Team:Hong Kong HKUST/pneumosensor/module two

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 a quorum-sensing system to control many competence-specific genes which play a role in DNA uptake, processing, and integration. There is a link between this quorum-sensing system and the competence-specific genes, which is an alternative sigma factor ComX. Two identical copies of gene (comX1 and comX2) encode a competence-specific alternative sigma factor, σ^x. Expression of ComX allows the 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.

The combox promoter is a promoter associated with initiating transcription of a class of genes (commonly referred to as late competence genes) coding for bacterial competence proteins and bacterial transformation in Streptococcus Pneumoniae strains. As described before, it contains a general consensus sequence of TACGAATA for recognition by its promoter-specific sigma factor X, which is a protein expressed from the early competence gene comX.

In fact, the combox promoter can be found on many different regions within the genomic DNA of Streptococcus Pneumoniae strains. Researches and investigations have also reported variants of the combox promoter having 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 full characterization including promoter activity, specificity, sequence, as well as the biomolecular mechanism can be greatly enhanced with further investigations and experiments. As part of module II, we were interested in reproducing this gene circuit with all the associated genes and promoters to be combined into a single transcriptional unit. To the best of our knowledge, the genes and promoters of this gene circuit for bacterial transformation are found at different locations of the long genomic DNA of many Streptococcus Pneumoniae strains. 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. With part I of module to focusing on ComX, part II of module II focused mainly on isolating the combox promoter, and also identifying the most probable length of the promoter. Characterization of the combox promoter was carried out together with the characterization of comX gene. Because combox promoter is highly specific to σ^x for activation, genes downstream of the combox promoter will be translated only if σ^x are present. Hence, by using fluorescence protein as a reporting mechanism, this comX-combox system could be further utilized as a specific reporter device that could be used by the iGEM community.

Besides ComX, another positive factor involved in competence regulation was later found out to be ComW. The comW gene (SP0018) is regulated by the quorum-sensing system and is required for a high-level of competence. Coexpression of comW with comX restores the accumulation of σ^x and the expression of late genes as ComW contributes to the stabilization of the alternative sigma factor σ^xagainst proteolysis and is required for full activity of σ^x in directing transcription of late competence genes. ComW functions to act as a barrier which covers comX protein from being degraded by the ClpXP degradation enzyme. Hence, ComW will be degraded instead of ComX, and the production of ComX protein will be increased.

Based on these findings, we would like to integrate this alternative sigma factor system from Gram-positive Streptococcus pneumoniae into Gram-negative Escherichia coli. Firstly, we cloned out the comX and comW genes from the genomic DNA of S. pneumoniae NCTC 7465 strain. We then used BioBrick BBa_K880005 (consisting of constitutive promoter J23100 and strong RBS B0034) to drive the expression of those genes. Lastly, we combined these constructs with the combox promoter and GFP generator to check the functionality of the system, and to calculate the relative promoter unit of combox promoter.