Team:Hong Kong HKUST/Achievements/medal requirement

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<div class='content_1'><h3> Module Two what'sthename Description </h3>
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<h3 class= "What_medal"> Bronze Medal</h3>
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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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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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<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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<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 <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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<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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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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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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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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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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Revision as of 19:24, 2 October 2014

Bronze Medal

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Silver Medal

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Gold Medal

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