Difference between revisions of "Part:BBa K3190103"

Revision as of 13:13, 16 October 2019

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.

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.

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.

Figure 1: Colony PCR of yeast transformed with GPER-Li-sfGFP

Specific yeast genotyping primers were used for the PCR reaction. PCR products were separated by electropheresis on 1% agarose gel. The sizes of the molecular weight standards are shown on the left. Lanes 1-8 correspond to individual colonies.

The band size on lanes 4 and 7 was observed to be of 1000 bp, which conformed that the construct has been integrated into the yeast genome.

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 determine the expression of GFP and intracellular localization of the receptor, confocal microscopy was performed with the positive colonies of yeast expressing GPER-Li-sfGFP.

Figure 3: Confocal microscopy of transformed yeast cells.

Figures 3a and 3b depict the yeast expressing empty vectors. Figures 3c and 3d depict the yeast expressing GPER-Li-sfGFP.

The images further confirmed the expression of the protein in cells expressing GPER-Li-sfGFP, 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, intracellular localization of the receptor can not be confirmed. While no fluorescence was observed in the cells transformed with the empty vector.