Difference between revisions of "Part:BBa K3190107"

Revision as of 08:18, 15 October 2019

Xenopus laevis lutropin-choriogonadotropic hormone receptor LHCGR CDS

Mammalian Luteinizing Hormones (LH) share structural similarity, functional equivalency, and bind the same receptor as hCG; this suggests that Xenopus LHCGR may serve as a good alternative to Homo sapiens LHCGR for the detection of the ligand i.e. luteinizing hormone as LH has been found to induce maturation of Xenopus oocytes in vitro (Wlizla et al., 2017). The gene encoding for the receptor was codon optimised and coupled to the strongest constitutive promoter pCCW12 (BBa_K3190002) for heterologous expression in S. cerevisiae, wherein we expect that upon binding of LH to the external part of the membrane spanning receptor, a transduction of the signal takes place that activates the G protein that is bound to the receptor internally. With LH attached, the receptor shifts conformation and thus mechanically activates the G protein, which detaches from the receptor and activates the MAPK system.

Usage and Biology

The receptor can be successfully expressed in S. cerevisiae as was shown by conjugating the part to superfolder GFP (BBa_K3190109).

The XLHCGR is used as the receptor in our minimal biosensor system. Known as the 5 modular system, it contains the following modules:

• Module 1: XLHCGR
• 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 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 (XLHCGR). We therefore expect a band of 2838 bp.

[INSERT GEL IMAGE!!] Figure Legend: Above gel electrophoresis image shows the positive colony PCR sample. A band was observed around 2800 bp, which correlates well to the expected band size for a construct of 2838 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.

Sequence and Features