Difference between revisions of "Team:Technion Israel/Results"
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future this ability can be used in the same manner to | future this ability can be used in the same manner to | ||
Revision as of 09:40, 19 October 2016
Histamine-Tar novel chemoreceptor
We successfully redesigned, clone and test a novel Histamine-Tar chemoreceptor. The design made with Rosetta bioinformatics tool, by following a protocol presented in "Rosetta and the Design of Ligand Binding Sites" (1), in order to design a binding site around a selected small molecule ligand. The Rosetta’s design process for the new ligand Histamine produced 870 results, out of which 11 variants remained after filtering. The 11 variants were cloned into the native Tar ligand-binding domain(LBD), out of them only 6 exhibited the expected sequences in sequencing and were subjected to chemotaxis tests. Out of 6 tested variants only one of Tar mutated variants (variant number 9) was successfully designed and verified to bind Histamine as a ligand, instead of Aspartate. The results of the chemotaxis test for variant number 9 are presented in figure 2.
Fig. 1: Sequencing Results. Query describes the native Tar LBD sequence and Sbjct describes the design mutations sequence. Each mutation regin marked with diffrent color (blue and red).
a.
b.
c.
d.
Fig. 2: microscope results of chemotaxis activity for variant His_9 with 10mM of Histamine. a. Tar-Histamine after 0 minutes (when the Histamine added). b. Tar-Histamine after 20 minutes. c. Control after 0 minutes (when the Histamine added). d. Control after 20 minutes.
As we showed, Rosetta provides us with the means to
redesign a chemoreceptor to bind new ligands. In the
future this ability can be used in the same manner to
design dozens of new receptors. The critical step of the
design process remains the lab work required to clone and
test the variants, this step can be optimized by using a high
throughput chemotaxis assay. Aside from this, any receptor designed
can be further improved by introducing a directed evolution step to
improve its specificity towards the new ligand.
In addition, we wrote “How to use” Rosetta guide with Rosetta developers and
iGEM TU Eindhoven team, and build a framwork software tool to allow fellow researchers the ability to redesign a binding site easlly.
FlashLab - microfluidic chip
We showed a working concept of the FlashLab project - a chip that serves as a detection tool based on the chemotaxis system of E. coli bacteria - by using a commercial ibidi chip filled with a suspension of bacteria expressing the desired receptor and chromoprotein. We successfully demonstrated this concept for both PctA-Tar chimera and Histamine-Tar variant of the Rosetta software.
In video 1, the displacement of the bacteria can be clearly seen in test chip (left chip, PctA-Tar with repellent), compared to he control chip (right chip, PctA-Tar with buffer).
Video 1: from left to right: (1) PctA-Tar chimera with Tetrachloroethylene repellent added. (2) PctA-Tar chimera with Motility buffer added (control).
To conclude, we demonstrated a working concept of the FlashLab project, by using a commercial ibidi chip filled with a suspension of bacteria expressing the chimera and chromoprotein. A solution of a repellent/attractant was added to the chip and the displacement of the bacteria was monitored and recorded.
PctA-Tar chimera
We have successfully confirmed the functionality of the hybrid PctA-Tar receptor using swarming assay. From the results seen below, figure 3, and compared to the negative control, it is clear that the chimera functions and controls the chemotactic ability of the bacteria and can lead to swarming response.
a.
b.
c.
Fig. 3: Swarming assay for attractant response of the PctA-Tar chimera.
a. PctA chimera, b. Negative control- UU1250 strain w/o the Tar expression plasmid, c. positive control - ΔZ strain expressing all chemoreceptors.
PctA-Tar fused with GFP marker:
In addition, the correct localization of the chimera on both poles of the bacteria was proven through fusion of GFP to the C- terminus of the chimera. The results of these tests as seen in figure 4, prove the correct localizations.
Fig. 4: Results of GFP fusion. (A) Positive control- E.Coli strain expressing GFP protein,(B) Negative control- UU1250 strain expressing Tar chemoreceptor, (C) UU1250 strain expressing Tar-GFP chemoreceptor, (D) UU1250 strain expressing PctA-Tar-GFP Chimera, Flourcense (490nm excitation).
References:
1. Moretti, R., Bender, B.J., Allison, B. and Meiler, J., 2016. Rosetta and the Design of Ligand Binding Sites. Computational Design of Ligand Binding Proteins, pp.47-62
