Team:Hong Kong HKUST/pneumosensor/module two

Figure 1. σ^x-Combox promoter mechanism

The reporter system contain a constitutive promoter BBa_J23100, which continuously expressing σ^x protein required for combox promoter induction. σ^x protein will then bind to combox promoter and express green fluorescence protein. The whole construct is built in E.Coli DH10B strain.

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 σ^x. Expression of σ^x 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 σ^x-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 σ^x 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 σ^x-combox system could be further utilized as a specific reporter device that could be used by iGEM communities.

Figure 2. σ^x - comW Interaction Diagram

ComX and ComW are both produced by a constitutive promoter J23100, which continuously expressing σ^x protein required for combox promoter induction, and ComW protein required for σ^x stabilization. ComW protein act as a barrier that protect σ^x from being degraded by ClpXP degradation enzyme, hence it increase the production of σ^x. The increase in σ^x production will increase the expression of green fluorescence protein by combox promoter.

Besides σ^x, 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 σ^x restores the accumulation of σ^x 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 σ^x 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 σ^x 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.