1.Construction of Logic Gate Circuits
1.1.1 Sequence Sources
We obtained the gene sequences of LldR, pCaCd, pPepT promoter, and CDD-iRGD from the iGEM website, and added the ssSTII1 localization sequence at the end of the CDD-iRGD sequence. The DNA sequences of CDD-iRGD and switch were synthesized by Sangon (China, Shanghai), and the remaining sequences were synthesized by General Biology (China, Anhui).
1.1.2 Plasmid Vectors
We purchased plasmids with pUC/pACYC177 as the backbone from MIAOLING BIOLOGY (China, Hubei), which have resistance to ampicillin (Amp) or kanamycin (kana). The antibiotics used for screening were purchased from Sangon. The amplification of the LldR gene used PCR technology, the Taq enzyme was purchased from Vazyme Biotech (China, Jiangsu), and primers were synthesized by Sangon.
1.1.3 Techniques Used to Construct Gate Circuits
Our experimental techniques involve plasmid amplification, extraction, enzyme digestion, ligation, and gel recovery. The E. coli we used all came from DH5α strains sourced from MIAOLING BIOLOGY. The adsorption columns used in plasmid extraction came from Biocomma (China, Guangdong). The enzymes used in enzyme digestion experiments all came from New England Biolabs (China, Beijing), and the equipment for gel recovery experiments all came from Vazyme Biotech.
1.2 Experimental Procedure
We amplified the synthesized sequences in E.coli using a plasmid as a vector, and then used enzyme digestion to obtain the target sequence. We constructed a "AND" gate circuit plasmid with pUC19 as the plasmid vector, inserting LldR promoter, trigger A1, T7 terminator, spacer sequence, pPepT promoter, trigger A2, T7 terminator, spacer sequence, pCaCd promoter, trigger A3, T7 terminator. At the same time, we also constructed a target protein plasmid with pU19 as a vector by inserting Switch and CDD-iRGD sequences. We transformed these two types of plasmids into E.coli and obtained the target E.coli that were successfully transformed through resistance screening.
2.In Vitro Verification of Promoters
2.1.1 Reporter Gene
We used luciferin purchased from Beyotime (China, Shanghai) to develop for the reporter gene LUC. We constructed three plasmids, each inserting a conditionally inducible promoter + reporter gene.
2.1.2 Simulation of Hypoxia, High Lactic Acid, Low PH
We purchased anaerobic jars (including anaerobic bags) from Thermo Fisher (USA, Massachusetts) to simulate an anaerobic environment. We used HCl and NaOH solutions to adjust the PH value of the liquid LB medium and added lactic acid to adjust the lactic acid concentration.
2.2.1 Verification of Hypoxic Promoter
We transformed E. coli with the plasmid containing pPepT+LUC. We cultured it in 15ml centrifuge tubes filled with LB liquid medium. We put these centrifuge tubes into an anaerobic bag and cultured them overnight in an anaerobic jar. We set up a control group that was not put into an anaerobic bag or jar. After culturing, we developed LUC and detected fluorescence intensity with an enzyme labeler.
2.2.2 Verification of High Lactic Acid Promoter
We transformed E. coli with the plasmid containing LldR+LUC. We pre-configured LB liquid medium with lactic acid concentrations of 0%, 1%, 3%, 5%, 10%, and inoculated the above-mentioned E.coli for overnight culture on a 37-degree 180rpm shaker bed. After culturing, we developed LUC and detected fluorescence intensity with an enzyme labeler.
2.2.3 Verification of Low PH Promoter
We transformed E. coli with the plasmid containing pCaCd +LUC. We inoculated the above-mentioned E.coli into LB liquid medium and divided it into two parts: one part was adjusted to PH 4.4 with HCl; another part was adjusted to PH 8.0 with NaOH solution after slight shaking; each part was divided into five tubes; we adjusted PH using LB liquid medium to form a gradient of PH 4.4, 4.8, 5.2, 5.6, 6.0, 6.4，6.8,7.2,7.6 and 8; we also used one tube of LB liquid medium with unadjusted PH as a control. After overnight culture on a 37-degree 180rpm shaker bed, we developed LUC and detected fluorescence intensity with an enzyme labeler.