The circuit we originally designed was mainly divided into two parts, consisting of a plasmid that converts glucose into the key precursor GPP and a multifunctional plasmid that synthesizes herbicides and EPS under the control of blue light commonly used in the greenhouse.
FIG.1 Map of precursor synthesis plasmid
FIG.2 Map of multifunctional plasmids that synthesizes herbicides and EPS
The precursor synthesis plasmid was designed according to the principle of the MVA pathway.
FIG.3 MVA pathway
FIG.4 The precursor synthesis circuit
Considering that multifunctional plasmids need to undertake many functions, the team decided to design multiple circuits to verify the implementation of different functions in a modular manner.
The first modularized circuit is the blue light inducible system modified from the arabinose operon. As shown in the figure, the Arabinose binding and dimerization domain of native AraC protein was replaced with blue light responsive VVD domain, generating VVD-AraC fusion regulatory protein. Then the modified arabinose operon will regulated by blue light (470nm) instead of arabinose. Three different promoters are selected for the expression of VVD-AraC fusion protein to find the optimal one.
FIG. 5 The principle of VVD mediated blue-light inducible system
FIG. 6 The blue light-inducible circuit
FIG .7 Map of the blue light-inducible plasmid
The second modular circuit mainly contains AstABC gene cluster for the synthesis of aspartic acid, under IPTG-inducible Ptrc promoter. It will convert the FPP generated by the precursor plasmid to aspartic acid. The broad-host-vector pBBRMCS1 is used as the backbone. Specific terminators（transcription terminator T1 from the E. coli rrnB gene）and three ribosome binding sites between AA gene clusters were added.
FIG.8 AA synthesis verification circuit
FIG.9 Map of AA synthesis plasmid
The third modular circuit is for the synthesis of exopolysaccharides (EPS), including the EPS synthesis gene pmgA and galU under the control of Ptrc promoter. This is our improved version of the EPS synthetic plasmid in team XJTU-iGEM in 2020.
FIG. 10 EPS synthesis circuit
FIG.11 EPS synthesis plasmid
We designed a fourth modular circuit for the biosafety and project feasibility. We synthesized the lytic gene and make it under the Pλ promoter to achieve the temperature control of bacterial lysis and product release. When the temperature is 42oC or higher, the regulatory protein of CI will degrade to allow the expression of lysis gene, and our engineered bacteria would break, releasing the product herbicide and EPS. About 10% of the engineered bacteria will escape the lysis process and recover, facilitating a new round of controlled production and release of herbicides and EPS.
FIG. 12 EPS synthesis circuit
FIG.13 EPS synthesis plasmid
By the concept of engineering and modular circuits design , we can realize the synthesis and detection of different functional genes more efficiently, fully reflecting the flexibility of synthetic biology.
Finally, we arrives our destination circuits that combines the whole functions of above modular circuits, which can achieve AA and EPS synthesis under the regulation of light, and cell lysis/product release controlled by temperature.
FIG.14 final destination circuit