Team:Hong Kong HKUST/pneumosensor/module two
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Revision as of 11:41, 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. 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: PchbB (BBa_K1379000) and PcomFA (BBa_K1379001).
PchbB 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. PchbB 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, PchbB and PcomFA promoters system could be further utilized as a specific reporter device 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 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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