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(lacZ), capable of producing a blue pigment in the presence of X-gal. 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). Repression of the operator within the reporter operon is made possible by the unique repressor proteins produced by the repressor gene of the repressor operon. 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 gene will act upon the operator of our reporter and prevent the transcription of lacZ). With the chromophore not being produced, the B. subtilis will refrain from exhibiting a colorimetric output and will remain in the "negative state". However, when a particular pathogen is introduced to our engineered 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 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 that might be present within the blood 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 engineered B. subtilis are harmless in the unlikely event that they escape the casing of our device. The reporter and repressor genes have been placed into the thrC locus of B. subtilis and effectively replace the genes that were originally present in that location. Because the thrC gene is needed for the synthesis of threonine, an essential amino acid, replacing it with our operon leaves B. subtilis incapable of producing endogenous threonine and thus renders it auxotrophic.

Figure 1: B. s Detector Schematic