THC-sensing
1 C-Phycocyanin extracellular secretion under IPTG induction
1.1 Synthesis of pathways to sense THC
We designed the Plasmids and had the company GENEWIZ synthesized them.
1.2 Construction of pathway to verify C-Phycocyanin extracellular secretion
We obtain the gene fragments of C-Phycocyanin, signal peptide and tags by PCR from the plasmid synthesized by the company GENEWIZ. After the completion of PCR, electrophoresis is performed to determine the size of DNA, and then DNA fragments are recovered. Finally we insert it into the PET-Duet1 downstream of the T7 promoter and upstream of the T7 terminator to verify the external secretion of C-Phycocyanin under IPTG induction.
1.3 Verification of imported gene pathways
We transformed E.coli BL21(DE3) competent cells with the prepared plasmid for overnight culture. Then, we confirmed that the constructed plasmid has been successfully transformed E.coli BL21(DE3) by colony PCR as well as PCR using the extracted plasmid as the template. Finally, we preserve the engineered bacteria in glycerol and store them at -80℃ for standby.
1.4 Feasibility experiments on IPTG induction
We select E.coli BL21(DE3) strain control, IPTG-induction E.coli BL21(DE3) strain, as the two groups of experiment strains. We inoculated them into two 50mL centrifuge tubes with 50uL Amp of 20mL LB under submerged cultivation(37℃,220rpm) for 6h. After that, we take back the shaken bacteria 1:100 inoculated in four conical flasks containing 250mL LB with 250uL Amp. We add 100μL 500mmol/L IPTG for each 50mL bacterium to induce the expression of C-Phycocyanin for 5h. The fusion expression proteins are extracted using his-tag protein extraction kit and verified by SDS-PAGE gel. In order to confirm the expression of C-phycocyanin, we decided to do western blot on the basis of SDS-PAGE.
2 C-Phycocyanin extracellular secretion under THC induction
2.1 Verification of imported gene pathways
We transformed E.coli BL21(DE3) competent cells with the prepared plasmid for overnight culture. Then, we confirmed that the constructed plasmid has been successfully transformed E.coli BL21(DE3) by colony PCR as well as PCR using the extracted plasmid as the template. Finally, we preserve the engineered bacteria in glycerol and store them at -80℃ for standby.
3 Further experiment
3.1 Feasibility experiments on THC sensation
We select E.coli BL21(DE3) strain control, THC-sensing E.coli BL21(DE3) strain, as the two groups of experiment strains.
Since Δ9-THC needs to be approved by the Public Security Bureau in China before it can be purchased, we chose the non-psychoactive 11-OH-Δ9-THC to test the normal function of the sensing protein. That's because we learned the Anti-Δ9-THC antibody fragment can also bind to 11-OH-Δ9-THC with higher affinity from the paper 2019 Queens_Canada referred to.[1]
We add 5mg 11-OH-Δ9-THC for each 10mL bacterium to reach 0.5mg/mL Δ9-THC and induce the expression of C-Phycocyanin downstream of the PmrC promoter for 5h. The fusion expression proteins are extracted using his-tag protein extraction kit and verified by SDS-PAGE gel.
3.2 Quantitative experiments on THC sensation
We hope to verify the induction efficiency of 11-OH-Δ9-THC by adding different amounts of 11-OH-Δ9-THC, forming different 11-OH-Δ9-THC concentrations, and measuring the expression efficiency of C-phycocyanin.
Therefore, We will add 0.5/1/5/10 mg 11-OH-Δ9-THC for each 10mL bacterium to reach 0.5mg/mL 11-OH-Δ9-THC and induce the expression of C-Phycocyanin downstream of the PmrC promoter for 5h. The fusion expression proteins will be extracted using his-tag protein extraction kit and verified by SDS-PAGE gel.
3.3 Further feasibility experiments on THC sensation
To more directly demonstrate the proper operation of the THC-sensing system, we want to induce our pathways with Δ9-THC rather than 11-OH-Δ9-THC. And we have been actively contacting the approval authorities to obtain the right to purchase Δ9-THC.
THC metabolism module
1. CYP2C9 extracellular secretion under induction
1.1 synthesis of pathways to metabolism THC
We designed the Plasmids and had the company GENEWIZ synthesized them.
1.2 Construction of pathway to verify CYP2C9 extracellular secretion
We obtain the gene fragments of CYP2C9, ompT and His-tag by PCR from the pCYP2C9. After the completion of PCR, electrophoresis is performed to determine the size of DNA, and then DNA fragments are recovered. Finally we insert it into the pET-Duet1 downstream of the T7 promoter and upstream of the T7 terminator to verify the external secretion of CYP2C9 under IPTG induction.
