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Team:Hong Kong HKUST/pneumosensor/module two
From 2014.igem.org
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| - | <div class='content_1'><h3> <i>comX</i> - | + | <div class='content_1'><h3> <i>comX</i> - P<sub>CelA</sub> - P<sub>helicase</sub> mechanism </h3> |
<table class="content_table" align= "center" > | <table class="content_table" align= "center" > | ||
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<p> | <p> | ||
| - | + | Combox promoters can be found on many different regions within the genomic DNA of <i>Streptococcus Pneumoniae</i> strains. The promoters | |
| - | 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> | + | 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, | |
| - | 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><br> | + | 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 | |
| - | Combox promoters have high specificity to | + | 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><br> | |
| - | </p> | + | Combox promoters have high specificity to σ<sup>x</sup> 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. | |
| + | </p> | ||
</div> | </div> | ||
</td> | </td> | ||
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| - | <div class='content_1'><h3> | + | <div class='content_1'><h3> ComX and ComW mechanism </h3> |
<table class="content_table" align= "center" > | <table class="content_table" align= "center" > | ||
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<img src= "https://static.igem.org/mediawiki/2014/f/fd/Diagram_text_2_edited_copy.png"/> | <img src= "https://static.igem.org/mediawiki/2014/f/fd/Diagram_text_2_edited_copy.png"/> | ||
</div> | </div> | ||
| - | <p>Besides | + | <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 σ<sup>x</sup> 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. | ||
</p> | </p> | ||
</div> | </div> | ||
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<p> | <p> | ||
| - | + | Based on these findings, we integrated this alternative sigma factor system from Gram-positive <i>Streptococcus pneumoniae</i> into | |
| - | + | Gram-negative Escherichia coli. We firstly cloned out the comX and comW genes from the genomic DNA of S. pneumoniae NCTC 7465 strain. | |
| - | 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> | + | We then used BBa_K880005 (consisting of constitutive promoter J23100 and strong RBS B0034) from the BioBricks to express those genes.<br><br> |
| - | + | Lastly, we combined these constructs with combox promoter and GFP generator to check the functionality of the system, and calculate the | |
| - | 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. | + | relative promoter unit of combox promoter. |
| - | + | ||
| - | + | ||
| - | + | ||
</p> | </p> | ||
| - | |||
</div> | </div> | ||
</td> | </td> | ||
Revision as of 15:41, 3 October 2014
comX - PCelA - Phelicase mechanism
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. |
ComX and ComW mechanism
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. |
|
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