Team:Brasil-SP/Project/DetectionModule

     The genetic circuit that is being developed by the Brasil-SP team is a biodetection system designed for Cystatin C. We will use the Quorum Sensing bacterial recognition system, based on communication between bacteria. This system consists in the recognition and release of substances that diffuse through the environment; those substances are called autoinducers. The autoinducers are responsible for the activation of their own synthesis, allowing the bacterial cells to respond appropriately to cell density. Thus, it is possible to control the expression of specific genes that are only activated when a certain cell concentration is achieved; as a result the behavior of the group and the formation of communities can also be controlled. In our biosensor, the Quorum Sensing substance is called AIP and its receiver is called ComD. Both AIP and ComD are anchored in the cell membrane. The AIP is attached to a linker that is cleaved in the presence of the protease cathepsin S, which can be inhibited by Cystatin C activity. After being cleaved and released from the membrane, AIP binds to the receptor triggering the phosphorylation of ComE (intracellular signalling molecule), which binds to a specific promoter sensitive to ComE, initiating the expression of the downstream gene. (lasR)

     One of the crucial steps in this project is to establish a threshold between normal and abnormal Cystatin C level. This threshold will be established using the threshold system constituted by Pseudomonas aeruginosa QteE and LasR genes. In P. aeruginosa, the expression of QteE is controlled by a constitutive promoter while LasR is indirectly induced by AIP through the action of ComE. We will also use a promoter inducible by isopropyl β-D-1-thiogalactopyranoside (IPTG) to control QteE transcription and translation. The QteE protein destabilizes LasR protein so that LasR cannot induce the expression of downstream genes. While the concentration of QteE is equal or greater than LasR, almost all LasR proteins are destabilized. Thus, QteE creates a barrier to cell response. By controlling expression levels of QteE gene, we manipulate the barrier to standardize the AIP concentration, that it is closely related to the Cystatin C concentration. If the concentration of LasR is high enough to overcome the barrier imposed by QteE, the LasR promoter is induced and the reporter gene Green Fluorescent Protein (GFP) is transcribed. However, the LasR and QteE proteins are part of the Quorum Sensing system of gram-negative bacteria, while our genetic circuit will be built in Bacillus subtilis, a gram-positive bacteria. To guarantee the appropriate folding of LasR protein and the induction of the promoter by LasR, substances called HSLs (homoserine lactone) must be added to the system. HSLs are intermediate products of a pathway triggered by the LasI gene. As these intermediates are found in Bacillus subtilis, the activation of transcription by a constitutive promoter meets the demand for HSLs. On the other hand, the aiiA gene in gram-positive bacteria leads to HSLs degradation. In order to guarantee a good performance of our circuit, the aiiA gene will be knocked out.