Part:BBa_K3190103
G protein-coupled estrogen receptor (GPER) CDS with Linker-superfolder GFP
G protein-coupled estrogen receptor (GPR30, also referred to as GPER), an intracellular transmembrane estrogen receptor, was identified in 2005 (Revankar, 2005). It is found to localise to the endoplasmic reticulum and specifically binds to estrogen and its derivatives (the ligand). The interaction between estradiol and the membrane-associated receptor triggers non-genomic signalling; intracellular calcium mobilization and synthesis of phosphatidylinositol 3,4,5-trisphosphate in the nucleus. The coding sequence of the GPER was fused with the nucleotides for the linker (BBa_K3190206) and superfolded GFP (BBa_K3190205) in the C-terminus (GPER-Li-sfGFP) to carry out localisation assay and characterise the expression and proper alignment of the receptor in the intercellular organelles.
Usage and Biology
GPER-Li-sfGFP can be successfully expressed in S. cerevisiae.
Using USER ligation, we assembled the receptor with sfGFP, and the pCCW12 promoter (BBa_K3190002), which is a strong constitutive promoter. We assembled the construct on a plasmid backbone compatible with multiplex integration cassette. The backbone used will integrate the construct in the yeast genome at chromosome 10, site 3.
E. coli cloning
The construct was successfully cloned in E. coli as confirmed by below gel image of colony PCR:
[INSERT GEL IMAGE from 6/6/19!!] Figure Legend: Above gel electrophoresis image shows the positive colony PCR sample. A band was observed around 1800 bp, which correlates well to the expected band size for a construct of 1866 bp.
Yeast transformation
For the yeast transformation, we picked the positive E. coli colonies and purified DNA from these. After confirming the sequence, we successfully transformed the construct into S. cerevisiae as depicted in below gel image from yeast colony PCR.
For the colony PCR, we used 2 primers, one in the forward direction for the backbone and one in the reverse direction for the yeast chromosome 10. In the presence of our construct, we expect to see a band at 1000 bp as, that is the size of the fragment between the two primer regions. In the absence of the constructs, we expect to see the bands at 1500 bp, as this is the size of site 3 of chromosome 10.
[INSERT GEL IMAGE FROM 29/7/19] Figure Legend: Above gel image shows the positive colony of yeast successfully transformed with GPER-Li-sfGFP. We see the expected band size of around 1000 bp.
Western blot
The expression of the GPER-li-sfGFP was confirmed by performing western blot, using anti GFP antibody. The results are depicted below:
[INSERT WB IMAGE HERE] Figure legend: Here is a nice gel image, hopefully
Microscopy
To further confirm that the receptor is expressed, we performed fluorescence microscopy. To determine where the receptor is locating within the cell, we also performed confocal microscopy.
Fluorescence and Confocal microscopy of intracellular sfGFP in the yeast cells transformed with GPER-Li-sfGFP. The images further confirm the expression of the protein, and also confirms the proper alignment of the receptor, as sfGFP is tagged to the C-terminus of the receptor, which is expressed inside the cell. However, from the images, we cannot confirm the intracellular localization of the receptor.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI.rc site found at 750
Illegal SapI.rc site found at 1162
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