iGEM 2024 — Team csmu-nchu-taiwan


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[Cycle 1](#1)
[Cycle 2](#2)
[Cycle 3](#3)

# Cycle 1: Testing the Performance of Different Vectors

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**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.

![](https://static.igem.wiki/teams/5355/wet/exp/12.png)

![](https://static.igem.wiki/teams/5355/wet/13-55.png)

# Cycle 2: Testing Yield from Different Tobacco Varieties

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**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.

![](https://static.igem.wiki/teams/5355/wet/exp/13.png)

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

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**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.

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[*Nicotiana benthamiana* Experiment](#1)
[Zebrafish Experiment](#2)
[Protocol](#3)

# *Nicotiana benthamiana* Experiment:

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The experiment began by extracting total RNAs from the hypocotyl tissue of beet and red-leaf beet with TriPure reagent, which was then used to synthesize complementary DNA (cDNA) through reverse transcription. Then we used RT-PCR for amplifying BvAD6 and BvADH, respectively, from the beet and red-leaf beet cDNA.

![](https://static.igem.wiki/teams/5355/wet/exp/1.png)

Figure.1 Total RNA preparation from cotyledon and hypocotyl of Beet and Redleaf Beet

![](https://static.igem.wiki/teams/5355/wet/exp/2.png)

Figure.2 BvAD6 and CTPBvADH amplification from hypocotyl cDNA of Beet and Redleaf Beet

The amplified BvAD6 and BvADH gene fragments were then cloned into the pKBdCAD6, pKBdTCP19AD6, pKCydCAD6, and pEAQCTPBvADH plasmid vectors to create recombinant plasmids. These recombinant plasmids were introduced into the Agrobacterium tumefaciens strain GV3850 in using electroporation.

![](https://static.igem.wiki/teams/5355/wet/exp/3.png)

Figure.3 Simplified diagrams of vectors

![](https://static.igem.wiki/teams/5355/wet/exp/4.png)

Figure.4 Use Western blot to determine the vector build

![](https://static.igem.wiki/teams/5355/wet/exp/5.png)

Figure.5 The simplified diagram of pEAQBvADH and the determination

![](https://static.igem.wiki/teams/5355/wet/exp/6.jpg)

Figure.6 The process of optical density test

![](https://static.igem.wiki/teams/5355/wet/exp/7.jpg)

Figure.7 The process of electroporation

The Agrobacterium strains carrying the desired gene constructs were then used to infiltrate Nicotiana benthamiana (benthamiana) plants through agroinfiltration. Leaf samples were collected from the infiltrated benthamiana plants at 3, 5, and 7 days post-agroinfiltration (dpai). Total protein was extracted from these samples and subjected to protein assays to analyze enzyme activity.

![](https://static.igem.wiki/teams/5355/wet/exp/8.jpg)

Figure.8 Tobacco after Agro-infiltration

![](https://static.igem.wiki/teams/5355/wet/exp/9.jpg)

Figure.9 Detect the infection of Agrobacterium

![](https://static.igem.wiki/teams/5355/wet/exp/10.jpg)

Figure.10 The infected leaves under normal light

![](https://static.igem.wiki/teams/5355/wet/exp/11.png)

Figure.11 Detection of AD6 and ADH expression in N. benthamiana

![](https://static.igem.wiki/teams/5355/wet/exp/12.png)

Figure.12 Detection of AD6 and ADH expression in N. benthamiana

Finally, we used high-performance liquid chromatography (HPLC) method for analyzing and quantifying the L-DOPA from leaves.

# Zebrafish Experiment:

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The aim of this experiment was to evaluate the therapeutic effect of different sources of L-DOPA (Stednitz et al., 2015) on 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) (Kim et al., 2023; Manasa, Chitra, & Tamilanban, 2020; Omar, Kumar, & Teoh, 2023; Stednitz et al., 2015) induced therapeutic effects in a zebrafish model of Parkinson's disease. The following experimental planning was used to investigate the dosage effect, neuroprotective effect (Cronin & Grealy, 2017) and neurorestorative capacity (Cronin & Grealy, 2017) of L-DOPA, as well as the dosage effect of MPTP.

**Danio Vision Setup**

Zebrafish embryos were placed individually into each well of a 24-well plate, with one embryo per well. The plate was then placed into the Danio Vision system for testing.
For the test, the embryos were first given a 5-minute acclimation and rest period. After this, a 3-minute observation period of free swimming behavior was conducted. The Danio Vision camera captured the embryos’ movements during these 3 minutes, recording data such as swimming distance, speed, and acceleration. Swimming distance was selected as the primary parameter for observation and analysis.

