iGEM 2022 — Team rs-joint-china

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CONTENTS

[2022/08/13](#a1)
[2022/08/14](#a2)
[2022/08/15](#a3)
[2022/08/16](#a4)
[2022/08/17](#a5)
[2022/08/17](#a6)
[2022/08/18](#a7)
[2022/08/19](#a8)
[2022/08/20](#a9)

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Notebook

2022/08/13

1.Break the ice

Get to know each other by asking questions and introduce oneself to the others

2.Run for the captain of the team

People who wished to be the captain each made a speech in front of all the members

3.Learn the basic knowledge

4.Design the name and icon of the team

![](https://static.igem.wiki/teams/4401/wiki/note/1.png)
![](https://static.igem.wiki/teams/4401/wiki/note/2.png)
![](https://static.igem.wiki/teams/4401/wiki/note/3.png)

2022/08/14

1.Learning all the instruments in the lab

2.Extract DNA from poplar leaves

3.Clone target genes by PCR

4.Run agarose gel electrophoresis to check the gene

5.Recycle PCR products in the gel

![](https://static.igem.wiki/teams/4401/wiki/note/4.png)
![](https://static.igem.wiki/teams/4401/wiki/note/5.png)
![](https://static.igem.wiki/teams/4401/wiki/note/6.png)
![](https://static.igem.wiki/teams/4401/wiki/note/7.png)
![](https://static.igem.wiki/teams/4401/wiki/note/8.png)

2022/08/15

1.Learn the knowledge about the vector construction

2.Construct the clone vector

3.Escherichia coli genetic transformation

4.Foster the cells in solid medium

![](https://static.igem.wiki/teams/4401/wiki/note/9.png)

2022/08/16

1.Use the colony PCR and run agarose gel electrophoresis to check whether the vector construction is successful

2.Foster the cells in LB liquid after checking

![](https://static.igem.wiki/teams/4401/wiki/note/10.png)
![](https://static.igem.wiki/teams/4401/wiki/note/11.png)
![](https://static.igem.wiki/teams/4401/wiki/note/12.png)

2022/08/17

1.Visit Beijing Zhongguancun Biology Bank

2.Run gel with the gene of interest and combine it with the expression vector

3.Foster the expression vector in the LB liquid

![](https://static.igem.wiki/teams/4401/wiki/note/13.png)
![](https://static.igem.wiki/teams/4401/wiki/note/14.png)
![](https://static.igem.wiki/teams/4401/wiki/note/15.png)
![](https://static.igem.wiki/teams/4401/wiki/note/16.png)
![](https://static.igem.wiki/teams/4401/wiki/note/17.png)

2022/08/17

1.Prepare the PCR system

2.Construct the gene of interest MYB-46 with expression vector

3.Foster the vector

![](https://static.igem.wiki/teams/4401/wiki/note/18.png)
![](https://static.igem.wiki/teams/4401/wiki/note/19.png)
![](https://static.igem.wiki/teams/4401/wiki/note/20.png)

2022/08/18

1.Extract plasmid

2.Transform the yeast receptive state.

3.Prepare the poplar leaves

4.Infect the poplar leaves with the expression vector

![](https://static.igem.wiki/teams/4401/wiki/note/21.png)
![](https://static.igem.wiki/teams/4401/wiki/note/22.png)
![](https://static.igem.wiki/teams/4401/wiki/note/23.png)

2022/08/19

1.Run pcr for the vectors

2.Run agarose gel electrophoresis to check the useful parts

3.Recycle them and use lead to check its ability of bearing the lead

![](https://static.igem.wiki/teams/4401/wiki/note/24.png)
![](https://static.igem.wiki/teams/4401/wiki/note/25.png)
![](https://static.igem.wiki/teams/4401/wiki/note/26.png)
![](https://static.igem.wiki/teams/4401/wiki/note/27.png)

2022/08/20

1.Learn how to get the gene sequence of the gene

2.Prepare for the online speech about our programme

3.Vote to choose the captain of the whole team and the captain of the wet lab and dry lab

![](https://static.igem.wiki/teams/4401/wiki/note/28.png)
![](https://static.igem.wiki/teams/4401/wiki/note/29.png)
![](https://static.igem.wiki/teams/4401/wiki/note/30.png)

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CONTENTS

[Design:](#a1)
[Build:](#a2)
[1.Acquire the gene of interest](#b1)
[1.1 Clone gene](#c1)
[1.2 Construction of clone vector](#c2)
[2.Construction of expression vector](#b2)
[2.1 We constructed PtoMYB46 gene expression vectors pBI121 and pYES2 using seamless cloning technique (Figure 3).](#c3)
[2.2 Transformation of Agrobacterium tumefaciens and yeast](#c4)
[Test:](#a3)
[1.The Genetic transformation of poplar](#b3)
[2.The Genetic Transformation of the Poplar](#b4)
[Synthesis：](#a4)
[1.Positive selection](#b5)

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Engineering Success

Design:

First, we amplified pto-MYB46 gene sequence, constructed overexpression vector, and transformed poplar leaves by leaf disk method to obtain transgenic poplar, and compared the lead ion absorption efficiency of transgenic plants and wild-type plants. At the same time, we constructed yeast expression vectors to detect the tolerance and growth curve of yeast cells under different lead ion concentrations.

