Cycle 1: Testing the Performance of Different Vectors


    Design

    We aim to produce L-DOPA using plant synthesis. To achieve this goal, we selected AD6, ADH, and P19 and tested their expressions and yields.

    Build

    We cloned AD6 and P19 into pKBdCGFP, constructing pKBdCAB6 and pKBdTCP19BvAD6. Additionally, we cloned AD6 into pKCydCV, assembling pKCydCBvAD6. We also chose pEAQ as the vector and assemble pEAQBvADH.

    Test

    Western Blot test results indicated that using pKBdCGFP as a vector had higher yields. The tobacco infected with pKBdTCP19BvAD6 produced the highest L-DOPA content.

    Learn

    The results demonstrate that construction of vector was successful, and we effectively expressed these in tobacco. Vector pKBdCGFP is suitable for our project, and the combination of AD6 and P19 have the highest yields.


Cycle 2: Testing Yield from Different Tobacco Varieties


    Design

    We aimed to test whether reducing the disease resistance mechanism in tobacco could effectively enhance Agrobacterium infection and target product yield. Therefore, we selected WT and RdR6i Nicotiana benthamiana for testing.

    Build

    We injected the constructed vectors into both WT and RdR6i Nicotiana benthamiana.

    Test

    We collected tobacco leaves and performed Western Blot analysis to detect L-DOPA yields.

    Learn

    Results showed that the yield of pKBdTCP19BvAD6 in WT tobacco and pKBdTCP19BvAD6 + pEAQBvADH in RdR6i tobacco were comparable. However, in RdR6i tobacco, the yield of pKBdTCP19BvAD6 was significantly lower compared to WT. This indicates that RdR6i tobacco did not perform as expected in this project.


Cycle 3: Testing the Movement of Zebrafish Larvae Under Different Drug Treatments


    Design

    We aimed to observe the movement of zebrafish larvae under different concentrations of L-DOPA and MPTP treatments. Through this testing, we found the optimal concentration for testing the protective and recovery effects of L-DOPA on MPTP-induced Parkinson’s disease symptoms in zebrafish.

    We treated zebrafish larvae at 20-120 hpf and 72-120 hpf with L-DOPA, and at 48-120 hpf and 48-72 hpf with MPTP.

    Build

    We cultured zebrafish embryos in egg water and then transferred them to a 24-well plate for treatment at the planned concentrations.

    Test

    We used Danio Vision to observe the swimming distance of zebrafish larvae at 120 hpf and conducted data analysis.

    Learn

    Through movement data analysis, we found that L-DOPA at specific concentrations did not significantly affect the movement of zebrafish larvae. Additionally, specific concentrations of MPTP performed expected Parkinson’s disease-like symptoms. Finally, we applied these results to protective and recovery testing.