Difference between revisions of "Part:BBa K3190101"

Revision as of 12:28, 20 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 localise to the endoplasmic reticulum and specifically binds to estrogen and its derivatives. 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. For our project, the gene encoding for the receptor was codon optimized and coupled to strong constitutive promoter pCCW12 for heterologous expression in S. 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 hormones are detected.

Chromosomal integration

Following transformation of our yeast strains, correct chromosomal integration was verified using the yeast colony PCR.

For the colony PCR, 3 specific yeast genotyping primers were used. In the presence of our construct, we expected bands at 1000 bp. In the absence of the constructs, we expected bands at 1500 bp, as this is the size of site 3 of chromosome 10.

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 were 1000 bp.

The band size on lane 3 was observed to be of 1000 bp, which confirmed that the construct had been integrated into the yeast genome.

Expression of G protein-coupled estrogen receptor

Figure 3: Western blot of insoluble vs soluble cellular protein | Western blot was carried out using anti-GFP antibodies. Yeast expressing empty vectors was used as negative control. Yeast expressing GFP was used as positive control. All blue prestained protein standards was used as ladder for comparison. Expected band sizes were 71 kDa.

Figure 4: 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.

We transformed yeast with GPER-sfGFP construct to verify receptor expression, where the sfGFP is tagged to the C-terminal of the receptor. First, we performed western blot to verify GPER expression, then we performed confocal microscopy to see intracellular localization of GPER-sfGFP. Results from western blot and confocal microscopy confirmed our GPER expression.

Bioactivity assay with estrogen

Inorder 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: Fluorescence intensity measurement in Relative Fluorescence Units (RFU) | Y axis indicates fluorescence intensity in RFU, while the X axis indicates the increasing estradiol concentrations in picomoles. 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. This indicates that the biosensor does not work as intended with the above mentioned estradiol concentrations, so it has to be further evaluated with even higher concentrations of the estradiol hormone.

Sequence and Features