Difference between revisions of "Part:BBa K3190101"

Revision as of 12:50, 18 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. 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

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.

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 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.

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

Bioactivity assay with estrogen

To test the functionality of the biosesnor, the positive yeast transformants were induced 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.

Figure 3: 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. A (orange line) indicates the fluorescence (RFU)from the yeast cells with the 5 assembler cassette, induced with increasing concentrations of estradiol. B (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.

There was no other data apart from the fluorescence intensity measurements for this part because the part did not contain myc tag or a GFP to confirm the protein expression.

Sequence and Features