Build:

1.Acquire the gene of interest

1.1 Clone gene

Gene of interest refers to the genes that we’re studying. We extracted the MYB46 DNA sequence, then we increased the tiny number of genes substantially (The Polymerase Chain Reaction Technology). The gene of interest repeats to make sure when experimenting genes and expression vectors combine, the rate of combination is faster. We gained the genes from recycling; we used the Gel Electrophoresis to detect the concentration and purity of the genes.

1.2 Construction of clone vector

We built a cloning vector (pEASY) which was in a cyclic form, we used it to connect to the product. pEasy was in a liquid state, and it had an unstable property. To make pEasy more stable, we transformed it into a germ which was the Escherichia Coli. The PCR product was connected with the clone vector using pEASY®-Blunt Cloning Kit, and then transformed into Trans1-T1 Chemically Competent Cell and sent to the company for sequencing. The result of sequencing is figure 3.

![](https://static.igem.wiki/teams/4401/wiki/engi/1.png)

Fig.1 Construction of clone vector. (A)The recycle of Gel Electrophoresis. (B) Clone vector profile of pEASY-PtoMYB46

![](https://static.igem.wiki/teams/4401/wiki/engi/2.png)

Fig.2 PtoMYB46 gene sequencing results

2.Construction of expression vector

2.1 We constructed PtoMYB46 gene expression vectors pBI121 and pYES2 using seamless cloning technique (Figure 3).

![](https://static.igem.wiki/teams/4401/wiki/engi/3.png)

Fig.3 Vector profile of pBI121-PtoMYB46-EGFP and pYES2- PtoMYB46

2.2 Transformation of Agrobacterium tumefaciens and yeast

The constructed expression vectors pBI121 and pYES2 were transformed into Agrobacterium tumefaciens and yeast receptive cells, respectively. Then PtoMYB46 gene was identified by colony PCR.

![](https://static.igem.wiki/teams/4401/wiki/engi/4.png)

Fig.4Identification ofPtoMYB46gene c byColonyPCR

Test:

1.The Genetic transformation of poplar

We needed to transform the genes onto the cells of the plants, T-DNA was a fragment of the plasmids. We used Agrobacterium to make the fragment of PtoMYB46 through T-DNA vector, then transformed the fragment of the PtoMYB46 onto the genome of the recipient plant, it could replicate as the plant replicates. The construct was mobilized into Agrobacterium tumefaciens strain GV3101, which was used for poplar transformation. We found that PtoMYB46 not only positively regulates plant growth, but also promotes Pb2+ uptake (Figure 6).

![](https://static.igem.wiki/teams/4401/wiki/engi/5.png)

Fig.5 Agrobacterium infected the tissues of the poplar leaves (A) Leaves of poplar infect in the infection solution inside the conical flask. (B) Filter paper absorbed the redundant Agrobacterium.

![](https://static.igem.wiki/teams/4401/wiki/engi/6.png)

Fig.6 PtoMYB46 positively regulates poplar growth and Pb2+ tolerance. (A) Phenotypes of PtoMYB46 overexpression and wild-type (WT) poplar lines under normalconditions. Scale bar 5 cm. (B) Phenotypes of PtoMYB46-OE and WT lines after 20 days of further growth under 600 mg⋅kg–1 Pb2+ treatment. Scale bar 5 cm.

2.The Genetic Transformation of the Poplar

We sampled some yeasts, and observed how much lead ion could they absorbed after a period of time. PtoMYB46 expression enhances Pb2+ sensitivity and increases Pb2+ content in yeast. To investigate the cellular function of PtoMYB46, the protein was expressed in BY4741 susceptible to Pb2+ excess. PtoMYB46 and empty vector-complemented BY4741 cells were grown in SD/Ura medium overnight. Cells grown overnight were used for spotting on SD/Ura agar plates supplemented with 0, 10, 20 and 30 μM (CH3COO)2Pb at indicated dilutions. The Pb2+ supplementation of the medium caused more considerable growth inhibition in yeast cells expressing PtoMYB46 than in the control (Fig. 7). We also analyzed the relative growth in liquid media in the presence of Pb2+ in yeast cells. The growth of BY4741 cells expressing PtoMYB46 were Higher than cells transformed with the empty vector (Fig. 8). The growth inhibition due to the functional PtoMYB46 in ycf1 suggested that PtoMYB46 may facilitate the import of Cd inside the yeast.

![](https://static.igem.wiki/teams/4401/wiki/engi/7.png)

Fig.7 Growth of yeast cells expressing PtoMYB46 on plates containing SD/-Ura without (CH3COO)2Pb.

![](https://static.igem.wiki/teams/4401/wiki/engi/8.png)

Fig.8Heavy metal tolerance analysis ofPtoMYB46in yeast cells

Synthesis：

1.Positive selection

PtoMYB46 protein sequences of multiple species were collected from NCBI, and then MAGE was used to perform multiple sequence alignment and draw phylogenetic evolutionary trees. Then, positive selected amino acid sites were analyzed by EasyCodeML. And the library of potential mutants was constructed according to the positive selected amino acid sites (Table 1). We used alphafold to predict protein structure (Figure 9).

Tab.1 Positive selection analysis of PtoMYB46 gene

![](https://static.igem.wiki/teams/4401/wiki/engi/table1.jpg)
![](https://static.igem.wiki/teams/4401/wiki/engi/9.png)

Fig.9 Prediction PtoMYB46 protein structure.

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