Difference between revisions of "Part:BBa K3190101"

Revision as of 10:07, 15 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 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 gene encoding for the receptor was codon optimised and coupled to the strongest constitutive promoter pCCW12 for heterologous expression in S. cerevisiae.

Usage and Biology

The receptor can be successfully expressed in S. cerevisiae as was shown by conjugating the part to superfolder GFP (BBa_K3190103).

The GPER is used as the receptor in our minimal biosensor system, known as a multiplex cassette, or 5 modular system. Below figure explains the concept of the modular system:

[INSERT OJAS' MODULE DRAWING HERE] Figure legend: Overview of the multiplex assembler system with both 3 and 5 modules.

Our biosensor contains 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.

Using USER ligation, we assembled the receptor with 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 contains a URA selection marker, and 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. We used forward primer for the promoter (pCCW12) and reverse primer for the gene (GPER). We therefore expect a band of 1818 bp.

[INSERT GEL IMAGE!!] 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 1818 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 our 5 modular system. We see the expected band size of around 1000 bp.

Bioactivity assay with estrogen

To test the functionality of our biosensor, we induced the positive yeast transformants with increasing concentrations of estrogen hormone, which is the ligand for the biosensor. In theory, induction with estrogen should lead to the production of fluorescence due to the activation of the reporter gene ZsGreen. The fluorescence was measured using a plate reader.

[INSERT GRAPH HERE] Figure legend: The blue line indicates the fluorescence from the yeast cells with empty vectors induced with increasing concentrations of estrogen. The orange line indicates the fluorescence from the yeast cells with the minimal biosensor induced with increasing concentrations of estrogen.

From the results of the bioactivity assay, no significant difference could be observed between the cells transformed with our biosensor, and empty vectors, when inducing with estrogen. This indicates that there is an issue with the biosensor, which would have to be further evaluated.

Sequence and Features