Difference between revisions of "Part:BBa K3190109"

Latest revision as of 00:12, 22 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 Human chorionic gonadotropin (hCG); this suggests that Xenopus laevis lutropin-choriogonadotropic hormone receptor (XLLHCGR) may serve as a good alternative to Homo sapiens LHCGR for the detection of LH, as LH has been found to induce maturation of Xenopus oocytes in vitro (Wlizla et al., 2017). For this biobrick (XLLHCGR-Li-sfGFP), 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

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 electrophoresis 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 unsuccessful chromosomal integration.

Expression of XLHCGR

In order to examine expression and localization of XLHCGR, we fused sfGFP to C-terminal of the receptor using a linker and transformed yeast with the same (XLHCGR-Li-sfGFP). First, we performed western blot to verify XLHCGR expression, then we performed confocal microscopy to see intracellular localization of XLHCGR-Li-sfGFP.

Figure 2: Western blot of insoluble vs soluble cellular protein | Western blot was carried out using anti-GFP antibodies. Yeast expressing empty vectors and XLHCGR-Li-sfGFP was used as negative and positive control respectively. Two replicate yeast cultures were used for the western blot. Expected band sizes are of 107 kDa.

XLHCGR-Li-sfGFP was found in the soluble fraction, indicating its presence within the cytosol and not anchored to the plasma membrane as expected. Also, the band size around 32 kDa indicates that the receptor might have been expressed in a truncated form.

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-Li-sfGFP. D) Fluorescence filter XLHCGR-Li-sfGFP.

As expected, a clear fluorescent signal was seen in yeast expressing XLHCGR-Li-sfGFP (Fig. 3C and D) confirming expression of XLHCGR-Li-sfGFP. However, localization of the fluorescent signal (Fig. 3D) was not sufficient to conclude on the specific location of XLHCGR within the cell.

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 3: / Line 3: @@
 <partinfo>BBa_K3190109 short</partinfo>
-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).
+Mammalian Luteinizing Hormones (LH) share structural similarity, functional equivalency, and bind the same receptor as Human chorionic gonadotropin (hCG); this suggests that <i> Xenopus laevis </i> lutropin-choriogonadotropic hormone receptor (XLLHCGR) may serve as a good alternative to <i> Homo sapiens </i> LHCGR for the detection of LH, as LH has been found to induce maturation of <i> Xenopus </i> oocytes <i> in vitro </i> (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 (XLLHCGR-Li-sfGFP), 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===
-XLHCGR-Li-sfGFP can be successfully expressed in S. cerevisiae.
+In our studies, XLHCGR-Li-sfGFP was used to examine expression and localization of XLHCGR (BBa_K3190107) in <i> S. cerevisiae. </i>
-Using USER ligation, we assembled the receptor with sfGFP, and the pCCW12 promoter (<partinfo>BBa_K3190002</partinfo>), 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.
-<b> <i>E. coli</i> cloning </b>
-The construct was successfully cloned in <i>E. coli</i> as confirmed by below gel image of colony PCR:
-<b>[RE-RUN COLONY PCR AN INSERT GEL IMAGE ON THURSDAY] </b>
+<b> <font size="4">Chromosomal integration</font> </b>
-<small> 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.</small>
+XLHCGR-Li-sfGFP was integrated into the yeast chromosome, and correct insertion was verified using colony PCR.
-<b> Yeast transformation </b>
-For the yeast transformation, we picked the positive <i>E. coli </i> colonies and purified DNA from these. After confirming the sequence, we successfully transformed the construct into <i>S. cerevisiae</i> as depicted in below gel image from yeast colony PCR.
+[[File:ovulaid27.png|300px]]
-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.
+<small> <b>Figure 1: 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 electrophoresis 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 unsuccessful chromosomal integration. </small>
-[[File:ovulaid8.png|400px]]
-<small> 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.</small>
+<b> <font size="4">Expression of XLHCGR</font> </b>
-<b> Western blot </b>
+In order to examine expression and localization of XLHCGR, we fused sfGFP to C-terminal of the receptor using a linker and transformed yeast with the same (XLHCGR-Li-sfGFP). First, we performed western blot to verify XLHCGR expression, then we performed confocal microscopy to see intracellular localization of XLHCGR-Li-sfGFP.
-The expression of the XLHCGR-Li-sfGFP was confirmed by performing western blot, using anti GFP antibody. The results are depicted below:
+[[File:ovulaid36.png|600px]]
-<b> [INSERT WB IMAGE HERE] </b>
-<small> Figure legend: Here is a nice gel image, hopefully </small>
+<small><b>Figure 2: Western blot of insoluble vs soluble cellular protein </b> | Western blot was carried out using anti-GFP antibodies. Yeast expressing empty vectors and XLHCGR-Li-sfGFP was used as negative and positive control respectively. Two replicate yeast cultures were used for the western blot. Expected band sizes are of 107 kDa.</small>
+XLHCGR-Li-sfGFP was found in the soluble fraction, indicating its presence within the cytosol and not anchored to the plasma membrane as expected. Also, the band size around 32 kDa indicates that the receptor might have been expressed in a truncated form.
-<b> Microscopy </b>
+<b> <font size="4"> Microscopy </font> </b>
-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.
+To further verify expression of XLHCGR-Li-sfGFP, and examine intracellular localization of the receptor, confocal microscopy was performed.
-[[File:ovulaid2.png|400px]]
+[[File:ovulaid28.png|400px]]
-[[File:ovulaid15.png|400px]]
+<small> <b>Figure 3: Confocal microscopy of transformed yeast cells </b> | A) Bright field empty vector. B) Fluorescence filter empty vector. C) Bright field XLHCGR-Li-sfGFP. D) Fluorescence filter XLHCGR-Li-sfGFP.  </small>
-<small> 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.</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. However, localization of the fluorescent signal (Fig. 3D) was not sufficient to conclude on the specific location of XLHCGR within the cell.