Engineering Success

Overview

We follow a Design-Build-Test-Learn cyclic pattern to move the project forward. This year's project was roughly divided into three cycles. In the first cycle, we constructed CsTBH and CsPAL overexpression vectors, which are the prerequisites for the synthesis of flavonoids in plants, and stored the constructed CsTBH-PCY and CsPAL-PCY in E. coli; in the second cycle, we constructed the CsTBH-PJG and CsPAL-lacZi vectors, which were used to verify the regulatory relationship between CsTBH and CsPAL, and to clarify the relationship between CsTBH and CsPAL. the regulatory relationship between CsTBH and CsPAL, and after clarifying that CsTBH positively regulates CsPAL, we entered the third cycle; we designed the bioreactor based on the results of the first two parts. CsTBH-PCY/CsPAL-PCY was cotransformed into cucumber plants, and the phenotypes of cucumber fruit spines were observed for the quantitative assay of in vivo plant bioreactors.

Fig.1: DBTL (design-build-test-learn) schema diagram for this project

Cycle1 Construction and preservation of CsTBH and CsPAL overexpression vectors in E.coli.

Design

In biological studies, E. coli often serves as a host for functional vectors. We constructed vectors for CsTBH-PCY, CsPAL-PCY by cloning the sequences of the coding regions of the genes CsTBH and CsPAL, ligating them into the overexpression vector PCY, and then preserving their plasmids in E. coli (DH5α).

Fig.2: Mapping of PAL-LacZi, TBH-PJG, PAL-PCY and TBH-PCY plasmid vectors

Build

1. Normal PCR

2. Gel Electrophoresis

3. Assembly

4. E. coli Transformation

5. Colony PCR

Fig. 3: Sequencing results of CsTBH-PCY vector

Fig. 4: Sequencing results of CsPAL-PCY vector

Fig. 5 PCR results of CsTBH-PCY E. coli colonies

Fig. 6 PCR results of CsPAL-PCY E. coli colonies

Test

The constructed PCY-TBH as well as PCY-PAL can grow on E. coli.

Fig.7 Growth of constructed E. coli colonies of TBH-PCY and PAL-PCY vectors on culture medium

Learn

Successful chassis synthesis vectors were found to be constructed by cloning the gene and culturing.

Cycle2 Validation of CsTBH regulation of CsPAL expression in yeast system

Design

To verify that the TBH gene is able to interact with the cis-acting regulatory elements on the promoter of the PAL gene, and thus to demonstrate that the two interact.

Build

1、Vector digestion

2、PCR cloning

3、Homologous recombination

4、Plasmid extraction

5、Yeast cell transformation

Test

Demonstrate the reliability of results using the blue color development system

Fig.8 Yeast color development in chromogenic plates and analysis of interactions

Learn

The yeast one-hybrid assay was able to demonstrate that CsTBH binds the promoter of CsPAL, theoretically validating the feasibility of using the CsTBH-CsPAL module as a flavonoid bioreactor.

Cycle3 Design of CsTBH -CsPAL Module in Plant Live Bioreactors

Design

In order to design a plant bioreactor that produces flavonoids as expected, CsTBH-PCY and CsPAL PCY were co expressed in plants using a system of Agrobacterium mediated infection of cucumber cotyledons based on the results of the first two experiments. The purpose is to expect that our bioreactor can truly produce more flavonoids, which can be better applied in human life.

Build

1、Fluorescence color development

2、Tissue culture

3、Phenotypic investigation

4、Liquid chromatography

Test

Validation of flavonoid production using transgenic phenotypes, substance assays

Fig.9 Fruit phenotypes of CsTBH overexpressing transgenic plants

Fig.10 Mass spectra and bar graphs of total flavonoids content in the fruits of CsTBH overexpression plants by LC-MS

Learn

The CsTBH-CsPAL module is feasible as a flavonoid bioreactor. Providing a basis for large-scale production in future plant factories.