iGEM 2022 — Team rs-united-china

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**Important Dates**

**August 3rd**

Objective:

-To study how to use machines involved in our experiment, including the PCR amplifier, the centrifuge, and the horizontal gel electrophoresis apparatus.

-To prepare the consumables for later experiment

-To extract the DNA genome out of the poplar leaf

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**August 4th**

Objective:

-To amplify our targeted DNA sequence (COMT)

-To connect COMT to pEASY vector

-To insert the plasmid into Escherichia coli

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**August 5th**

Objective

-To determine the presense of COMT in Escherichia coli using colony PCR

-To learn seamless cloning for later experiment

-To check the sequence of the plasmid

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**August 6th**

Objective

-To extract the plasmid from Escherichia coli colony

-To test the concentration of plasmid using OD 260/OD 280

-To clone plasmid using PCR

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**August 7th**

Objective

-To reclaim plasmid using gel electrophoresis

-To connect the targeted DNA to vector PBI121

-To insert PBI121into competent Escherichia coli

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**August 8th**

Objective

-To check the presence of the vector using colony PCR

-To insert PBI121 into Agrobacterium tumefaciens

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**August 9th**

Objective

-To pre-culture poplar leaf allowing effective infestation of Agrobacterium tumefaciens

-To conduct Agrobacterium infestation

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**August 10th**

Objective

-To extract melatonin from poplar saplings

-To check the concentration of melatonin in each group of saplings

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CONTENTS

[Design:](#a1)
[Build:](#a2)
[Test:](#a3)

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Design:

We first amplified the COMT gene sequence, constructed the overexpression vector, and transformed the night disk into transgenic poplars by the leaf disk method. The endogenous melatonin content of transgenic poplars was measured, and the difference of melatonin between transgenic plants and wild-type plants was compared.

Build:

a )PtoCOMT was cloned successfully

Specific primers, enzymes and cDNA were reacted in PCR to obtain gene fragments ligated to the pEASY vector. The sequencing results showed that we successfully cloned PtoCOMT. And the length of this gene is 1098bp.

![](https://static.igem.wiki/teams/4402/wiki/engi/1.png)

Figure 1. (a) Electrophoregram of PtoCOMT. (b) PtoCOMT gene sequence.

b)Construction of expression vector and genetic transformation in populus

The gene was successfully attached to the PBI121 vector by seamless cloning technology. The recombinant PBI121-PtoCOMT-GFP vector was transformed into Agrobacterium tumefaciens and then transformed by leaf disc method. And transgenic plants were obtained.

![](https://static.igem.wiki/teams/4402/wiki/engi/2.png)

Figure 2. (a) PBI121-PtoCOMT vector diagram. (b) Wild-type populus and transgenic populus.

Test:

Over-expression on endogenous melatonin contents in populus

To ascertain the direct involvement of PtoCOMT in melatonin biosynthesis, transgenic populus with overexpression of PtoCOMT were generated. We used Melatonin ELISA Kit to detect melatonin in wild-type and transgenic plants. A standard curve needs to be constructed based on the standards in the kit at concentrations of 0,1,2,4,8,16 pg/ml. The contents of melatonin in transgenic populus increased by 1.5-fold compared to that in wild-type populus. Therefore, we found that COMT was not efficient in catalyzing the synthesis of melatonin, so we wanted to develop a method to improve the synthesis efficiency.

![](https://static.igem.wiki/teams/4402/wiki/engi/3.png)

Figure 3. Melatonin content in wild-type and transgenic populus

design and test:

Bioinformatics modification of PtoCOMT to improve the efficiency of melatonin synthesis

We identified positive selection sites of PtoCOMT by bioinformatics analysis. These sites were then mutated to construct a mutant library. The results showed that mutations at positions 48, 49, 89, 92 position have high degree of significance. And we used alphafold to predict protein structure and it turns out that the structure does change at these sites. So we sort of figured out a way to design proteins.

Table 1. Positive selection sites calculated by bioinformatics.

![](https://static.igem.wiki/teams/4402/wiki/engi/table1.jpg)
![](https://static.igem.wiki/teams/4402/wiki/engi/4.png)

Figure 4. Protein structure before and after mutation at 48, 49, 89, 92.

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