We transform E.coli BL21(DE3) competent cells with the prepared plasmid for overnight culture and preserve the engineered bacteria in glycerol and stored them at -80℃ for standby.
1.4 Feasibility experiments on IPTG induction
We select E.coli BL21(DE3) IPTG-induction E.coli BL21(DE3) strain and add 100μL 500mmol/L IPTG for each 50mL bacterium to induce the expression of CYP2C9 overnight. The fusion expression proteins are purified by his-tag protein extraction kit and verified by SDS-PAGE gel.
2 CYP2C19 extracellular secretion under induction
2.1 synthesis of pCYP2C19 to metabolism THC
We design the pCYP2C19 and had the company GENEWIZ synthesized them
2.2 Construction of pathway to verify CYP2C19 extracellular secretion
We obtain the gene fragments of CYP2C19, ompT and His-tag by PCR from the pCYP2C19. After the completion of PCR, electrophoresis is performed to determine the size of DNA, and then DNA fragments are recovered. Finally we insert it into the pET-Duet1 downstream of the T7 promoter and upstream of the T7 terminator to verify the external secretion of CYP2C19 under IPTG induction.
2.3 Verification of imported gene pathways
We transform E.coli BL21(DE3) competent cells with the prepared plasmid for overnight culture and preserve the engineered bacteria in glycerol and stored them at -80℃ for standby.
2.4 Feasibility experiments on IPTG induction
We select E.coli BL21(DE3) IPTG-induction E.coli BL21(DE3) strain and add 100μL 500mmol/L IPTG for each 50mL bacterium to induce the expression of CYP2C19 overnight. The fusion expression proteins are purified by his-tag protein extraction kit and verified by SDS-PAGE gel.
3 UGT1A3 extracellular secretion under induction
3.1 synthesis of pUGT1A3 to metabolism THC
We design the pUGT1A3 and had the company GENEWIZ synthesized them
3.2 Construction of pathway to verify UGT1A3 extracellular secretion
We obtain the gene fragments of UGT1A3, ompT and His-tag by PCR from the pUGT1A3. After the completion of PCR, electrophoresis is performed to determine the size of DNA, and then DNA fragments are recovered. Finally we insert it into the pET-Duet1 downstream of the T7 promoter and upstream of the T7 terminator to verify the external secretion of UGT1A3 under IPTG induction.
3.3 Verification of imported gene pathways
We transform E.coli BL21(DE3) competent cells with the prepared plasmid for overnight culture and preserved the engineered bacteria in glycerol and stored them at -80℃ for standby.
3.4 Feasibility experiments on IPTG induction
We select E.coli BL21(DE3) IPTG-induction E.coli BL21(DE3) strain and add 100μL 500mmol/L IPTG for each 50mL bacterium to induce the expression of UGT1A3 overnight. The fusion expression proteins are purified by his-tag protein extraction kit and verified by SDS-PAGE gel.
4 Future experiment
To further verify the feasibility of our project, we are planning to measure the activity of the enzyme.
We find an kit, Enzyme-linked immunoassay of human cytochrome P450 family member 2C9 (CYP2C9) test kit, which can be used to figure out CYP2C9's enzyme activity to further our experiment. The experimental principle of this kit is that the level of human cytochrome P450 family member 2C9 (CYP2C9) in specimens is determined by double antibody sandwich method. The microwells are coated with purified human cytochrome P450 family member 2C9 (CYP2C9) antibody making solid phase antibody. The microwells coated with antibody are successively added with cytochrome P450 family member 2C9 (CYP2C9). Then they are combined with HRP labeled cytochrome P450 family member 2C9 (CYP2C9) antibody to form antibody-antigen-HRP-conjugated antibody complex. After thorough washing, TMB substrate is added and then color develops. TMB is converted to blue under the catalysis of HRP enzyme and to the final yellow under the action of acid. There is a positive correlation between color depth and cytochrome P450 family member 2C9 (CYP2C9). The absorbance (OD) is measured with a microplate reader at 450nm wavelength, and the activity concentration of CYP2C9 (CYP2C9) can be calculated by standard curve.
The future experiments of CYP2C19 and UGT1A3 are as same as the CYP2C9.
Suicide Module
1 Arabinose-induced killing switch
1.1 Synthesis of pathways
We designed the plasmid and had the company AZENDA synthesized it.
1.2 Verification of Arabinose-induced suicide pathway
The pathway is designed to enable users to kill engineered bacteria inside their intestines whenever needed. We use arabinose to specifically induce suicide pathways in engineered bacteria. On the one hand, it must eliminate engineered bacteria efficiently. On the other hand, it should be harmless to other microbes in the intestine or human body.
