Difference between revisions of "Team:Technion Israel/Modifications/EstoTar"
| Line 198: | Line 198: | ||
<h2 class="">Introduction</h2> | <h2 class="">Introduction</h2> | ||
<p class="text-justify"> | <p class="text-justify"> | ||
| − | + | Our attempts to fuse two segments originating from different organisms to design a new receptor was met with great challenges. These specific segments were the LBD of the Human Estrogen Receptor α (hERa) and the cytoplasmic domain of Tar.<br><br> | |
| − | + | ||
| + | |||
| + | hERɑ is a human nuclear receptor that induces signal transduction in response to estrogenic compounds. Despite the fact that bacterial chemoreceptors are comprised of a two component system and the hERα is not, we assumed that hERa will trigger the phosphorylation cascade of the chemotaxis system, due to the conformational changes caused by the estrogen binding to its domain. This led us to design and construct the new hybrid: hERa-Tar (1). | ||
| + | |||
<br> | <br> | ||
<b>hERɑ</b> is a human nuclear receptor that induces signal transduction in response to estrogenic compounds. | <b>hERɑ</b> is a human nuclear receptor that induces signal transduction in response to estrogenic compounds. | ||
Revision as of 19:40, 16 October 2016
hERɑ - Introduction
Introduction
Our attempts to fuse two segments originating from different organisms to design a new receptor was met with great challenges. These specific segments were the LBD of the Human Estrogen Receptor α (hERa) and the cytoplasmic domain of Tar.
hERɑ is a human nuclear receptor that induces signal transduction in response to estrogenic compounds. Despite the fact that bacterial chemoreceptors are comprised of a two component system and the hERα is not, we assumed that hERa will trigger the phosphorylation cascade of the chemotaxis system, due to the conformational changes caused by the estrogen binding to its domain. This led us to design and construct the new hybrid: hERa-Tar (1).
hERɑ is a human nuclear receptor that induces signal transduction in response to estrogenic compounds.
Design and Implementation
As we designed intein-gBlock (see page) containing the
LBS
of hERɑ, this LBD was used in this approach as well, by isolation of the LBD rom the intein.
The hERɑ-Tar chimera was created by using the cDNA sequence coding for the LBD of hERɑ and fusing it to the C terminus of tar. The chimera was clone into
UU1250
to generate strain
UERT
As far as we know, this move has never been done before.
In order to predict whether it can be possible, we modeled the structure of hERɑ-Tar chimera, comparing
to the native Tar chemoreceptor, using Phyre2 online modeling server (2)
====================**3d modeling of est-tar**====================
In order to verify that hERɑ-Tar chimera migrated to the proper location in the cell membrane - GFP was fused to the C terminus of Tar,
and the protein was tracked by using fluorescence microscope. The proper location of Tar is in the membrane poles (3).
To validate whether the chemotaxis ability can be restored - chemotaxis assay Link to microscope assay
was performed with 17-ꞵ-estradiol as a chemical, to check whether the strain has a attractant/repellent response to it.
Results
The clone was sequenced successfully. However, when testing the chemotaxis ability of our new strain, we had difficulties dissolving
the estrogenic compound in a solvent that does not kill our bacteria. The compound we tried to use is 17-β-estradiol, which is a
hydrophobic substance that dissolves in hydrophobic solvents, such as Ethanol and DMSO. These solvents are lethal for bacteria. When
estradiol stock was diluted to a concentration that did not kill the bacteria - 0.1% DMSO in the solution, they showed no response whatsoever.
When the location of hERɑ-Tar chimera in the cell was examined, under a Link to microscope assay, there was green fluorescence
in the entire cell, which indicates that the chimera accumulated inside the cytoplasm.
a.
b.
Fig. 2: UERTG under a fluorescence microscope. a. Under fluorescence light. b. Under white light.
Apparently, the human estrogen-LBD causes disruption in the hERɑ-Tar chimera structure that leads to disconnection between
the binding region and the signaling region. The position of this chimera, viewed via fluorescence microscope, support this
assumption, since it did not reach its proper location in the membrane, instead it stayed in the cytoplasm, cannot sense the environment.
An interesting phenomena happened when a solution of 17-β-estradiol dissolved in DMSO (concentration 10-5 mg/ml) was added to
UERT strain.
The bacteria stopped moving, and after a few minutes returned to move. As mentioned, DMSO is lethal to bacteria,
however the bacteria managed to survive. It might be the estradiol that “helped” the bacteria to recover. Comparing
to the control - when only DMSO was added to the bacteria, they were dead.
====================** Video #1 **====================
Video 1: Link to microscope assay. a. Adding 17-β-estradiol in DMSO to UERT. b. Control - Adding DMSO to UERT.
Following, a range of 17-β-estradiol concentration in DMSO was added to
UERT,
in order to find the concentration range of estradiol that affect the bacetria. Unfortunately, we couldn’t repeat the
results to raise a reasonable hypothesis.
====================** Video #2 **====================
Video 2: Microscope assay
Add the link.
PctA-Tar and NarX chimera had more potential to succeed, due to their structure similarity to the native Tar chemoreceptor (they all contain
HAMP,
domain, which is located invariably at the C-terminal end of the last transmembrane segment). These chimeras were built using the foreign
HAMP rather than the native HAMP of Tar. In contrast to the human hERɑ receptor, which does not contain HAMP region. This chimera was built
using the native HAMP of Tar. This fact indicates that in order to connect unnatural LBD to the C terminus of Tar - this LBD must be
naturally connected to HAMP domain for functional chemoreceptors.
Most of chemotaxis receptors contain HAMP domain (two-thirds) , though, Sequence conservation of the domain is not strong and includes no
invariant residues (1). This fact support our assumption.
Outlook
A further research is need to be done, regarding dissolving estrogen derivatives in a non-fatal solvent.
In addition, another attempt of fusing GFP to hERɑ-Tar chimera is necessary to conclude it definitely as a failure.
Next chemoreceptors will be generated by using Binding region contain HAMP domain.
1. HULKO, Michael, et al. The HAMP domain structure implies helix rotation in transmembrane signaling. Cell, 2006, 126.5: 929-940.
2. Phyre2 modeling server.
3. SHIOMI, Daisuke, et al. Helical distribution of the bacterial chemoreceptor via colocalization with the Sec protein translocation
machinery. Molecular microbiology, 2006, 60.4: 894-906.
