Contributions

1. We provide a detailed protocol for IPTG induced protein expression and affinity chromatography purification. See Methods
2. We provide experimental procedures and methods related to tobacco cultivation, Agrobacterium mediated transient transformation etc.
3. We provide a complete molecular cloning experimental procedure and a directed evolution experimental method based on Error Prone PCR.
4. We provide a complete analysis and design process for enzyme Semi-Rational Design, including molecular docking, molecular dynamics simulation, etc.
5. We provide compound extraction methods and specific techniques for phaselic acid, resveratrol, and crocin.

See Results

1. 1 Improved Part, 3 Composite Parts and 12 Basic Parts were uploaded.
2. We provide four experimentally validated sgRNA design combinations, and have optimized codons for the four enzymes used to synthesize Crocin, two enzymes used to synthesize Resveratrol, and the enzyme used to synthesize Phaselic Acid, all of which are specific to the chassis of Nicotiana benthamiana. We also conducted codon optimization for VvSTS and HCT-M in Escherichia coli.
3. We optimized the sequence of the 35s Minimal Promoter for plant expression vectors using machine learning methods.
4. The three Composite Parts we provide correspond to the heterologous synthesis circuit design of Crocin, Resveratrol, and Phaselic Acid.
5. All of our parts have also been uploaded to the iGEM Plant Parts Collection, please refer to the following link for details. See Parts

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1. The model framework for metabolic flow analysis in the project, including the construction of genome-scale metabolic network model, flux balance analysis, knockout strategy selection and target product yield prediction, provides theoretical support for the feasibility of the project, which is closely related to the substantive advancement of the project and provides a feasible modeling framework for metabolic analysis in plant synthetic biology.
2. We have designed a business model for the analysis of the return on investment and net present value of VersaTobacco target products, providing a feasible model framework for market prospect analysis.
3. Deep Learning-Based Conformer Architecture Provides Solution for tobacco promoter Optimization.
4. Through Directed Evolution and Semi-Rational Design of HCT-M and VvSTS, a series of mutants with improved catalytic activity and thermal stability were successfully screened and constructed, providing new options for efficient synthesis of Phaselic Acid and Resveratrol.

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1. Our software, the Nicotiana Promoter Optimizer with the Conformer model (NPC), offers an integrated solution for optimizing tobacco promoters, designed for future iGEMers utilizing tobacco or VersaTobacco as their chassis.

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1. We developed a comprehensive HP narrative framework focused on the efficiency and safety of plant synthetic biology chassis: bridging the gap from public awareness to academia with demand-driven designs targeting industry needs and addressing biosafety concerns.
2. Through our HP, we explored the biosafety challenges commonly associated with the N. benthamiana chassis and offered perspectives on enhancing project safety from legal and regulatory viewpoints.
3. We also discussed universal strategies to increase heterologous synthesis yields in N. benthamiana.
4. Education: We organized the Sichuan University Applied Cellular and Molecular Activities Competition, establishing it as a highly academic and widely praised school-featured competition. This event provided a new approach to sustainably promoting synthetic biology on university campuses.

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