Difference between revisions of "Part:BBa K3852002"

Latest revision as of 17:39, 21 October 2021

Pfba1+T2R1+eGFP

Introduction

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.

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

Assembly Compatibility:

10
INCOMPATIBLE WITH RFC[10]
Illegal XbaI site found at 638
Illegal XbaI site found at 1345
12
COMPATIBLE WITH RFC[12]
21
COMPATIBLE WITH RFC[21]
23
INCOMPATIBLE WITH RFC[23]
Illegal XbaI site found at 638
Illegal XbaI site found at 1345
25
INCOMPATIBLE WITH RFC[25]
Illegal XbaI site found at 638
Illegal XbaI site found at 1345
1000
COMPATIBLE WITH RFC[1000]

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 <partinfo>BBa_K3852002 short</partinfo>
-This gene encodes a member of a family of candidate taste receptors that are members of the G protein-coupled receptor superfamily and that are specifically expressed by taste receptor cells of the tongue and palate epithelia. This intronless taste receptor gene encodes a 7-transmembrane receptor protein, functioning as a bitter taste receptor. This gene is mapped to chromosome 5p15, the location of a genetic locus (PROP) that controls the detection of the bitter compound 6-n-propyl-2-thiouracil.
+===Introduction===
-Bitter taste signaling in humans is mediated by a group of 25 bitter receptors (T2Rs) that belong to the G-protein coupled receptor (GPCR) family. However, the molecular targets or receptors in humans for these bitter peptides were poorly characterized and least understood. The molecular models showed that the bitter peptides bind within the same binding pocket on the receptor. The ligand binding pocket in T2R1 is present on the extracellular surface of the receptor, and is formed by the transmembrane helices 1, 2, 3 and 7 and with extracellular loops 1 and 2 forming a cap like structure on the binding pocket. Experiments show that bitter taste receptor T2R1 can be activated by dipeptides and tripeptides.In our experiments, we used this gene to express bitter taste receptors in Saccharomyces cerevisiae.
+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.
-===Usage and Biology===
+[[File:T--BIT-China--Engeering 6.png|550px|thumb|center|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.
+[[File:T--BIT-China--Engeering 7.png|550px|thumb|center|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.
+[[File:T--BIT-China--Engeering 8.png|550px|thumb|center|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.
+[[File:T--BIT-China--Engeering 9.png|550px|thumb|center|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.
+[[File:T--BIT-China--Engeering 10.png|550px|thumb|center|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.
+[[File:T--BIT-China--Engeering 11.png|550px|thumb|center|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.
-Receptor that may play a role in the perception of bitterness and is gustducin-linked. May play a role in sensing the chemical composition of the gastrointestinal content. The activity of this receptor may stimulate alpha gustducin, mediate PLC-beta-2 activation and lead to the gating of TRPM5. We acquire it from synthesis company.
-===Reference===
-[1] C Conte, M Ebeling, A Marcuz et al. Identification and characterization of human taste receptor genes belonging to the TAS2R family[J] Cytogenet Genome Res. 2002,98(1):45-53.
-[2] Upadhyaya J, Pydi SP, Singh N et al. Bitter taste receptor T2R1 is activated by dipeptides and tripeptides[J] Biochem Biophys Res Commun.2010,398(2):331-5.
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