Team:BIT-China/Engineering

Test Animation

Template

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:

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.

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

Agarose gel electrophoresis

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

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.

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.

Fig5.T1R1 verification gel

Electrotransformation of Saccharomyces cerevisiae

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

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.

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.

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.

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.

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.

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.

Fig12. Pfba1+T2R1+eGFP verification gel

Saccharomyces cerevisiae transformation experiment

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

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.

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.

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.

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.