iGEM 2021 — Team BIT-China

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# Team:BIT-China/Notebook

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# Team:BIT-China/Engineering

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Content

[**Abstract**](#Abstract)
[**Engineering Success**](#Engineering-Success)

[Receptor expression system](#受体表达系统--T1R1（BBa_K3852005）)

[Modification and synthesis of T1R1 gene](#T1R1基因的改造与合成（T1R1的片段长度）)
[Agarose gel electrophoresis](#琼脂糖凝胶电泳)
[****E. coli**** transformation experiment](#大肠杆菌转化实验)
[Electrotransformation](#酿酒酵母的电转化)
[Homologous recombination](#酿酒酵母的同源重组)
[Detection Systems](#检测系统--Pfba1+T2R1+eGFP(-BBa_K3852002))

[OE-PCR (connect Pfba1 and eGFP)](#OE-PCR（连接Pfba1与eGFP）)
[Gibson connection and ****E. coli**** transformation experiment](#吉布森连接与大肠杆菌转化实验)
[Construction of Pfba1+T2R1+eGFP](#酶切酶连法构建Pfba1+T2R1+eGFP)
[*****Saccharomyces cerevisiae***** transformation experiment](#酿酒酵母转化实验)
[Improvement](#改进)

[Modified
Gal promoter（BBa\_K3852001）](#Modified-Gal-promoter（BBa_K3852001）)
[PHO3 (BBa\_K3852003)](#PHO3-(BBa_K3852003))

#### Abstract

        Our goal is to construct a kind of ******Saccharomyces cerevisiae****** whose function is to detect taste, and finally to **judge the proportion of various tastes** in food through fluorescence intensity. This target involves the expression of the following taste receptors: umami taste receptors (**T1R1, T1R3**), sweet taste receptors **(T1R2, T1R3)**, bitter taste receptors **(T2R1, T2R4, T2R46)**. These three receptor proteins all come from the human genome, the purpose is to ensure that their detection is **more suitable for human taste perception**.

        The biosensor we constructed with ******Saccharomyces cerevisiae****** as the chassis cell is mainly divided into two parts: **1. Receptor expression system 2. Detection system.** In building the above system, we have successfully completed the construction and verification of many BioBricks. Take the following four BioBricks as examples:

(1)
**BBa\_K3852005(T1R1)**:

Function as receptor protein, as part of umami taste receptor

(2)
**BBa\_K3852002(Pfba1+T2R1+eGFP)**:

The bitter receptor gene is followed by the fluorescent protein gene, which plays a role in judging whether the receptor protein is positioned correctly.

(3)
**BBa\_K3852001(Modified Gal promoter)**:

It functions as an inducible promoter for expressing receptor proteins.

(4)
**BBa\_K3852003(PHO3)**:

Overexpression of this gene in *****Saccharomyces cerevisiae***** can achieve a certain degree of high temperature tolerance.

Relevant data will be recorded below, hoping to make some contributions to the iGEM community.

#### Engineering Success

#### Receptor expression system T1R1(BBa\_K3852005)

        The protein encoded by this gene is a G protein coupled receptor, which is a component of the heterodimeric amino acid taste receptor **T1R1+3**.

        According to literature research, it is found that the umami taste receptor in the human body is composed of **T1R1** and **T1R3**, so we plan to introduce the two receptor proteins **T1R1** and **T1R3** into *****Saccharomyces cerevisiae*****, and integrate these two genes into **pESC-LEU**, using galactose Induction, two genes can be expressed in *****Saccharomyces cerevisiae***** at the same time.

The plasmid design is as follows:

![pic](https://static.igem.org/mediawiki/2021/d/da/T--BIT-China--e1.png)

##### Fig1. Plasmid pESC-LEU carrying T1R1 and T1R3

Below we will focus on the basic situation of T1R1 gene synthesis and expression:

#### Modification and synthesis of T1R1 gene

        We first obtain the sequence of **T1R1** from **NCBI**, and then use **Snap Gene** to optimize the sequence. The sequence optimization is mainly to modify the signal peptide of the heterologous receptor. The method is to use the first **17 Aa** of **Ste2** to the first **20 Aa** of mGluRα of **T1R1** ( The signal peptide of the protein) is replaced, and the purpose is to increase the content of **T1R1** in the endoplasmic reticulum, thereby increasing the probability of its positioning on the membrane. After entrusting Jinweizhi Company to optimize the codons, we can synthesize the target gene we need.

![pic](https://static.igem.org/mediawiki/2021/2/21/T--BIT-China--e2.png)

##### Fig2. Design (where SS is the signal peptide sequence of Ste2)

#### Agarose gel electrophoresis

Result: The target gene was successfully designed, as shown below.

![pic](https://static.igem.org/mediawiki/2021/7/7a/T--BIT-China--e3.jpg)

##### Fig3. T1R1 gel picture

#### ****E. coli**** transformation experiment

        After successfully completing the synthesis of the **T1R1** gene and plasmid construction, we introduced it into ****E. coli**** via an ****E. coli**** transformation experiment, and then cultivated it in a medium with ampicillin, and observed colonies growing on the medium.