In order to demonstrate that the induction of CcdB expression by arabinose induction can remarkably accelerate the suicide process of engineered bacteria, we adopted the method of colony counting to conduct our experiment. We cultured the strain at 37°C overnight, bacteria were then divided into groups and obtained by centrifugation, after which we resuspended each group of bacteria by LB medium with different concentrations of arabinose(0%, 0.05%, 0.10%, 0.15%, 0.20%)and added the corresponding medium to make the final volume 20mL[2]. Following which we continued to incubate groups of engineered bacteria and coated the diluted bacterial solution on solid LB-kanamycin medium plates every hour. Colonies were counted after 37℃ incubation overnight.
At the same time, to prove that CcdB kills cells without lysing them, we measured the OD600 of each sample of groups to characterize unruptured bacteria cells, while the initial OD600 was measured as the blank sample.
2 Cold-triggered toxin suicide switch
2.1 Synthesis of pathways
We designed the plasmid and had the company AZENDA synthesized it.
2.2 Verification of cold-triggered toxin suicide pathway
This pathway is designed to kill engineered bacteria that escape the human body through a cold-triggered toxin-antitoxin system, preventing engineered bacteria from polluting the environment.
MazF can recognize mRNA sites and cut mRNA so that it can block translation. MazF is invalid when MazE and MazF are expressed at the same time. However, the degradation efficiency of MazE is faster than MazF, so when MazE is not expressed, the content of MazF increases, resulting in the death of engineering bacteria.
The strains are cultured overnight at 37°C, and then aliquoted into pre-heated flasks containing 37°C and 28°C medium to make a final volume of 20 mL, and the OD600 is measured. The two flasks are then incubated at 37°C and 28°C, respectively. Samples are taken every 0.5 hours, the diluted bacterial solution is coated on solid LB-kanamycin medium plates, cultured at 37°C overnight, and the colonies are counted.
The previous effect didn't meet our expectations. In order to explore our design, MazF inhibited the growth of bacteria instead of causing the programmed death of engineered bacteria, the engineered bacteria were diluted and coated on LB-Kanr solid medium and cultured at 28°C, and then a petri dish was taken every hour and placed at 37°C for a total of 8 hours. After growing colonies, the number of colonies was recorded to see if there was a significant change.[3][4]
2.3 RNA Thermometer Verification
2.3.1 Construction of pathways
We obtain the gene fragment of GFP by PCR from the plasmid of our lab, and insert the required enzyme digestion sites at both ends. After the completion of PCR, electrophoresis is performed to determine the size of DNA, and then DNA fragments are recovered. Finally, we use the restriction endonucleases to digest GFP and digest Psuicide plasmid to remove mazE, RBS and mazF fragments, and then ligate the GFP fragment into Psuicide plasmid using T4 ligase. Then, we sequenced the obtained plasmid to verify that the plasmid was correctly constructed.
2.3.2 Verification of RNA thermometer verification pathway
In order to verify the function of RNA thermometers, We culture the strain at 37°C overnight, and then aliquots, adding to prepared flasks filled with 37°C and 28°C LB medium to make the final volume 20mL, then measure the initial OD600 and GFP fluorescence intensity. Samples are taken once every half-hour, OD600 and GFP fluorescence intensity are measured. Besides, we use fluorescence microscope to characterize the fluorescence results.
References
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Brennan, J. (2005)
The production of recombinant single chain antibody fragments for the detection of illicit drug residues.
doctoral thesis, Dublin City University.
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Ariff RM, Fitrianto A, Abd Manap MY, Ideris A, Kassim A, Suhairin A, Hussin AS.
Cultivation Conditions for Phytase Production from Recombinant Escherichia coli DH5α.
Microbiol Insights. 2013 Apr 9;6:17-28. doi: 10.4137/MBI.S10402. PMID: 24826071; PMCID: PMC3987752.
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Amitai S, Yassin Y, Engelberg-Kulka H. MazF-mediated cell death in Escherichia coli a point of no return. J Bacteriol. 2004 Dec;186(24):8295-300. doi: 10.1128/JB.186.24.8295-8300.2004. PMID: 15576778; PMCID: PMC532418
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Tripathi A, Dewan PC, Sid （dique SA, Varadarajan R. MazF-induced growth inhibition and persister generation in Escherichiea coli. J Biol Chem. 2014 Feb 14;289(7):4191-205. doi: 10.1074/jbc.M113.510511. Epub 2013 Dec 27. PMID: 24375411; PMCID: PMC3924284.
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