Difference between revisions of "Team:Technion Israel/Modifications/EstoTar"
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| − | under a fluorescence microscope. <b>a.</b> Under fluorescence light. <b>b.</b> Under white light. | + | under a fluorescence microscope. <b>a.</b> Under fluorescence light at 490nm excitation. <b>b.</b> Under white light. |
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Revision as of 05:15, 17 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).
Procedure:
The intein-gBlock was designed with the estrogen
LBD
site as the splicing inducer. The cDNA sequence was the source of said
LBD in the intein gBlock.
This design provided the team an opportunity to easily extract the
LBD and fuse it to HAMP region of Tar to get a final hybrid product hERa-Tar.
The new chimera was cloned to UU1250 to generate the new strain:
UERT. To the best of our knowledge, this design and cloning has never been reported before.
In order to predict the feasibility of this new hybrid, a 3D model was made using the Phyre2 Fold Recognition server (3).
Later, in order to confirm the correct localization at both poles of the bacterial membrane (4), a GFP reporter protein was fused to the hERa-Tar chimera and tested with fluorescence microscopy.
Finally, a “Chip Microscope assay” was conducted to study the effects of 17- β-estradiol on the chemotaxis system of the
UERT strain. In short, a suspension of the
UERT strain was added to an ibidi microchannel chip and the bacterial concentration was monitored in a fixed point for the whole experiment as the estradiol was added to the channel.
Results
The 3D structure of the hERa-Tar, as can be seen in figure 1, clearly indicates an incorrect folding of the HAMP region and thus an overall incorrect structure. Nevertheless, the rest of the tests were conducted in hope for successful results. ====================**3d modeling of est-tar**====================
The results of the fluorescence microscopy were not promising neither. That is due to the fact that although the GFP was expressed, the signal indicated that the chimera failed to localize at the poles and stayed in the cytoplasm, figure 2. In other words, in case that the chimera is expressed it will not be able to cross the membrane and thus the chemotaxis system will not function correctly.
a.
b.
Fig. 2: UERTG under a fluorescence microscope. a. Under fluorescence light at 490nm excitation. 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. CHEN, Dongsheng, et al. Phosphorylation of human estrogen receptor α by protein kinase A regulates dimerization. Molecular and cellular biology, 1999, 19.2: 1002-1015.
2. HULKO, Michael, et al. The HAMP domain structure implies helix rotation in transmembrane signaling. Cell, 2006, 126.5: 929-940.
3. Phyre2 modeling server.
4. 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.
5. WADHAMS, George H.; ARMITAGE, Judith P. Making sense of it all: bacterial chemotaxis. Nature Reviews Molecular Cell Biology, 2004, 5.12: 1024-1037.