**Immunohistochemical staining**

Parkinson's disease patients experience degeneration of the substantia nigra in the brain, leading to a deficiency of dopaminergic neurons. In zebrafish, the human substantia nigra corresponds to the bilateral ventral brain. Therefore, we aimed to observe the number of dopaminergic neurons in the zebrafish bilateral ventral brain through immunostaining to assess the damage caused by MPTP to these dopaminergic cells.

However, due to insufficient technical expertise and time constraints, we were unable to quantify the results and can only provide photos for reference.

## Neuroprotection Experiment

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![](https://static.igem.wiki/teams/5355/wet/exp/danio1-1.png)

### L-DOPA dose test

* L-DOPA (24~120 hpf) : 0.5 mM / 1.0 mM
* Test: Fertilized zebrafish embryos were cultured in egg water in a petri dish. After 24 hours, the embryos were transferred to a 24-well plate, with 8 embryos per well. The embryos were immersed in 2 mL baths of 0.5 mM and 1.0 mM L-DOPA, with each concentration occupying two wells. At 120 hpf, swimming distance and survival rates were observed using Danio Vision.

### MPTP Dose Test

* MPTP (48~120 hpf) : 50 μM / 100 μM / 200 μΜ
* Fertilized zebrafish embryos were cultured in egg water in a petri dish. At 48 hours post-fertilization (hpf), the embryos were transferred to a 24-well plate, with 8 embryos per well. Each well was filled with 2 mL of 50 μM, 100 μM, or 200 μM MPTP, with each concentration occupying two wells. At 120 hpf, swimming distance and survival rates were observed using Danio Vision.

### Neuroprotection Experiment

* MPTP : 100μM , L-DOPA : 0.5mM
* Fertilized zebrafish embryos were cultured in egg water in a petri dish. At 24 hours post-fertilization (hpf), the embryos were transferred to a 24-well plate. For the Test group, each well received 2 mL of 0.5 mM L-DOPA, with three wells per concentration and 8 embryos per well. For the positive control group, each well was filled with 2 mL of egg water.
  At 48 hpf, the Test wells were changed to a solution of 2 mL 0.5 mM L-DOPA + 100 μM MPTP, while the positive control wells were changed to 2 mL 100 μM MPTP. Swimming distance was observed at 120 hpf using Danio Vision.
* Due to time constraints and uncontrollable factors such as the occasional low yield or lack of egg production in zebrafish, we have only completed the neuroprotective testing of the chemically synthesized standards. We have not yet been able to conduct comparative testing between the plant-synthesized samples and the chemically synthesized standards.

## Recovery Experiment

![](https://static.igem.wiki/teams/5355/wet/exp/danio2.png)

### L-DOPA dose test

* L-DOPA (72~120 hpf) : 0.5 mM / 1.0 mM
* Test : Fertilized zebrafish embryos were cultured in egg water in a petri dish. At 72 hours post-fertilization (hpf), the embryos were transferred to a 24-well plate, where they were immersed in 2 mL baths of 0.5 mM and 1.0 mM L-DOPA, with each concentration occupying two wells and 8 embryos per well. At 120 hpf, swimming distance and survival rates were observed using Danio Vision.

### MPTP Dose Test

* MPTP (48~72 hpf) : 200 μM / 400 μM / 600 μΜ
* Fertilized zebrafish embryos were cultured in egg water in a petri dish. At 48 hours post-fertilization (hpf), the embryos were transferred to a 24-well plate, with each well containing 2 mL of 50 μM, 100 μM, or 200 μM MPTP, using two wells per concentration and 8 embryos per well. At 72 hpf, the MPTP bath was replaced with egg water. At 120 hpf, swimming distance and survival rates were observed using Danio Vision.

### Recovery Experiment

* MPTP : 400 μM , L-DOPA : 0.5 mM / 1 mM
* Fertilized zebrafish embryos were cultured in egg water in a petri dish. At 48 hours post-fertilization (hpf), the embryos were transferred to a 24-well plate, with each well receiving 2 mL of 400 μM MPTP, using three wells for this concentration and 8 embryos per well. At 72 hpf, the Test group had their bath replaced with L-DOPA, while the positive control group had their bath replaced with egg water.
  At 120 hpf, swimming distance was observed using Danio Vision, and immunofluorescence staining was performed for further analysis. Additionally, swimming distance and survival rates were recorded at 120 hpf using Danio Vision.
* Due to time constraints and uncontrollable factors such as the occasional low yield or lack of egg production in zebrafish, we have not yet been able to complete the recovery testing of the standards.

