Team:Hong Kong HKUST/pneumosensor/module two

Figure 1. σ^x-Com-Box promoter mechanism

The reporter system contains a constitutive promoter BBa_J23100, which continuously expresses σ^x required for Com-Box promoter induction. σ^x will then bind to Com-Box promoter and express green fluorescence protein. The whole construct was built in E. coli DH10B strain.

In order to achieve the functionality of pneumosensor, we must have a highly specific reporting system which will only give fluorescent signal under the presence of S. pneumoniae. In search for the suitable gene circuit, the discovery by Prof. Morrison on the competence for genetic transformation in S. pneumoniae which depends on quorum-sensing system to control many competence-specific genes acting in DNA uptake, processing, and integration has provided the ideal framework for this module. (Lee and Morrison, 1999) There is a link between this quorum-sensing system and the competence-specific genes, which is an alternative σ^x (ComX protein) that serves as a competence-specific global transcription modulator. (Luo and Morrison, 2003) 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, σ^x will be expressed and associated with RNA polymerase apoenzyme. The resulting holoenzyme will then be guided by σ^x to initiate transcription of a set of “late” genes enabling genetic transformation and other unknown functions. Characterized genes regulated by σ^x were found to contain an 8 base pairs consensus sequence TACGAATA known as the Cin-Box or the Com-Box. (Piotrowski, Luo, & Morrison, 2009). Taking advantage of this competence-specific mechanism, it is now able to produce the S. pneumoniae sensing device of high specificity by incorporating this system into E. coli.

iGEM 2014 Hong_Kong_HKUST Team has cloned σ^x from S. pneumoniae strain NCTC7465 and characterized its ability to initiate transcription of two downstream promoters with different lengths: P_celA (BBa_K1379000) and P_comFA (BBa_K1379001), which have the consensus Com-Box sequence. Though much information about 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. 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.

P_celA and P_comFA promoters have high specificity to σ^x for activation, so genes downstream the promoters will be translated only if σ^x is present. Hence, by using fluorescence protein as a reporting mechanism, this σ^x, P_celA and P_comFA promoters system could be further utilized as a specific reporter device in E. coli DH10B strain that could be used by iGEM communities.

Figure 2. σ^x - comW Interaction Diagram

σ^x and ComW protein are both produced by a constitutive promoter BBa_J23100, which continuously expresses σ^x required for P_celA and P_comFA promoters induction, and ComW protein is required for σ^x stabilization. ComW protein acts as a barrier that protects σ^x from being degraded by ClpXP degradation enzyme, hence it increases the production of σ^x. The increase in σ^x production will increase the expression of green fluorescence protein by P_celA and P_comFA promoters.

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 X 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 comX gene expressing σ^x, and comW genes from the genomic DNA of S. pneumoniae NCTC 7465 strain. We then used BBa_K880005 (consisting of constitutive promoter BBa_J23100 and strong RBS BBa_B0034) from the BioBricks to express those genes.

Lastly, we combined these constructs with P_celA and P_comFA promoters and GFP generator to check the functionality of the system, and calculate the Relative Promoter Unit (R.P.U) of the promoters.