Difference between revisions of "Part:BBa K3190103"

Latest revision as of 21:10, 21 October 2019

G protein-coupled estrogen receptor (GPER) CDS with Linker-superfolder GFP

G protein-coupled estrogen receptor (GPR30, also referred to as GPER), an intracellular transmembrane estrogen receptor, was identified in 2005 (Revankar, 2005). It is found to localize to the endoplasmic reticulum and specifically binds to estrogen and its derivatives. The interaction between estradiol and the membrane-associated receptor triggers non-genomic signaling; intracellular calcium mobilization and synthesis of phosphatidylinositol 3,4,5-trisphosphate in the nucleus. For this biobrick (GPER-Li-sfGFP), the C-terminal end of GPER (BBa_K3190101) was fused with superfolder GFP (BBa_K3190205) using a linker (BBa_K3190206).

Usage and Biology

In our studies, GPER-Li-sfGFP was used to examine expression and localization of GPER (BBa_K3190101) in S. cerevisiae.

Chromosomal integration

GPER-Li-sfGFP was integrated into the yeast chromosome, and correct insertion was verified using colony PCR.

Figure 1: Colony PCR of yeast transformed with GPER-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 G protein-coupled estrogen receptor

Expression of GPER-Li-sfGFP was confirmed by performing western blot, using anti GFP antibody. The results are depicted below:

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-Li-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. However, GPER-Li-sfGFP band size of 32 kDa indicated that the receptor might have been in its truncated form.

Microscopy

To further verify expression of GPER-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 GPER-Li-sfGFP. D) Fluorescence filter GPER-Li-sfGFP.

As expected, a clear fluorescent signal was seen in yeast expressing GPER-Li-sfGFP (Fig. 3C and D) confirming expression of GPER-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
COMPATIBLE WITH RFC[21]
23
COMPATIBLE WITH RFC[23]
25
COMPATIBLE WITH RFC[25]
1000
INCOMPATIBLE WITH RFC[1000]
Illegal BsaI.rc site found at 750
Illegal SapI.rc site found at 1162

@@ Line 3: / Line 3: @@
 <partinfo>BBa_K3190103 short</partinfo>
-G protein-coupled estrogen receptor (GPR30, also referred to as GPER), an intracellular transmembrane estrogen receptor, was identified in 2005 (Revankar, 2005). It is found to localise to the endoplasmic reticulum and specifically binds to estrogen and its derivatives (the ligand). The interaction between estradiol and the membrane-associated receptor triggers non-genomic signalling; intracellular calcium mobilization and synthesis of phosphatidylinositol 3,4,5-trisphosphate in the nucleus.
+G protein-coupled estrogen receptor (GPR30, also referred to as GPER), an intracellular transmembrane estrogen receptor, was identified in 2005 (Revankar, 2005). It is found to localize to the endoplasmic reticulum and specifically binds to estrogen and its derivatives. The interaction between estradiol and the membrane-associated receptor triggers non-genomic signaling; intracellular calcium mobilization and synthesis of phosphatidylinositol 3,4,5-trisphosphate in the nucleus.
-The coding sequence of the GPER was fused with the nucleotides for the linker (<partinfo>BBa_K3190206</partinfo>) and superfolded GFP (<partinfo>BBa_K3190205</partinfo>) in the C-terminus (GPER-Li-sfGFP) to carry out localisation assay and characterise the expression and proper alignment of the receptor in the intercellular organelles.
+For this biobrick (GPER-Li-sfGFP), the C-terminal end of GPER (<partinfo>BBa_K3190101</partinfo>) was fused with superfolder GFP (<partinfo>BBa_K3190205</partinfo>) using a linker (<partinfo>BBa_K3190206</partinfo>).
 ===Usage and Biology===
-GPER-Li-sfGFP can be successfully expressed in S. cerevisiae.
+In our studies, GPER-Li-sfGFP was used to examine expression and localization of GPER (<partinfo>BBa_K3190101</partinfo>) 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>
+<b> <font size="4">Chromosomal integration</font> </b>
-The construct was successfully cloned in <i>E. coli</i> as confirmed by below gel image of colony PCR:
+GPER-Li-sfGFP was integrated into the yeast chromosome, and correct insertion was verified using colony PCR.
-<b>[INSERT GEL IMAGE from 6/6/19!!] </b>
-<small> Figure Legend: Above gel electrophoresis image shows the positive colony PCR sample. A band was observed around 1800 bp, which correlates well to the expected band size for a construct of 1866 bp.</small>
-<b> Yeast transformation </b>
+[[File:ovulaid26.png|300px]]
-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.
+<small><b>Figure 1: Colony PCR of yeast transformed with GPER-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>
-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.
-<b> [INSERT GEL IMAGE FROM 29/7/19] </b>
-<small> Figure Legend: Above gel image shows the positive colony of yeast successfully transformed with GPER-Li-sfGFP. We see the expected band size of around 1000 bp.</small>
-<b> Western blot </b>
+<b> <font size="4">Expression of G protein-coupled estrogen receptor</font> </b>
-The expression of the GPER-li-sfGFP was confirmed by performing western blot, using anti GFP antibody. The results are depicted below:
+Expression of GPER-Li-sfGFP was confirmed by performing western blot, using anti GFP antibody. The results are depicted below:
-<b> [INSERT WB IMAGE HERE] </b>
-<small> Figure legend: Here is a nice gel image, hopefully </small>
+[[File:ovulaid21.png|500px]]
-<b> Microscopy </b>
+<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>
-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.
+As expected, GPER-Li-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. However, GPER-Li-sfGFP band size of 32 kDa indicated that the receptor might have been in its truncated form.
-[[File:ovulaid1.png|500px|left|alt text]]
-<small> Fluorescence and Confocal microscopy of intracellular sfGFP in the yeast cells transformed with GPER-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>
+<b> <font size="4">Microscopy</font> </b>
+To further verify expression of GPER-Li-sfGFP, and examine intracellular localization of the receptor, confocal microscopy was performed.
+[[File:ovulaid19.png|500px]]
+<small><b>Figure 3: Confocal microscopy of transformed yeast cells | </b> A) Bright field empty vector. B) Fluorescence filter empty vector. C) Bright field GPER-Li-sfGFP. D) Fluorescence filter GPER-Li-sfGFP.  </small>
+As expected, a clear fluorescent signal was seen in yeast expressing GPER-Li-sfGFP (Fig. 3C and D) confirming expression of GPER-Li-sfGFP. In addition, the localization of fluorescent signal (Fig. 3D) suggests localization in the endoplasmic reticulum (ER).