Streptococcus pneumoniae 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 factor ComX, σx. Expression of ComX allows transcription of many genes that are involved in transformation and specifically expressed during competence. These late genes share a conserved 8-bp sequence in their promoter regions, TACGAATA (combox) which is specifically induced by σx-containing RNA polymerase.

Combox promoters can be found on many different regions within the genomic DNA of Streptococcus Pneumoniae strains. The promoters were reported to have variants with different lengths and consensus sequences, but generally the range of variety has been kept small. Though much information about the combox promoter 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.

Combox promoters have high specificity to σx for activation, so genes downstream the combox promoter will be translated only if σx are present. Hence, by using fluorescence protein as a reporting mechanism, this comX-combox system could be further utilized as a specific reporter device that could be used by iGEM communities.

Reporting Module Description

Besides comX, another positive factor involved in competence regulation was later found out to be ComW. The gene comW (SP0018) is regulated by the quorum-sensing system and is required for a high-level of competence. Coexpression of comW with comX restores the accumulation of σx and the expression of late genes as ComW contributes to the stabilization of the alternative sigma factor σx against proteolysis by ClpXP and is required for full activity of σx in directing transcription of late competence genes.

Based on these findings, we integrated this alternative sigma factor system from Gram-positive Streptococcus pneumoniae into Gram-negative Escherichia coli. We firstly cloned out the comX and comW genes from the genomic DNA of S. pneumoniae NCTC 7465 strain. We then used BBa_K880005 (consisting of constitutive promoter J23100 and strong RBS B0034) from the BioBricks to express those genes.

Lastly, we combined these constructs with combox promoter and GFP generator to check the functionality of the system, and calculate the relative promoter unit of combox promoter.

References

Andrew Piotrowski et al. (2009) "Competence for Genetic Transformation in Streptococcus pneumoninae: Termination of Activity of the Alternative Sigma Factor ComX Is Independent of Proteolysis of ComX and ComW" Journal of Bacteriology.

Ping Luo et al. (2003) "Transient Association of an Alternative Sigma Factor, ComX, with RNA Polymerase during the Period of Competence for Genetic Transformation in Streptococcus pneumonia" Journal of Bacteriology.

Sung CK et al. (2005) "Two distinct functions of ComW in stabilization and activation of the alternative sigma factor ComX in Streptococcus pneumoniae. " Journal of Bacteriology.

Haiying Li et al. (2004) "Identification of ComW as a new component in regulation of genetic transformation in Streptococcus Pneumoniae" Molecular Biology.

Myeong S.Lee et al. (1999) "Identification of a New Regulator in Streptococcus Pneumoniae Linking Quorum Sensing to Competence for Genetic Transformation"


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