Team:Hong Kong HKUST/pneumosensor/module two
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<img src= "https://static.igem.org/mediawiki/2014/archive/7/73/20141008143022!Diagram_text_2_edited_without_text.png"/> | <img src= "https://static.igem.org/mediawiki/2014/archive/7/73/20141008143022!Diagram_text_2_edited_without_text.png"/> | ||
<h5>Figure 2. σ<sup>x</sup> - <i>comW</i> Interaction Diagram</h5><br> | <h5>Figure 2. σ<sup>x</sup> - <i>comW</i> Interaction Diagram</h5><br> | ||
- | <h6>σ<sup>x</sup> and ComW are both produced by a constitutive promoter J23100, which continuously expressing σ<sup>x</sup> protein required for P<sub>celA</sub> and P<sub>comFA</sub> promoters induction, and ComW protein required for σ<sup>x</sup> stabilization. ComW protein act as a barrier that protect σ<sup>x</sup> from being degraded by ClpXP degradation enzyme, hence it increase the production of σ<sup>x</sup>. The increase in σ<sup>x</sup> production will increase the expression of green fluorescence protein by P<sub>celA</sub | + | <h6>σ<sup>x</sup> and ComW are both produced by a constitutive promoter J23100, which continuously expressing σ<sup>x</sup> protein required for P<sub>celA</sub> and P<sub>comFA</sub> promoters induction, and ComW protein required for σ<sup>x</sup> stabilization. ComW protein act as a barrier that protect σ<sup>x</sup> from being degraded by ClpXP degradation enzyme, hence it increase the production of σ<sup>x</sup>. The increase in σ<sup>x</sup> production will increase the expression of green fluorescence protein by P<sub>celA</sub> and P<sub>comFA</sub> promoters.</h6> |
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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 | + | 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 σ<sup>x</sup>, and <i>comW</i> genes from the genomic DNA of <i>S. pneumoniae</i> 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> |
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. | 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. |
Revision as of 14:44, 10 October 2014
S. pneumoniae σx promoters module
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 σx. σX (ComX) serve as a competence-specific global transcription modulator. 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 associate 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 a 8 base pairs consensus sequence TACGAATA known as the Cin-Box or the Com-Box. (Piotrowski, Luo, & Morrison, 2009) 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: PcelA (BBa_K1379000) and PcomFA (BBa_K1379001).
PcelA and PcomFA promoters can be found on many different regions within the genomic DNA of Streptococcus Pneumoniae 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. PcelA and PcomFA promoters have high specificity to σx for activation, so genes downstream the promoters will be translated only if σx are 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
Besides σx, 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 σx 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 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 J23100 and strong RBS B0034) from the BioBricks to express those genes. |
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