in vivo Detection

Our pathogen detection system consists of two collections of interconnected genes (operons) placed inside specific regions (loci) of the chromosome. The first operon is designated the "reporter" and consists of a constitutive promoter (Pveg), a ribosomal binding site, a repressible operator(C2P22), and a chromophore (lacZ). Our "reporter" operon works in conjunction with our "repressor" operon, which consists of the same promoter, ribosomal binding site, and a repressor gene designed to negate the function of aforementioned operator (C2P22). The repressor gene will be flanked by sequences homologous to a conserved target sequence of our intended pathogen of interest, such that we can identify any strain present. By default, when the two operons are placed inside B. subtilis, the repressor operon will act upon the operator of the reporter operon and prevent the translation of the chromophore. With the chromophore not being produced, the B. subtilis will refrain from exhibiting a colorimetric output and remain in what we would call the "negative state". However, when a particular pathogen is introduced to the B. subtilis, it will uptake the target sequence contained within the pathogen through homologous recombination. Essentially, the repressor gene, which is flanked by DNA regions homologous to the target sequence, will be replaced by the target sequence and "knocked off" the chromosome. With the repressor gene absent, the reporter operon will be at liberty to produce the chromophore and cause the B. subtilis to yield a colorimetric output. A major strength of our project design lies in its high level of customization and modularity. Using our reporter and repressor operons, we can in theory facilitate the detection of any pathogen with an adequately sequenced genome simply by replacing the homologous flanking regions of our repressor gene with the proper sequences. We have also taken cautious measures to ensure that our B. subtilis colonies are harmless in the unlikely event that they escape the casing of our device. The reporter and repressor genes will be placed into the thrC locus of B. subtilis and effectively replace any genes that were originally present in that location. Because the thrC gene is essential for the synthesis of threonine, an essential amino acid, replacing it with our operons will effectively make the B. subtilis incapable of producing endogenous threonine and render it auxotrophic.