![pic](https://static.igem.org/mediawiki/2021/b/bc/T--BIT-China--e4.png)

##### Fig4. Colonies grown after transferring the umami taste receptor expression plasmid

        In order to verify whether the plasmid was successfully introduced into ****E. coli****, we performed colony PCR, and then ran the verification gel and **observed the correct band**, which proved the successful construction of the plasmid.

![pic](https://static.igem.org/mediawiki/2021/5/53/T--BIT-China--e5.jpg)

##### Fig5.T1R1 verification gel

#### Electrotransformation of *****Saccharomyces cerevisiae*****

        We successfully introduced the plasmid extracted from ****E. coli**** into *****Saccharomyces cerevisiae***** through **electrotransformation**.

![pic](https://static.igem.org/mediawiki/2021/0/03/T--BIT-China--i12456.png)

##### Fig6.*****Yeast***** expressing T1R1 receptor

#### Homologous recombination in *****Saccharomyces cerevisiae*****

        After realizing the *****yeast***** electrotransformation method to successfully introduce the plasmid into *****Saccharomyces cerevisiae*****, we then wanted to adopt a relatively simple and new way to realize the gene introduction: the specific idea is to divide the **YPRC3** binding site into two parts and add it to the left and right sides of the target gene, that is, the left and right homology arms, are then transferred to *****Saccharomyces cerevisiae*****, after which **homologous recombination** occurs with the *****yeast***** genome.

#### Detection Systems--Pfba1+T2R1+eGFP( BBa\_K3852002)

        In the human body, the taste receptor protein will be expressed and located on the **cell membrane** and perform normal functions. In order to verify whether the expressed protein is located on the **cell membrane** after the gene is transferred into *****Saccharomyces cerevisiae*****, we connect the taste receptor through the link sequence. After using fluorescent proteins of different colors, the positioning of the receptors on the cell membrane can be observed through a fluorescence microscope. The following will specifically show the experimental data related to the construction and verification of **Pfba1+T2R1+eGFP**.

![pic](https://static.igem.org/mediawiki/2021/5/50/T--BIT-China--e6.png)

##### Fig7. The design of the receptor followed by the fluorescent protein

#### OE-PCR (connect Pfba1 and eGFP)

        We obtained the **Pfba1+eGFP** fragment by **OE-PCR**. The following is a picture obtained after agarose gel electrophoresis. By comparing the length of the fragment, the success of the synthesis of the fragment is verified.

![pic](https://static.igem.org/mediawiki/2021/2/26/T--BIT-China--e7.jpg)

##### Fig8. Pfba1+eGFP gel picture

#### Gibson connection and ****E. coli**** transformation experiment

        After achieving the successful connection of **Pfba1** and **eGFP**, we integrated the fragment into **pESC-LEU**, and then introduced it into ****E. coli**** by the ****E. coli**** competent transformation experiment, and cultivated it in a medium containing ampicillin. We can observe there are colonies growing on the base.

![pic](https://static.igem.org/mediawiki/2021/e/e3/T--BIT-China--e8.jpg)

##### Fig9. Colonies grown after transfer to a plasmid containing T2R1+eGFP

        In order to verify whether the plasmid was successfully introduced into ****E. coli****, we carried out colony PCR, and then ran the verification gel and observed the correct band. After sequencing, the successful construction of the plasmid was proved.

![pic](https://static.igem.org/mediawiki/2021/a/aa/T--BIT-China--e9.jpg)

##### Fig10. eGFP verification gel

#### Construction of Pfba1+T2R1+eGFP by enzyme digestion method

        After picking the bacteria, extract the plasmid, perform **Swa1** single-enzyme digestion, then **AvrⅡ** single-enzyme digestion, and then enzyme-linked to obtain **pESC-LEU** containing **Pfba1+T2R1+eGFP**. Then the plasmid is sensed by ****E. coli**** In the state transformation experiment, it was introduced into *E.coli*, and then cultured in a medium containing ampicillin, and colonies were observed to grow on the medium.

![pic](https://static.igem.org/mediawiki/2021/8/86/T--BIT-China--e10.jpg)

##### Fig11. Colonies grown after transferring a plasmid containing Pfba1+T2R1+eGFP

        In order to verify whether the plasmid was successfully introduced into ****E. coli****, we carried out **colony PCR**, and then ran the verification gel and observed the correct band. After sequencing, the successful construction of the plasmid was proved.

![pic](https://static.igem.org/mediawiki/2021/3/33/T--BIT-China--e11.jpg)

##### Fig12. Pfba1+T2R1+eGFP verification gel

#### *****Saccharomyces cerevisiae***** transformation experiment

        We successfully introduced the plasmids extracted from ****E. coli**** into *****Saccharomyces cerevisiae***** by electrotransformation.

