Difference between revisions of "Part:BBa K3190109"

Revision as of 22:09, 15 October 2019

Xenopus laevis lutropin-choriogonadotropic hormone receptor LHCGR CDS with Linker-superfolder GF

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 coding sequence for the receptor XLHCGR was codon optimised and fused with the nucleotides for the linker (BBa_K3190206) and superfolded GFP (BBa_K3190205) in the C-terminus (XLHCGR-Li-sfGFP) and coupled to the strongest constitutive promoter pCCW12 (BBa_K3190002) for heterologous expression in S. cerevisiae. The construct was important to carry out localisation assay and characterise the expression and proper alignment of the receptor in the intercellular organelles.

Usage and Biology

XLHCGR-Li-sfGFP can be successfully expressed in S. cerevisiae.

Using USER ligation, we assembled the receptor with sfGFP, and 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:

[RE-RUN COLONY PCR AN INSERT GEL IMAGE ON THURSDAY]

Figure Legend: Above gel electrophoresis image shows the positive colony PCR sample. A band was observed around XXXX bp, which correlates well to the expected band size for a construct of XXXX 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.

Figure Legend: Above gel image shows the positive colony of yeast successfully transformed with XLHCGR-Li-sfGFP. We see the expected band size of around 1000 bp.

Western blot

The expression of the XLHCGR-Li-sfGFP was confirmed by performing western blot, using anti GFP antibody. The results are depicted below:

[INSERT WB IMAGE HERE]

Figure legend: Here is a nice gel image, hopefully

Microscopy

To further confirm that the receptor is expressed, we performed fluorescence microscopy. To determine where the receptor is locating within the cell, we also performed confocal microscopy.

Fluorescence and Confocal microscopy of intracellular sfGFP in the yeast cells transformed with XLHCGR-Li-sfGFP. The images further confirm the expression of the protein, and also confirms the proper alignment of the receptor, as sfGFP is tagged to the C-terminus of the receptor, which is expressed inside the cell. However, from the images, we cannot confirm the intracellular localization of the receptor.

Sequence and Features