Part:BBa_K3190109
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 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
Chromosomal integration
Following transformation of our yeast strains, correct chromosomal integration was verified using the yeast colony PCR.
For the colony PCR, 3 specific yeast genotyping primers were used. In the presence of our construct, we expect to see a band at 1000. 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 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. Expected band sizes are of 1000bp.
The band size on lane 3 was observed to be of 1000 bp, which confirmed that the construct has been integrated into the yeast genome.
Expression of XLHCGR
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]
Image to be developed
Figure 3: 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 yeast expressing XLHCGR-Li-sfGFP and yeast expressing empty vectors (negative control).
Figure 4: Confocal microscopy of transformed yeast cells | A and B depict bright field vs fluorescence filter showing yeast expressing empty vector backbones. C and D depict bright field vs fluorescence filter showing yeast expressing XLHCGR-sfGFP.
Yeast strain containing empty vectors was visible on the bright field (A) but not on fluorescence filter (B) as expected as there is no sfGFP. Whereas, yeast strain containing XLHCGR-sfGFP was visible on both bright field (C) and fluorescence filter (D) due to the expression of sfGFP, which confirms the expression of XLHCGR as GFP is tagged to the C-terminal of the receptor.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 428
Illegal BglII site found at 1682 - 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal SapI.rc site found at 2179
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