![pic](https://static.igem.org/mediawiki/2021/9/99/T--BIT-China--222.png)

##### Fig13. *****Yeast***** expressing T2R1 receptor with fluorescent protein

#### Improvement

#### Modified Gal promoter (BBa\_K3852001)

        At the beginning, when we constructed the receptor expression system, the promoters used were all **Pfba1**, but if the plasmid expressing the umami taste receptor, the plasmid expressing the sweet taste receptor, and the plasmid expressing the bitter taste receptor were all introduced into *****Saccharomyces cerevisiae***** at the same time , may cause difficulty in the growth of *****Saccharomyces cerevisiae*****, and aggravate its metabolic burden. After discussion and literature reading, we decided to add different inducible promoters to the plasmid expressing taste receptors, so that when different inducers are added, *****Saccharomyces cerevisiae***** can express different taste receptor proteins, thereby detect different tastes.

        We chose three inducible promoters, namely **Modified Gal promoter**, PADH7, PCUP1. Modified Gal promoter is induced by estradiol, PADH7 is induced by vanillin, and PCUP1 is induced by copper ions. The following will focus on the experiments conducted on **Modified Gal promoter**.

![pic](https://static.igem.org/mediawiki/2021/5/5f/T--BIT-China--e12.png)

##### Fig14. Gene circuit design diagram

#### **Promoter strength measurement**

        We designed the following plasmid, introduced it into the *****Saccharomyces cerevisiae***** strain, and prepared to measure the fluorescence intensity with a microplate reader, and use the fluorescence **intensity/OD600** to characterize the promoter intensity.

![pic](https://static.igem.org/mediawiki/2021/6/65/T--BIT-China--e13.png)

##### Fig15. Plasmid design diagram

        Plot the measured data into the following figure. It can be seen from the figure that within a certain range, increasing the amount of the inducer estradiol can enhance the strength of the **Modified Gal promoter**, which is conducive to subsequent gene expression.

![pic](https://static.igem.org/mediawiki/2021/4/4d/T--BIT-China--e14.png)

##### Fig16. Promoter strength measurement chart

#### PHO3 (BBa\_K3852003)

        The expression of **PHO3** gene in *****Saccharomyces cerevisiae***** can hydrolyze thiamine phosphate in the pericytoplasmic space and increase the uptake of thiamine by cells. We have found in our experiments that overexpression of this gene can promote *****Saccharomyces cerevisiae***** to withstand high temperatures, and if the sample we tested is hot soup, it may inactivate the *****Saccharomyces cerevisiae***** used in the test.

        If you wait for the sample to become cold, The detection is not only time-consuming, but may also change the flavor of the sample. Therefore, we plan to overexpress the **PHO3** gene of our strain to improve its heat resistance and make it possible to detect a wider range of samples. Below we will explain in detail how to conduct experiments to verify that overexpression of the **PHO3** gene can improve the heat tolerance of *****Saccharomyces cerevisiae*****.

#### *******Yeast***** growth status comparison**

        Our experiment is divided into two groups, one is the strains that overexpress **PHO3** (experimental group), the other is the normal strains (control group), using the same conditions at **37 degrees**, cultivation in the environment of **20g/L glucose solution**, co-cultivation for 36h, draw the following figure from the measured data. It is not difficult to see that the growth of **PHO3** overexpression strains is obviously better than that of normal strains. It can be seen that the overexpression of **PHO3** gene has high temperature tolerance for *****Saccharomyces cerevisiae*****. Later, we will overexpress this gene in our *****Saccharomyces cerevisiae***** strain to make it resistant to high temperatures.

![pic](https://static.igem.org/mediawiki/2021/5/51/T--BIT-China--e15.png)

##### Fig17. Growth curve of *****Saccharomyces cerevisiae*****

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# Team:BIT-China/Experiments

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# Team:BIT-China/Results

# Results

You can describe the results of your project and your future plans here.

### What should this page contain?

* Clearly and objectively describe the results of your work.
* Future plans for the project.
* Considerations for replicating the experiments.

### Describe what your results mean

* Interpretation of the results obtained during your project. Don't just show a plot/figure/graph/other, tell us what you think the data means. This is an important part of your project that the judges will look for.
* Show data, but remember **all measurement and characterization data must also be on the Part's Main Page on the [Registry](http://parts.igem.org/Main_Page).** Otherwise these data will not be in consideration for any medals or part awards!
* Consider including an analysis summary section to discuss what your results mean. Judges like to read what you think your data means, beyond all the data you have acquired during your project.

### Project Achievements

You can also include a list of bullet points (and links) of the successes and failures you have had over your summer. It is a quick reference page for the judges to see what you achieved during your summer.

* A list of linked bullet points of the successful results during your project
* A list of linked bullet points of the unsuccessful results during your project. This is about being scientifically honest. If you worked on an area for a long time with no success, tell us so we know where you put your effort.

### Inspiration

See how other teams presented their results.

* [2019 Newcastle](https://2019.igem.org/Team%3ANewcastle/Results)
* [2019 Munich](https://2019.igem.org/Team%3AMunich/Results)
* [2019 Tec Chihuahua](https://2019.igem.org/Team%3ATec-Chihuahua/Results)
* [2020 Aalto Helsinki](https://2020.igem.org/Team%3AAalto-Helsinki/Results)
* [2020 GreatBay SCIE](https://2020.igem.org/Team%3AGreatBay_SCIE/Results)
* [2020 Queens Canada](https://2020.igem.org/Team%3AQueens_Canada/Results)