### Assessing Indicators:

1. behavioral analysis (Zhao et al., 2020): we will use Daniovision to assess the swimming behavior of zebrafish, including total path length and swimming speed.

2. Histological analysis (Cronin & Grealy, 2017; Stednitz et al., 2015): Immunohistochemical staining will be performed to assess morphological changes in neurons and expression levels of relevant proteins.

Through this series of experiments, it is expected that the therapeutic effects of L-DOPA can be comprehensively evaluated, including its dose dependence, neuroprotective effects and neurorestorative capacity. Differences in the therapeutic effects of L-DOPA from different sources will also be explored, as well as the possible presence of novel therapeutic compounds in Nicotiana benthamiana extract. This study is expected to provide new insights and potential therapeutic options for the treatment strategy of Parkinson's disease.

# Total RNA extraction with Trizol reagent

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# Western blotting

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# Agrobacterium transformation

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# Inoculation by agro-infiltration

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# UV Spectrophotometer Extraction Procedure

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# HPLC

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# Immunohistochemical staining

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[HPLC](#Neurodegenerative)
[Neuroprotection Experiment](#Platform)
[Recovery Experiment](#Zebrafish)
[Immunohistochemical Staining](#AI)

# HPLC

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Five days after inoculation with bacterial suspension, leaf samples were harvested and 0.1 g of leaf tissue was extracted with 800 μL of 80% methanol for HPLC quantification.
Among all test groups, the highest yield was observed in WT expressing BvAD6,BvADH,P19, with an L-DOPA yield of 2068.02 ng/20 μL, equivalent to 80.396 mg L-DOPA per 100 g of leaf tissue.

In terms of plant synthesis, a yield of 80 mg per 1 g of leaf tissue is considered remarkably high, indicating a highly efficient production pathway for synthesizing compounds in other plants. Beyond the high yield, our production cycle is relatively short—requiring only one month of cultivation before bacterial inoculation, followed by just five days until harvest. This approach has the potential to offer a new method for high-yield, short-cycle plant-based drug synthesis in the future.

![](https://static.igem.wiki/teams/5355/wet/result/hplc-result.png)

## Neuroprotection Experiment

![](https://static.igem.wiki/teams/5355/wet/exp/danio1-1.png)

**L-DOPA Dose Test**

Zebrafish embryos were immersed in 0.5 mM and 1.0 mM L-DOPA bath treatments 24 hours post-fertilization (hpf), and their behavior was observed at 120 hpf using Danio Vision. Swimming distance was recorded and analyzed.

Zebrafish embryos immersed in 0.5 mM and 1.0 mM L-DOPA from 24 to 120 hours post-fertilization (hpf) showed a significant decrease in swimming distance, with the reduction being more pronounced at 1.0 mM. When zebrafish embryos are healthy, immersion in L-DOPA at concentrations of 0.5 mM and 1.0 mM negatively impacts their motor abilities, with the 1.0 mM concentration causing more severe damage. Therefore, in protective assays, only 0.5 mM L-DOPA was used for testing.

|  | Control | 0.5mM | 1.0mM |
| --- | --- | --- | --- |
| Distance Moved (cm) | 47.151 | 10.239 | 5.498 |
| Death rate | 1/16 | 1/16 | 1/16 |

![](https://static.igem.wiki/teams/5355/wet/result/21.jpg)

**MPTP Dose Test**

MPTP is a drug used to establish a Parkinson’s disease (PD) model in zebrafish. We immersed zebrafish embryos in 50 μM, 100 μM, and 200 μM MPTP baths starting 48 hours post-fertilization (hpf), and observed their behavior at 120 hpf using Danio Vision. Swimming distance was recorded and analyzed.

