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Revision as of 11:36, 18 October 2021

Pfba1+T2R1+eGFP

Introduction

Umami receptors in the human body protein expressed after positioning on the cell membrane and the exercise of normal function, in order to verify the gene into Saccharomyces cerevisiae, the expression of protein is located on the cell membrane, we connected to the link sequence after taste receptors, connected to a different color fluorescent protein, and observed by fluorescence microscope can locate receptors on the membrane. Experimental data related to Pfba1+T2R1+eGFP construction and validation are presented below.

Figure1. Receptors are followed by fluorescent proteins

Experimental

1. OE-PCR (connecting Pfba1 with eGFP)

We obtained the Pfba1-eGFP fragment by OE-PCR, and the following is a picture from the agar gel electrophoresis, which verifies the success of the synthesis of the fragment by comparing the length of the fragment.

Figure2. the far right band is Pfba1 plus eGFP

2. Gibson Assembly and E. coli conversion experiment

After the successful connection between Pfba1 and eGFP, we integrated the fragment into pESC-LEU, then introduced it into E. coli by E. coli receptor transformation experiment, cultured with a medium containing ampicillin, and observed that the colonies on the medium grew.

Figure3. A colony that grows after transferring to a plasmid containing the expression T2R1 + eGFP

In order to verify the successful introduction of plasmids into E. coli, we carried out a bacterial liquid PCR, and then by running the verification glue, observed the correct strip, proving the successful construction of plasmids.

Figure4. The stripe is an eGFP fragment

3. Enzyme-cutting and enzyme-joint method to build Pfba1+T2R1+eGFP

We culled the bacteria and then extracted the plasmid. The plasmids were firstly digested by Swa1 single enzyme, then digested by Avr 11 single enzyme, and then linked by enzyme.At last we obtained the pESC-LEU containing Pfba1+T2R1+eGFP. After that, the plasmids were introduced into E. coli through the receptive state transformation experiment, and then cultured in the medium containing ampicillin. Colony growth was observed on the medium.

Figure5. The colonies grew after transfection with Pfba1+T2R1+eGFP plasmid

In order to verify whether the plasmid was successfully introduced into E. coli, we carried out bacterial liquid PCR. After that, correct bands were observed by running the verification gel, which proved the successful construction of the plasmid.

Figure6. The stripe is a Pfba1-T2R1-eGFP fragment

4. Saccharomyces cerevisiae conversion experiment

The plasmid extracted from E. coli was successfully introduced into S. cerevisiae by electric transformation.

Sequence and Features