Difference between revisions of "Part:BBa K3190109"

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<partinfo>BBa_K3190109 short</partinfo>
 
<partinfo>BBa_K3190109 short</partinfo>
  
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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).  
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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).  
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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>.
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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===
 
===Usage and Biology===
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<b> <font size="4">Chromosomal integration</font> </b>
 
<b> <font size="4">Chromosomal integration</font> </b>
  
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For our studies, XLHCGR-Li-sfGFP was integrated into the yeast chromosome, and correct insertion was verified using colony PCR.  
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XLHCGR-Li-sfGFP was integrated into the yeast chromosome, and correct insertion was verified using colony PCR.  
  
  
 
[[File:ovulaid27.png|300px]]
 
[[File:ovulaid27.png|300px]]
  
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<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 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. </small>
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<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>
  
  
 
<b> <font size="4">Expression of XLHCGR</font> </b>
 
<b> <font size="4">Expression of XLHCGR</font> </b>
  
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Expression of the XLHCGR-Li-sfGFP was confirmed by performing western blot, using anti GFP antibody. The results are depicted below:
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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.
  
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[[File:ovulaid36.png|600px]]
  
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<b> [INSERT WB IMAGE HERE] </b>
 
  
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<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>
  
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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.  
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<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 GFP was used as negative and positive control respectively. Two replicate yeast cultures were used for the western blot.</small>
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As expected, XLHCGR-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.  
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<b> <font size="4"> Microscopy </font> </b>  
 
<b> <font size="4"> Microscopy </font> </b>  
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To further verify expression of XLHCGR-Li-sfGFP, and examine intracellular localization of the receptor, confocal microscopy was performed.
 
To further verify expression of XLHCGR-Li-sfGFP, and examine intracellular localization of the receptor, confocal microscopy was performed.
  
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[[File:ovulaid22.png|400px]]
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[[File:ovulaid28.png|400px]]
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<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-sfGFP. D) Fluorescence filter XLHCGR-sfGFP.  </small>
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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).
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<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>
  
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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.
  
  

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.


Ovulaid27.png

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.

Ovulaid36.png


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.

Ovulaid28.png

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