RIOT invader is activated by the RIOT responder, specifically by the hasS-hasI complex that is formed in the presence of hasA. RIOT responder makes use of an hasA-CD44v6 antibody conjugate, in which the CD44v6 antibody will bind to CD44v6 on the surface of cancer cells, while hasA will stick out from the cancer cells, allowing our genetically modified bacteria (RIOT sensor) to bind to hasA via hasR. This leads to a series of events that results in the activation of RIOT invader. RIOT invader makes use of LuxR and LuxI to activate the production of invasin, listeriolysin O (LLO) and green fluorescent protein (GFP) in high cell density. Invasin is derived from Yersinia pestis, and is a virulence factor that allows the bacterium to bind to β1 integrin present on mammalian cells and enter the cells. LLO is derived from Listeria monocytogenes, and allows the bacterium to escape from the endosome, which has a lower pH. LLO is activated in the low pH of the endosome, at around pH 5.5. LLO forms pores in the membrane of the endosome and results in the lysis of the endosome and the escape of the bacterium into the cytoplasm. The combination of both invasin and LLO allows the bacterium to invade mammalian cells and escape from the endosome into the cytoplasm of the cell.

RIOT invader is thus essentially split into 5 constructs as seen in Figure 1: (1) LuxR (BBa_K1897008), (2) LuxI (BBa_K1897009), (3) invasin (BBa_K1897011), (4) LLO (BBa_K1897013), (5) GFP (BBa_K1897014). Each part is made up of a front fragment (promoter and ribosome binding site (RBS)), a middle fragment (the coding region of the gene), as well as a terminator (transcriptional and translational terminator).

Figure 1: Overall cloning design. (A) shows the 3 parts that each construct is split into, while (B) shows what each part of each construct is made up of. pConst indicates the constitutive promoter BBa_J23119, pLuxR indicates the promoter BBa_R0062, RBS indicates the ribosome binding site BBa_B0030, and the terminator indicates BBa_B0015.

The first part of our cloning involves the stitching (via overlap PCR) of the three fragments (front, middle and terminator) together and putting them into the biobrick plasmid. The second part of the cloning involves putting the 5 parts together into a single plasmid via successive 3A assembly. On our end, we have only managed to put LuxR and LuxI together (BBa_K1897015), as well as invasin and GFP together (BBa_K1897017). We have also managed to put together another variation for testing of the quorum sensing system, which is LuxR and GFP (BBa_K1897016).

The size of each of the constructs made were verified by gel electrophoresis, and the protein expression of certain constructs were also characterised via western blot (Figure 2A, 2B, 2C). We also characterised the quorum sensing system of LuxR+GFP, and external N-(3-oxo-hexanoyl)-homoserine lactone (AHL) was added since LuxI was not present. We looked at the fluorescence of the bacteria under the microscope (Figure 2D) when 0 or 100 μM of AHL was added. There were minimal fluorescence observed at 0 μM AHL, but the green fluorescence can be seen clearly at 100 μM AHL. GFP quantifications via microplate reader of LuxR+GFP was also done for 6 hours (Figure 2E), and the level of fluorescence is higher for induced (100 μM AHL) than non induced (0 μM AHL), indicating that the addition of AHL induced the expression of GFP from pLuxR.

Figure 2: Characterisation of LuxR, LuxI+LuxR and LuxR+LLO. Western blot was done to check for protein expression of (A) LuxR, (B) LuxR+LuxI, (C) LuxR+LLO. The quorum sensing system of LuxR was also characterised, with LuxI being replaced by external AHL. (D) Induction of GFP production with/without AHL. Left Column: Green fluorescence microscopy images of LuxR+GFP bacteria of control with 0 μM AHL added (top) and with 100 μM AHL added (bottom). Right Column: Bright field fluorescence microscopy images of LuxR+GFP bacteria of control with 0 μM AHL added (top) and with 100 μM AHL added (bottom). (E) GFP quantifications via microplate reader of LuxR+GFP for 6 hours is shown in the presence of 0 or 100 μM AHL incubation. The level of fluorescence is higher for induced (100 μM AHL) than non induced (0 μM AHL), indicating that the addition of AHL induces expression of GFP from pLuxR.

Due to the time frame of this project, we would like to wrap up the comprehensive design and proof-of-concept of our RIOT system from here. In perspective, more functional assays of RIOT Invader and full-fledged co-expression with Sensor and Responder could be tested in vivo.