Part:BBa_K1992001
PctA-Tar hybrid receptor
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 270
Illegal NgoMIV site found at 351
Illegal NgoMIV site found at 355
Illegal NgoMIV site found at 374
Illegal AgeI site found at 474
Illegal AgeI site found at 685
Illegal AgeI site found at 1540 - 1000COMPATIBLE WITH RFC[1000]
Introduction
PctA is a chemoreceptor found in the bacterium Pseudomonas Aeruginosa which mediates chemotaxis towards amino acids and away from organic compounds. It is sensitive to all amino acids except for Aspartate (1). A PctA-Tar chimera was formed by the replacement of the Tar ligand binding domain (LBD) with the one of PctA. A procedure which previously was conducted in the literature was repeated (1), in order to prove the concept of the S. Tar platform.
Usage and Biology
Integration of the new chemoreceptor with the chemotaxis system of E. coli has been enabled thanks to our chimera design. Hence, a chemotactic attractant response to new amino acids and a repellent response to organic compounds, which weren't detected by E. coli before, are now mediated. This property is influential and can be used in various applications, including detection of water pollutants.
Design considerations
When designing the gBlock for the PctA-Tar Chimera an incompatible part is obtained, this according to the IDT gBlock sequence online analyzer (2). As a result, a split part has to be designed. The two parts have an overlap sequence, allowing to connect them using Gibson assembly. In addition, in the designing process a silent mutation was performed in order to delete PstI and EcoRI restriction sites and to lower GC content (Changes in the sequence can be seen in the design section and in features above). Due to its problematic synthesis, it is recommended to use the part itself.
Methods
The sequence for the desired segment of PctA was taken from the Pseudomonas genome database (3). The second part of the chimera, Tar’s cytoplasmatic region, was obtained from the iGEM parts catalog (BBa_K777000). Amino acids sequence of the LBD and HAMP (Histidine kinases, Adenylate cyclases, Methyl accepting proteins) of PctA was determined to be from 1 to 134 and this of the signaling region of Tar was determined to be 296-553 (Figure 1). See sequence and features.
A high expression plasmid carrying the chimera (BBa_K1992007) was transformed into bacteria lacking chemoreceptors- UU1250 strain (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 chemotaxis motility of our strain, chemotaxis assays were performed. First, a [http://2016.igem.org/Team:Technion_Israel/Experiments swarming assay] was conducted using a poor [http://2016.igem.org/Team:Technion_Israel/protocols BA medium]. Note that with a TB medium the strain failed to show decisive results, probably due to high concentration of amino acids which resulted in no movement.
Following the [http://2016.igem.org/Team:Technion_Israel/Experiments swarming assay], a GFP gene was fused to the chimera (BBa_K1992010) to verify the migration of the chemoreceptor to the poles of the bacterial membrane. This property is critical for signal amplification and adaptation of the cell (4).
In addition, a protocol for a [http://2016.igem.org/Team:Technion_Israel/Experiments chemotaxis assay on chip] for repellent response was constructed, making us the first group to ever show this kind of assay. A receptorless strain containing a PctA-Tar chimera plasmid (pSB1C3 was served as vector) and a blue chromoprotein plasmid (pSB1A2 was served as a vector with BBa_J23100+BBa_K592009 BioBricks) was cloned. This clone resulted in blue bacteria expressing the PctA-Tar chimera. The strain was confined into a commercial ibidi microchannel while Trichloroethylene (TCE) served as a repellent. TCE in the concentration of 10-3M (0.13 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 2 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.
As for the GFP fusion, the clone exhibited fluorescence on the polar parts of the bacterial membrane (Figure 3), indicating for a proper migration of the chimera to the poles of the membrane, as expected.
Moreover, a repellent response was shown while using our new chemotaxis assay for repellent response. The bacterial movement away from the TCE was distinguished to the naked eye and significant.
Conclusion
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.
References
(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) https://eu.idtdna.com/site/order/gblockentr
(3) http://www.pseudomonas.com/feature/show?id=111456
(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.
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