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===
-Through below experiments we confirm that GPER-Li-sfGFP can be successfully expressed in <i>S. cerevisiae</i>. We used GPER-Li-sfGFP to verify the expression of another of our submitted biobricks, the GPER (<partinfo>Ba_K3190101</partinfo>) used in a multiplex cassette 5-modular system, which makes up an estrogen-sensing biosensor.
+In our studies, GPER-Li-sfGFP was used to examine expression and localization of GPER (<partinfo>BBa_K3190101</partinfo>) in <i>S. cerevisiae</i>.
-This part, however, we expressed in a simpler multiplex cassette, with only 3 modules. The GPER conjugated to sfGFP was cloned into module 1, while the other two modules were kept empty.
-[[File:3-module-system.jpeg|800px]]
+<b> <font size="4">Chromosomal integration</font> </b>
-<small><b> Figure 1: Overview of the multiplex assembler system with 3 modules</b></small>
+GPER-Li-sfGFP was integrated into the yeast chromosome, and correct insertion was verified using colony PCR.
-<b> <font size="4">Chromosomal integration</font> </b>
-Following the 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.
+[[File:ovulaid26.png|300px]]
-[[File:ovulaid6.png|100px]]
+<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>
-<small><b>Figure 2: 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 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. </small>
-The band size on lanes 4 and 7 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 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:
 [[File:ovulaid21.png|500px]]
-<small><b>Figure 3: Western blot of insoluble vs soluble cellular protein  | </b> Western blot was carried out using anti-GFP antibodies. Yeast expressing empty vectors was taken  as negative control. Yeast expressing GFP was taken as positive control. All blue prestained protein standards was the ladder used for comparison. </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 GFP was used as negative and positive control respectively. Two replicate yeast cultures were used for the western blot. </small>
-The positive and negative control have worked as expected. GFP was so strongly expressed in the positive control cells that is was even seen in the insoluble fraction. Vice versa, GPER-sfGFP  was predominant in the insoluble fraction, which we expected since it is a membrane protein. A small band can however also be seen in the soluble fraction, indicating that the protein is very abundant in the respective cells.
+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.
 <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 GPER-Li-sfGFP and yeast expressing empty vectors (negative control).
+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 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 GPER-sfGFP.  </small>
+<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>
-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 sfGFP was visible on both bright field (C) and fluorescence filter (D) due to the expression of sfGFP, which confirms the expression of GPER as GFP is tagged to the C-terminal of the receptor. From (D) it also looks like GPER-sfGFP might have been expressed in the endoplasmic reticulum (ER).
+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).