Difference between revisions of "Part:BBa K3190109"

Revision as of 14:13, 20 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). For this biobrick, the C-terminal end of XLHCGR was fused with superfolder GFP (BBa_K3190205) using a linker (BBa_K3190206.

Usage and Biology

In our studies, XLHCGR-Li-sfGFP was used to examine expression and localization of XLHCGR (BBa_K3190107) in S. cerevisiae.

Chromosomal integration

For our studies, XLHCGR-Li-sfGFP was integrated into the yeast chromosome, and correct insertion was verified using colony PCR.

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. Expected band sizes are of 1000 bp, indicating successful chromosomal integration. Band sizes of 1500 bp indicate unsuccesful chromosomal integration.

<b> Expression of XLHCGR

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 insoluble vs soluble cellular protein | Western blot was carried out using anti-GFP antibodies. Yeast expressing empty vectors and GFP was used as negative and positive control respectively. Two replicate yeast cultures were used for the western blot. As expected, GPER-sfGFP was predominantly found in the insoluble fraction, suggesting possible membrane localization. The existence of a small band in the soluble fraction indicates that the protein was very abundant in the respective cells. Similarly, the presence of GFP in the insoluble fraction can be attributed to very high expression levels. </small>

<b> Microscopy

To further verify expression of XLHCGR-Li-sfGFP, and examine intracellular localization of the receptor, confocal microscopy was performed.

Figure 3: Confocal microscopy of transformed yeast cells | A) Bright field empty vector. B) Fluorescence filter empty vector. C) Bright field XLHCGR-sfGFP. D) Fluorescence filter XLHCGR-sfGFP. </small>

As expected, a clear fluorescent signal was seen in yeast expressing XLHCGR-Li-sfGFP (Fig. 3C and D) confirming expression of XLHCGR-Li-sfGFP. In addition, the localization of fluorescent signal (Fig. 3D) suggests localization in the endoplasmic reticulum (ER)..

Sequence and Features

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]

12
COMPATIBLE WITH RFC[12]

21
INCOMPATIBLE WITH RFC[21]
Illegal BglII site found at 428
Illegal BglII site found at 1682

23
COMPATIBLE WITH RFC[23]

25
COMPATIBLE WITH RFC[25]

1000
INCOMPATIBLE WITH RFC[1000]
Illegal SapI.rc site found at 2179

@@ Line 4: / Line 4: @@
 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 (<partinfo>BBa_K3190206</partinfo>) and superfolded GFP (<partinfo>BBa_K3190205</partinfo>) in the C-terminus (XLHCGR-Li-sfGFP) and coupled to the strongest constitutive promoter pCCW12 (<partinfo>BBa_K3190002</partinfo>) 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.
+For this biobrick, the C-terminal end of XLHCGR was fused with  superfolder GFP (<partinfo>BBa_K3190205</partinfo>) using a linker (<partinfo>BBa_K3190206</partinfo>.
 ===Usage and Biology===
-Through below experiments we confirm that XLHCGR-Li-sfGFP can be successfully expressed in <i>S. cerevisiae</i>. 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.
+In our studies, XLHCGR-Li-sfGFP was used to examine expression and localization of XLHCGR (BBa_K3190107) in S. cerevisiae.
-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.
-[[File:3-module-XLHCGR.jpeg|800px]]
-<small><b> Figure 1: Overview of the multiplex assembler system with 3 modules</b></small>
 <b> <font size="4">Chromosomal integration</font> </b>
-Following transformation of our yeast strains, correct chromosomal integration was verified using the yeast colony PCR.
+For our studies, XLHCGR-Li-sfGFP was integrated into the yeast chromosome, and correct insertion was verified using 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.
 [[File:ovulaid27.png|300px]]
-<small> <b>Figure 2: Colony PCR of yeast transformed with XLHCGR-Li-sfGFP |</b> 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. </small>
+<small> <b>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. Expected band sizes are of 1000 bp, indicating successful chromosomal integration. Band sizes of 1500 bp indicate unsuccesful chromosomal integration.
-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.
 <b> <font size="4">Expression of XLHCGR</font> </b>
-The expression of the XLHCGR-Li-sfGFP was confirmed by performing western blot, using anti GFP antibody. The results are depicted below:
+Expression of the XLHCGR-Li-sfGFP was confirmed by performing western blot, using anti GFP antibody. The results are depicted below:
 <b> [INSERT WB IMAGE HERE] </b>
-Image to be developed
-<small><b>Figure 3: Western blot of XLHCGR-Li-sfGFP using anti-sfGFP | </b> Here is a nice gel image, hopefully </small>
+<small><b>Figure 2: Western blot of insoluble vs soluble cellular protein | Western blot was carried out using anti-GFP antibodies. Yeast expressing empty vectors and GFP was used as negative and positive control respectively. Two replicate yeast cultures were used for the western blot.
+As expected, GPER-sfGFP was predominantly found in the insoluble fraction, suggesting possible membrane localization. The existence of a small band in the soluble fraction indicates that the protein was very abundant in the respective cells. Similarly, the presence of GFP in the insoluble fraction can be attributed to very high expression levels. </small>
 <b> <font size="4"> Microscopy </font> </b>
-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).
+To further verify expression of XLHCGR-Li-sfGFP, and examine intracellular localization of the receptor, confocal microscopy was performed.
 [[File:ovulaid22.png|400px]]
-<small> <b>Figure 4: Confocal microscopy of transformed yeast cells |</b>  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.
+<small> <b>Figure 3: Confocal microscopy of transformed yeast cells | A) Bright field empty vector. B) Fluorescence filter empty vector. C) Bright field XLHCGR-sfGFP. D) Fluorescence filter XLHCGR-sfGFP.  </small>
-  </small>
+As expected, a clear fluorescent signal was seen in yeast expressing XLHCGR-Li-sfGFP (Fig. 3C and D) confirming expression of XLHCGR-Li-sfGFP. In addition, the localization of fluorescent signal (Fig. 3D) suggests localization in the endoplasmic reticulum (ER)..
-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.