Difference between revisions of "Team:Technion Israel/Modifications/pcta"
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cytoplasmatic region, was obtained from the iGEM parts catalog.<br> | cytoplasmatic region, was obtained from the iGEM parts catalog.<br> | ||
The plasmid carrying the chimera was transformed into receptorless bacteria- UU1250 (Parkinson J S, University of Utah) | The plasmid carrying the chimera was transformed into receptorless bacteria- UU1250 (Parkinson J S, University of Utah) | ||
| − | and the | + | and the presence of the DNA sequence was verified by a colony PCR and sequencing.<br> |
In order to test the motility of our strain, chemotaxis assays were performed. First, swarming assay was conducted using a poor BA medium. | In order to test the motility of our strain, chemotaxis assays were performed. First, swarming assay was conducted using a poor BA medium. | ||
Note that with a TB medium the strain failed to show decisive results, provably due to high concentration of amino acid which resulted in no movement.<br> | Note that with a TB medium the strain failed to show decisive results, provably due to high concentration of amino acid which resulted in no movement.<br> | ||
Revision as of 09:20, 14 October 2016
PctA-Tar - Introduction
Introduction
PctA is a chemoreceptor found in the bacterium Pseudomonas Aeruginosa which mediates chemotaxis toward amino
acids and away from organic compounds. It is sensitive to all amino acids accept for Aspartate (1).
A PctA-Tar chimera was formed by the replacement of the Tar LBD with the one of PctA. A procedure which previously was
conducted in the literature was recovered (1), in order to prove the concept of the S. Tar platform.
Design and Implementation
The sequence for the desired segment of PctA was taken from the Pseudomonas genome database (2). The second part of the chimera, Tar’s
cytoplasmatic region, was obtained from the iGEM parts catalog.
The plasmid carrying the chimera was transformed into receptorless bacteria- UU1250 (Parkinson J S, University of Utah)
and the presence of the DNA sequence was verified by a colony PCR and sequencing.
In order to test the motility of our strain, chemotaxis assays were performed. First, swarming assay was conducted using a poor BA medium.
Note that with a TB medium the strain failed to show decisive results, provably due to high concentration of amino acid which resulted in no movement.
Following the swarming assay, a GFP gene was fused to the chimera to verify the migration of the chemoreceptor to the poles of the bacterial membrane.
In addition, a protocol for a chemotaxis assay on chip for repellent response was constructed by us and we are the first group to show this
kind of assay. A receptorless strain containing a PctA-Tar chimera plasmid (pSB1A2 was served as vector) and a blue chromoprotein plasmid
(pSB1C3 was served as a vector with J23100+K592009 biobricks). This clone resulted in blue bacteria expressing the PctA-Tar chimera. The
strain was confined into a commercial ibidi microchannel while Tetrachloroethylene (PCE) served as a repellent. PCE in the concentration
of 9×10-5M (0.015 g/lit) was inserted through the well and the bacterial movement was monitored throughout the entire experiment.
Results
The strain containing the PctA-Tar chimera showed a significant movement on the swarming assay with a BA medium. On figure 1 the swarming assay of our strain is shown compared to our negative control (UU1250) and our positive control (∆Z). Hence, the PctA-Tar chimera is expressed and bacterial movement is mediated by it.
a.
b.
c.
Fig. 1: Swarming assay for attractant response of the PctA-Tar chimera. a. PctA chimera, b. UU1250, c.∆Z.
As for the GFP fusion, the clone exhibited fluorescence on the polar parts of the bacterial membrane (figure 2), indicating for a proper migration of the chimera to the poles of the membrane, as expected.
a.
b.
Fig. 2: GFP fused to the PctA-Chimera is located in the polar part of the bacterial membrane. a. PctA-Tar strain, b. Cloned Tar strain.
Moreover, a repellent response was shown while using our new chemotaxis assay for repellent response.
The bacterial movement away from the PCE was distinguished to the naked eye and significant.
Outlook
Using our strain containing the PctA-Tar chimera we showed a modified response of the Tar chemoreceptor to substances which are natural attractants and repellents of PctA as it found originally in the bacterium Pseudomonas Aeruginosa. Therefore, this work serves as a decisive proof of concept for our S. Tar platform and it has valuable scientific meanings for our project.
1. Reyes-Darias, J.A., Yang, Y., Sourjik, V., and Krell, T. (2015). Correlation between signal input and output in PctA and PctB amino acid chemoreceptor of Pseudomonas aeruginosa. Mol. Microbiol. 96, 513–525.
2. Pseudomonas genome database
