Part:BBa_K3852002

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

挑菌培养后提取质粒，先进行Swa1单酶酶切，再进行AvrⅡ单酶酶切，之后进行酶连，即得到了含有Pfba1+T2R1+eGFP的pESC-LEU，之后将该质粒通过大肠杆菌感受态转化实验导入大肠杆菌中，再利用含有氨苄的培养基进行培养，观察到培养基上有菌落长出。

Figure5. 转入含表达Pfba1+T2R1+eGFP的质粒后长出的菌落

为了验证质粒是否成功导入大肠杆菌，我们进行了菌液PCR，之后通过跑验证胶，观察到了正确的条带，证明了质粒的成功构建。

Figure6. 为了验证质粒是否成功导入大肠杆菌，我们进行了菌液PCR，之后通过跑验证胶，观察到了正确的条带，证明了质粒的成功构建。

4.酿酒酵母转化实验

我们将从大肠杆菌中提取的质粒通过电转化的方式成功导入了酿酒酵母中。

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]