Part:BBa_K4117888:Experience

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Applications of BBa_K4117888

“T7 promoter+ PmrA+ RBS+ PmrB+ rnaB T1 terminator+ pmrC promoter” is the THC sensing pathway. To alert vaccinators that they have ingested THC, we design a THC sensing system. In this pathway, anti-THC can specifically bind to THC, and then promote PmrB phosphorylation of PmrA. PmrC promoters activate phosphorylated PmrA proteins and initiate transcription to express pigment proteins. Pigment proteins can be excreted in the stool, acting as a warning.

Outer membrane protease T (OmpT) is a 33.5 kDa endoprotease located on the outer membrane of Escherichia coli. OmpT signal peptide is an excretion tag linked on the N-terminus of protein. It can transport proteins across the outer membrane with the help of OmpT.

UGT1A3 in this pathway plays an significant role in metabolic section, working upon the sensing section. Tetrahydrocannabinol (Δ9 -THC), the primary psychoactive ingredient in marijuana, is subject to cytochrome P450 oxidation and subsequent UDP-glucuronosyltransferase(UGT)-dependent glucuronidation. Many studies have shown that CYP2C9 and CYP2C19 are the primary enzymes responsible for these cytochrome P450-dependent oxidations. In Mazur’s study, the specific human UGTs are responsible for classic cannabinoid metabolism, and among them, the highest activity toward THC-COOH was observed with UGT1A3.

Thus, in our system, Δ-9-ThC is hydroxylated by CYP2C9(BBa_K4117002) and CYP2C19(BBa_K4117022), remaining psychoactive. The major product of CYP2C9 and CYP2C19 metabolism, 11-OH-Δ-9-ThC, was further oxidized to THC-COOH. Then UGT1A3, which catalyzes the glucuronidation of the former substrate, increases the metabolite's water solubility, thereby facilitating excretion into the urine.

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