Difference between revisions of "Part:BBa K1992001"

Revision as of 23:58, 19 October 2016

PctA-Tar hybrid receptor

Sequence and Features

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 except 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.

Usage and Biology

Integration of the new chemoreceptor with the chemotaxis system of E. coli is 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.

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 part have overlap sequence allowing the connect them using Gibson assembly. In the design process silent mutation performed in order to to delete PstI and EcoRI restriction sites and to lower GC contect (changes in the sequence can be seen in the design section and in features above). Therefore it is recommended to use the part itself, since its synthesis is problematic.

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 (ligand binding domain) 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.

Figure 1: PctA-Tar chimera crystallography. Red - PctA LBD and HAMP, gray - Tar signaling domain.

A high expression plasmid carrying the chimera (BBa_K1992007) 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,[http://2016.igem.org/Team:Technion_Israel/Experiments swarming assay] was conducted using a poor 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.

Figure 2: Swarming assay for attractant response of the PctA-Tar chimera. From left to right: PctA chimera, UU1250, ∆Z.

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.

Figure 3: 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, fluorescence (490nm excitation).

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.

Video 1: from left to right: (1) PctA-Tar chimera with Tetrachloroethylene repellent added. (2) PctA-Tar chimera with Motility buffer added (control).

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