Difference between revisions of "Part:BBa K3190109"

Revision as of 16:56, 17 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

Through below experiments we confirm that XLHCGR-Li-sfGFP can be successfully expressed in S. cerevisiae. We used the successful expression of XLHCGR-Li-sfGFP to verify the expression of the XLHCGR used in a multiplex cassette 5-modular system, which makes up an LH-sensing biosensor.

This part, however, we expressed in a simpler multiplex cassette, with only 3 modules. The XLHCGR conjugated to sfGFP was cloned into module 1, while the other two modules were kept empty.

Figure 1: Overview of the multiplex assembler system with 3 modules

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 1: Colony PCR of yeast transformed with XLHCGR-Li-sfGFP | Specific yeast genotyping primers were used for the PCR reaction. PCR products were separated by electropheresis on 1% agarose gel. The sizes of the molecular weight standards are shown on the left. Lanes 1-8 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.

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 2: Western blot of XLHCGR-Li-sfGFP using anti-sfGFP | Here is a nice gel image, hopefully

Microscopy

To determine the expression of GFP and intracellular localization of the receptor, confocal microscopy was performed with the positive colonies of yeast expressing XLHCGR-Li-sfGFP.

Figure 3: Confocal microscopy of transformed yeast cells | Figures 3a and 3b depict the yeast expressing empty vectors. Figures 3c and 3d depict the yeast expressing XLHCGR-Li-sfGFP.

The images further confirmed the expression of the protein in cells expressing XLHCGR-Li-sfGFP, 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, intracellular localization of the receptor can not be confirmed. While no fluorescence was observed in the cells transformed with the empty vector.

Sequence and Features