We have achieved following experiment results as our PROOF OF CONCEPT.
(A) We have constructed a signal pathway (in principle) in which bacteria detects Nitric Oxide (NO) released in the inflammation sites in IBD patients, expresses therapeutic molecule - Epidermal Growth Factors (EGF) and releases them back to inflammation sites to relieve IBD symptoms. This is illustrated in Figure 1.
Specifically, 1) we mimicked NO release at pathological concentration by SNP (see NO release).
2) we successfully used NO (released by SNP) to induce expression of a demo protein (GFP), under control of a NO sensitive promoter (pNorV) (see NO sensing). The demo protein can be easily replaced by the therapeutic molecule – EGF.
3) we successfully demonstrated that our bacterial expressed EGF could relieve the symptoms in IBD mice (see collaboration). EGF suppressed the reduction of colon length and rescued weight loss phenotype.
(B) We have constructed a therapeutic biofilm system (in principle) for potential delivery of therapeutic molecules, in which we successfully fused a demo protein (EGF) to the biofilm protein CsgA in CsgA null bacteria (see therapeutic biofilm). The demo protein can be easily replaced by other therapeutic molecules which can function remotely (such as enzymes) without detaching from the biofilm, and thereby helping patients in long term. This is illustrated in Figure 2.
(C). We have constructed a “warship” to protect and confine our engineered bacteria, attempting to increase the surviving power of introduced bacteria against native microbiota and biosafety levels of using engineered bacteria, but still allow exchanging of nutrient and chemical through semi-permeable membranes (see warship). This is illustrated in Figure 3.
(D). We have constructed two kill switches which can inhibit bacteria growth under our control (see kill switch). We can either use light (via toxin fluorescent protein killer red) or a small molecule (via cell lysis protein gene E) to switch on the kill switch to terminate them when necessary. This is illustrated in Figure 4.