Embryos treated with 100 μM and 200 μM MPTP from 48 to 120 hpf exhibited a significant decrease in swimming distance. At 100 μM MPTP, zebrafish behavior was already impaired, indicating that this concentration is sufficient to cause damage. Therefore, 100 μM MPTP was selected as the concentration for establishing a PD model in protective assays.

|  | Control | 0.5mM | 1.0mM |
| --- | --- | --- | --- |
| Distance Moved (cm) | 47.151 | 10.239 | 5.498 |
| Death rate | 1/16 | 1/16 | 1/16 |

![](https://static.igem.wiki/teams/5355/wet/result/22.jpg)

**Neuroprotection Experiment**

At 24 hours post-fertilization (hpf), zebrafish embryos were immersed in a 2 mL bath of 0.5 mM L-DOPA. At 48 hpf, the solution was replaced with 2 mL of 0.5 mM L-DOPA + 100 μM MPTP, and their behavior was observed at 120 hpf using Danio Vision. Swimming distance was recorded and analyzed.

The group treated with MPTP alone and the group treated with MPTP + L-DOPA showed a significant increase in swimming distance, suggesting that L-DOPA at a concentration of 0.5 mM provides a protective effect on the motor abilities of zebrafish embryos.

|  | Control | MPTP | MPTP L-DOPA |
| --- | --- | --- | --- |
| Distance Moved (cm) | 34.810 | 2.188 | 11.006 |
| Death rate | 1/16 | 0/16 | 0/16 |

![](https://static.igem.wiki/teams/5355/wet/result/23.jpg)

## Recovery Experiment

---

![](https://static.igem.wiki/teams/5355/wet/exp/danio2.png)

**L-DOPA Dose Test**

At 72 hours post-fertilization (hpf), zebrafish embryos were immersed in 0.5 mM and 1.0 mM L-DOPA bath treatments, and their behavior was observed at 120 hpf using Danio Vision. Swimming distance was recorded and analyzed.

Zebrafish embryos immersed in 0.5 mM and 1.0 mM L-DOPA from 72 to 120 hours post-fertilization (hpf) showed no significant difference in swimming distance, indicating that these concentrations do not impair zebrafish motor abilities. Therefore, both concentrations will be used in subsequent therapeutic tests for further experimentation.

|  | Control | 0.5 mM | 1.0 mM |
| --- | --- | --- | --- |
| Distance Moved (cm) | 15.489 | 17.500 | 16.445 |
| Death rate | 1/16 | 0/16 | 0/16 |

![](https://static.igem.wiki/teams/5355/wet/result/24.jpg)

**MPTP Dose Test**

Zebrafish embryos were immersed in 200 μM, 400 μM, and 600 μM MPTP baths from 48 to 72 hours post-fertilization (hpf), and their behavior was observed at 120 hpf using Danio Vision. Swimming distance was recorded and analyzed.

Embryos treated with 400 μM and 600 μM MPTP from 48 to 72 hpf showed a significant decrease in swimming distance. At 400 μM MPTP, zebrafish behavior was already impaired, indicating that this concentration is sufficient to cause damage. Therefore, 400 μM MPTP was chosen as the concentration for establishing a Parkinson's disease (PD) model in therapeutic assays.

|  | Control | 200 μM | 400 μM | 600 μM |
| --- | --- | --- | --- | --- |
| Distance Moved (cm) | 33.672 | 41.320 | 19.757 | 15.681 |
| Death rate | 0/16 | 0/16 | 0/16 | 0/16 |

![](https://static.igem.wiki/teams/5355/wet/result/25.jpg)

**Recovery Experiment**

At 48 hours post-fertilization (hpf), zebrafish embryos were immersed in a 2 mL bath of 400 μM MPTP. At 72 hpf, the MPTP bath was replaced with L-DOPA, and their swimming distance was observed at 120 hpf using Danio Vision.

## Immunohistochemical Staining

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![](https://static.igem.wiki/teams/5355/wet/result/ihc1.jpg)

control

![](https://static.igem.wiki/teams/5355/wet/result/ihc2.jpg)

L-DOPA (0.5 mM)

![](https://static.igem.wiki/teams/5355/wet/result/ihc3.jpg)

control

![](https://static.igem.wiki/teams/5355/wet/result/ihc4.jpg)

MPTP (100 mM)

![](https://static.igem.wiki/teams/5355/wet/result/ihc5.png)

MPTP (100 µM)+L-DOPA(0.5mM)

![](https://static.igem.wiki/teams/5355/wet/result/ihc6.png)

MPTP (100 µM)+L-DOPA(1.0 mM)

![](https://static.igem.wiki/teams/5355/wet/result/ihc7.png)

MPTP (200 µM)

![](https://static.igem.wiki/teams/5355/wet/result/ihc8.png)

MPTP (400 µM)

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