Difference between revisions of "Part:BBa K3190101"

Latest revision as of 23:09, 21 October 2019

G protein-coupled estrogen receptor (GPER/GPR30) CDS

G protein-coupled estrogen receptor (GPR30, also referred to as GPER), an intracellular transmembrane estrogen receptor, was identified in 2005 (Revankar, 2005). It has been found to localize to the endoplasmic reticulum and specifically binds to estrogen and its derivatives. The interaction between estradiol and the membrane-associated receptor triggers non-genomic signaling; intracellular calcium mobilization and synthesis of phosphatidylinositol 3,4,5-trisphosphate in the nucleus. For our project, the gene encoding for the receptor was codon optimized and coupled to a strong constitutive promoter pCCW12 for heterologous expression in Saccharomyces cerevisiae.

Usage and Biology

For our study, we used the GPER to construct a biosensor. Here the GPER was integrated into the chromosome of S. cerevisiae , along with three other genes (see below) using a multiplex assembler system, allowing for simultaneous integration of multiple modules at the same time (Figure 1). The backbones we used were designed to integrate into S. cerevisiae chromosome 10, site 3. Below figure explains the concept of the modular system:

Figure 1: Overview of the multiplex assembler system with 5 modules | The system is designed such that it integrates into the genome of S. cerevisiae chromosome 10, site 3 by homologous recombination.

In total our biosensor contained the following modules:

• Module 1: GPER
• Module 2: Chimeric Gαs (BBa_K3190201)
• Module 3: Transcription factor STE12 (BBa_K3190203)
• Module 4: This module was kept empty in this construct
• Module 5: Reporter module ZsGreen (BBa_K3190204)

This construct is our biosensor, which should produce a signal when estrogen is detected.

Chromosomal integration

For our studies, GPER along with all other modules (5-assembler system) was integrated into the yeast chromosome, and correct insertion was verified using colony PCR.

Figure 2: Colony PCR of yeast transformed with 5 assembler construct | Specific yeast genotyping primers were used for the PCR reaction. PCR products were separated by electrophoresis on 1% agarose gel. The sizes of the molecular weight standards are shown on the left. Lanes 1-10 correspond to individual colonies. Expected band sizes are of 1000 bp, indicating successful chromosomal integration. Band sizes of 1500 bp indicate unsuccessful chromosomal integration.

Expression of G protein-coupled estrogen receptor

In order to examine expression and localization of GPER, we fused sfGFP to C-terminal of the receptor using a linker and transformed yeast with the same (GPER-Li-sfGFP; (BBa_K3190103). First, we performed western blot to verify GPER expression, then we performed confocal microscopy to see intracellular localization of GPER-sfGFP.

Figure 3: Western blot of insoluble vs soluble cellular protein | Western blot was carried out using anti-GFP antibodies. Yeast expressing empty vectors and GFP was used as negative and positive control respectively. Two replicate yeast cultures were used for the western blot. Expected band sizes are of 71 kDa.

Since sfGFP is fused to the C-terminal end of the receptor, GFP expression confirms receptor expression. However, the band size of 32 kDa indicates that the receptor might have been in its truncated form. To further verify expression of GPER-Li-sfGFP, and examine intracellular localization of the receptor, confocal microscopy was performed.

Figure 4: Confocal microscopy of transformed yeast cells. | A) Bright field empty vector. B) Fluorescence filter empty vector. C) Bright field GPER-sfGFP. D) Fluorescence filter GPER-sfGFP.

As expected, a clear fluorescent signal was seen in yeast expressing GPER-Li-sfGFP (Fig. 4C and D) confirming expression of GPER-Li-sfGFP. In addition, the localization of fluorescent signal (Fig. 4D) suggests localization in the endoplasmic reticulum (ER).

Bioactivity assay with estrogen

In order to test the functionality of our GPER biosensor, we induced our cells with increasing concentrations of estrogen hormone. Here, successful induction leads to activation of the reporter gene ZsGreen, resulting in a fluorescent signal. The fluorescence was measured using a plate reader.

Figure 5: Bioactivity assay of GPER biosensor using estradiol | Y axis indicates fluorescence intensity in RFU, while the X axis indicates the increasing estradiol concentrations in picomolar. GPER biosensor (orange line) indicates the fluorescence (RFU) from the yeast cells with the 5 assembler cassette, induced with increasing concentrations of estradiol. Empty vector (blue line) indicates the fluorescence (RFU) from the yeast cells with empty vectors induced with increasing concentrations of estradiol.

From the results of the bioactivity assay, not much could be concluded because there was no significant difference in fluorescence between the cells transformed with empty vector and the biosensor when induced with estradiol. The biosensor did not work as intended as there was no fluorescent signals of ZsGreen. The reason might be that the truncated GPER could not sense estradiol.

Sequence and Features