Difference between revisions of "Part:BBa K3190103"

Revision as of 12:06, 18 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

Through below experiments we confirm that GPER-Li-sfGFP can be successfully expressed in S. cerevisiae. We used the successful expression of GPER-Li-sfGFP to verify the expression of another of our submitted biobricks, the GPER (BBa_K3190101) used in a multiplex cassette 5-modular system, which makes up an estrogen-sensing biosensor.

This part, however, we expressed in a simpler multiplex cassette, with only 3 modules. The GPER conjugated to sfGFP was cloned into module 1, while the other two modules were kept empty.

Figure 1: Overview of the multiplex assembler system with 3 modules

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.

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:

Figure 2: Western blot of insoluble vs soluble cellular protein | Western blot was carried out using anti-GFP antibodies. Yeast expressing empty vectors was taken as negative control. Yeast expressing GFP was taken as positive control. All blue prestained protein standards was the ladder used for comparison.

The positive and negative control have worked as expected. GFP was so strongly expressed in the positive control cells that is was even seen in the insoluble fraction. Vice versa, GPER-sfGFP was predominant in the insoluble fraction, which we expected since it is a membrane protein. A small band can however also be seen in the soluble fraction, indicating that the protein is very abundant in the respective cells.

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 and yeast expressing empty vectors as negative control.

Figure 3: Confocal microscopy of transformed yeast cells. A and B depict bright field vs fluorescence filter showing yeast expressing empty vector backbones. C and D depict bright field vs fluorescence filter showing yeast expressing GPER-sfGFP.

Yeast strain containing empty vectors was visible on the bright field (A) but not on fluorescence filter (B) as expected as there is no sfGFP. Whereas, yeast strain containing sfGFP was visible on both bright field (C) and fluorescence filter (D) due to the expression of sfGFP, which confirms the expression of GPER as GFP is tagged to the C-terminal of the receptor. From (D) it also looks like GPER-sfGFP might have been expressed in the endoplasmic reticulum (ER).

Sequence and Features