Team:Hong Kong HKUST/pneumosensor/modules

Figure 1. Detection Module Diagram

CSP released by S. pneumoniae is detected by its receptor ComD, which autophosphorylates at the expenditure of ATP. ComD~P then phosphorylates ComE. ComE~P then induces the promoter P_comCDE, which drives the expression of GFP.

Transformation in Streptococcus pneumoniae, like many other species, depends on specialized state called competence. Competence is achieved during the exponential growth stage of pneumococcal culture by the secretion of competence-stimulating peptide (CSP), which is a 17- residue long pheromone that is species-specific.

Competence development controlled by a two-component regulatory system (TCS), which consists of the histidine kinase (HK) ComD and its cognate response regulator (RR) ComE. When CSP binds to it, ComD autophosphorylates to become phospho-ComD, ComD~P; at the expenditure of ATP. The cytosolic protein ComE is then phosphorylated by ComD~P through transphosphorylation reactions, producing ComE~P.

These two genes are part of the comCDE operon, which includes the comC gene (Pestova et al., 1996; Cheng et al., 1997) which encodes a prepeptide that is exported and matured by a dedicated ATP-binding cassette (ABC) transport protein, into CSP. The ABC transport protein is encoded by the comAB operon.

Interestingly, ComE~P binds to repeat sites adjacent to both comCDE and comAB (Ween et al., 1999), thereby creating a positive feedback loop, producing both pre-CSP and its required machinery for maturation and transport. The signal is thus amplified and competence is coordinated throughout the population. comD and comE genes were cloned out from the genomic DNA of the NCTC strain of S. pneumoniae. They would be contitutively expressed in our Pneumosensor.

As mentioned above, ComE~P induces several promoters, one of which is P_comCDE. In part 2 of our detecting circuit design, we make use of the inducible promoter P_comCDE. We obtain the sequence by oligos, and will characterize it with the help of green fluorescence protein (GFP) that we constructed in the downstream of the promoter by BioBrick RFC10. Rather than using ComE~P that has to be phosphorylated by ComD~P, which involves a chain of reactions, we use a phosphorylmimetic ComE mutant, ComE^D58E, gratefully shared by Bernard Martin et al. After characterization of P_comCDE, we hope to put P_comCDE together with Module 2 of our project, as the ultimate goal of our project is to make a system of automatic detection and lysis of S. pneumoniae.

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.

To complete the story of competence regulation mechanism from S. Pneumoniae, we would also like to integrate another positive factor involved in competence regulation which was later found out to be ComW. Prof. Morrison's lab released another research paper on the identification of a new component in the regulation of genetic transformation in S. Pneumoniae. The gene comW (SP0018) was found to be regulated by the quorum-sensing system and is required for a high-level of competence (Luo, Li, and Morrison, 2004). 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 (Piotrowski, Luo, and Morrison, 2009).

Based on these findings, we tried to integrate this ComW into the mechanism to see whether and how the presence of ComW affects σ^x. 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.