Team:Technion Israel/Modifications/EstoTar
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.
Despite the discouraging results from the GFP assay we attempted the “Chip microscope assay” to test for any real time response. Our first tests ended abruptly as we discovered that the estrogenic solution kills the bacteria almost immediately. Later we concluded that the fault was in the solvent that was used to dissolve the compound, 17-β-estradiol. Since this is a hydrophobic substance, it is only possible to dissolve it in hydrophobic solvents such as Ethanol and DMSO which are lethal for bacteria. When the stock solution, of 17-β-estradiol in DMSO, was diluted to a concentration that was not lethal for the bacteria - 0.1% DMSO content, no signs of response were visible.
A noteworthy phenomenon that occurred during one of the microscope tests, was when a solution of 17-β-estradiol dissolved in DMSO (concentration 10-5 mg/ml) was added to the
UERT strain suspension and completely halted the bacterial movement but several minutes later the bacteria regained viability. This did not occur when tried on the control strain.
====================** Video #1 **====================
Video 1: Link to microscope assay. a. Adding 10-5[mg/ml] 17-β-estradiol in DMSO to UERT. b. Control - Adding DMSO to UERT.
In attempts to understand and repeat this phenomenon, a range of estradiol concentrations was tested but none of them succeeded.
====================** Video #2 **====================
Video 2: Microscope assay.
Add the link.a. Adding 10-1[mg/ml] 17-β-estradiol in DMSO to
UERT.
b. Adding 10-2[mg/ml] 17-β-estradiol in DMSO to
UERT.
c. Adding 10-3[mg/ml] 17-β-estradiol in DMSO to
UERT.
d. Adding 10-5[mg/ml] 17-β-estradiol in DMSO to
UERT.
e. Adding 10-7[mg/ml] 17-β-estradiol in DMSO to
UERT.
f. Control - Adding DMSO to
UERT.
Outlook
Although the concept of this idea seemed promising, but the PctA-Tar and NarX chimeras 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.). Both mentioned chimeras were built using their own HAMP rather than Tars’ native HAMP. In contrast the hERɑ does not contain HAMP region, thus its chimera was built using the Tar’s native HAMP. We believe that this is the main reason of the chimera failure, and results of all tests support this assumption. For an example, in the modeled 3D structure, it is clear that HAMP region is the problematic, as there it is clear the HAMP is out of place.
Furthermore, the results of the GFP also fit this assumption, as the chimera was unsuccessful at crossing the membrane, probable due to the HAMP.
Lastly, most of the reported chemotaxis receptors naturally contain a HAMP domain, and seems to important in regulating the coiled-coil interactions that seem to be involved in signal propagation, which further supports our assumption. (5,2)
To conclude, further research is needed in order to fully understand the cause of failure and overcome the obstacles faced in this attempt of generating a new chemoreceptor which reacts to estrogen derivatives.
The main problems and a way to overcome them are as follows:
-The estrogen derivatives used were solubile only in hydrophics solvents which are lethat to the bacteria. The use of estrogen soluble in water or other non-lethal solvents should solve this problem.
-The 3D model did indicated incorrect folding of the newly designed receptor. A new design focused more on the Tar might aid in solving this problem.
-The hERa does not naturally contain a HAMP region. Using a chemoreceptor that does not have a HAMP domain might help overcoming this obstacle.
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.
