Overview

Our goal is to construct a comprehensive THC detection-degradation system. Thus, our project has four modules, namely THC-sensing module, metabolic module, suicide module and quorum sensing module. We used the E.coli substrain Nissle 1917 as our chassis and added several modules to it to fulfill our goal.

1. THC-sensing module

To alert users that they have ingested THC, we modified prokaryotic PmrA/PmrB system which initially uses the PmrB receptor on the cell membrane to sense the iron level in the environment by using the THC binding motif to sense THC. Inspired by 2021 BNU-China, we replaced the iron (III) binding motif with the anti-Δ9-THC motif(from iGEM19_Queens_Canada), which can specifically bind to Δ9-THC, and then promote PmrB to phosphorylate PmrA. The phosphorylated PmrA activates the PmrC promoter and starts the expression of C-phycocyanin^[1]. (reporter protein)and degradation enzymes.

Fig.1 THC-sensing pathways

2.THC-metabolizing module

On the basis of THC-sensing system, we developed a THC-metabolizing pathway that works upon sensing Δ-9-THC. We adopted three enzymes, CYP2C9, CYP2C19 and UGT1A3, which are necessary to metabolize the psychoactive Δ-9-THC in Cannabis.

Δ-9-THC is hydroxylated by CYP2C9 and CYP2C19, which produces a product that remains psychoactive. Then, the major product of CYP2C9 and CYP2C19 metabolism, 11-OH-Δ-9-THC, was further oxidized to THC-COOH. Finally, UGT1A3 catalyzes the glucuronidation of the former substrate, which promotes the water solubility of the metabolite, thereby facilitating its excretion into the urine. All these genes have been specifically designed to meet codon-bias of our chassis, and coupled to the signal peptide OmpT for stronger protein secretion. ^[2][3]

Fig.2 THC-metabolizing pathways

3.Biosafety module

As a project aimed at implanting engineered bacteria into human body, biosafety has always been the foremost issue. So we designed and tested two security modules for different purposes. First, we expect that our users can stop using our engineered bacteria at any time. Second, we don't want our engineered bacteria to pollute the environment outside the human body.

1) The kill switch controlled by L-arabinose

To enable our users to specifically eliminate the engineered bacteria, we added the L-arabinose inducible promoter, AraC gene, and the gene encoding DNA replication inhibitor (ccdB)^[4] into our pathway. L-arabinose is an inducer that is harmless to human and can be ingested directly. In addition, the content of arabinose in human diet is relatively low, so it will not accidentally trigger the killing switch. And the human body cannot digest arabinose. AraC protein is bifunctional. AraC protein binds to araO1 (-100 ~ -144) and acts as a repressor; when AraC protein combines with inducer L-arabinose to form a complex, it binds to araI region (-40 ~ -78) to bind RNA Pol to pBAD site^[5], and initiates the expression of ccdB. As a specific DNA replication inhibitor, CcdB will not cause bacterial lysis, so activating this "kill switch" will not cause damage to native microorganisms. Besides, there is enough arabinose in the soil, which can effectively prevent engineered bacteria from leaking into the soil.^[6]

Fig.3 kill switch pathways (controlled by L-arabinose)

2) The kill switch that prevent escape

We introduced mazF-mazE toxin-antitoxin system^[7] and an RNA thermometer^[8] to prevent environment leakage of our engineered bacteria.  When it is 37 ℃(in human body), the stem-ring structure of is the RNA thermometer opened, thus, RBS is exposed, the MazE(antitoxin protein) could be normally expressed and can bind MazF(toxin protein) protein makes MazF ineffective.  As a result, the engineered bacteria could survive.  When our engineered bacteria leak out of the body, the temperature is low, and the RNA thermometer retains the stem-ring structure at this time, the mazE gene cannot be expressed, the content of MazF increases, leading to the death of the engineered bacteria.

MazF can recognize mRNA sites and cut mRNA so that it can block translation, but does not cause bacterial lysis, native microorganisms will not be affected.

Fig.4 kill switch pathways (controlled by temperature)

4.Quorum sensing module

In order to ensure that the metabolism of engineered bacteria can efficiently perform their specific functions and not disrupt the local microbial community after colonization, we referred to 2021 BNUZ-China’s quorum sensing design which is orthogonal to the native microorganisms. When the engineered bacteria quantity is small, the expression of GDH by engineered bacteria will enhance their competitiveness, allowing them to rapidly colonize and reach a certain population quantity. Then, due to the limitations of the quorum sensing mechanism, the engineered bacteria will slow down their proliferation rate to ensure that they do not interfere with the intestinal microbiota. Meanwhile, our engineered bacteria start to express β-galactosidase to catalyze the production of galactose which regulate the colony structure of the intestines and promote the proliferation of probiotic bacteria.

Fig.5 Quorum sensing pathways. A)When the number of engineered bacteria is small, expressing GDH promotes the proliferation of engineered bacteria; B)The engineered bacteria express β-galactosidase to regulate intestinal colony structure after colonization

References

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8. Sen S, Apurva D, Satija R, Siegal D, Murray RM. Design of a Toolbox of RNA Thermometers. ACS Synth Biol. 2017;6(8):1461-1470. doi:10.1021/acssynbio.6b00301
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