Difference between revisions of "Part:BBa K1992001"
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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).
 
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 proof the concept of the S. Tar platform.
+
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
 
Usage and biology
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Design considerations
 
Design considerations
  
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When designing a 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. Therefore it is recommended to use the part itself, since its synthesis is problematic.  
+
When designing a 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. Therefore it is recommended to use the part itself, since its synthesis is problematic.  
  
 
   
 
   
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Figure 1: (a) Amino acids sequence of PctA-Tar chimera (1) (b) PctA-Tar chimera crystalogaphy
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Figure 1: (a) Amino acids sequence of PctA-Tar chimera (1) (b) PctA-Tar chimera crystallography
  
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The plasmid carrying the chimera was transformed into receptorless bacteria- UU1250 (Parkinson J S, University of Utah) and the presents of the DNA sequence was verified by a colony PCR and sequencing.
+
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.
+
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, probably due to high concentration of amino acids 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.
 
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 (TCE) served as a repellent. TCE 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.
+
In addition, a protocol for a 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 (pSB1A2 was served as vector) and a blue chromoprotein plasmid (pSB1C3 was served as a vector with J23100+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
 
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.   
+
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.   
  
 
   
 
   
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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.
 
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.
  
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Figure 3: GFP fused to the PctA-Chimera is located in the polar part of the bacterial membrane. From left to right: PctA-Tar strain, Cloned Tar strain.
+
Figure 3: GFP fused to the PctA-Chimera is located in the polar part of the bacterial membrane. From left to right: PctA-Tar strain, cloned Tar strain.
 
   
 
   
 
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.
 
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.
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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
 
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.  
+
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.  
  
  

Revision as of 10:29, 14 October 2016

PctA-Tar

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.

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 a 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. 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. Amino acids sequence of the LBD and HAMP 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 1a).


Figure 1: (a) Amino acids sequence of PctA-Tar chimera (1) (b) PctA-Tar chimera crystallography

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, probably due to high concentration of amino acids 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, making us the first group to ever 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) 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.


Figur 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: GFP fused to the PctA-Chimera is located in the polar part of the bacterial membrane. From left to right: PctA-Tar strain, cloned Tar strain.

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.

conclution

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) https://eu.idtdna.com/site/order/gblockentry 3) http://www.pseudomonas.com/feature/show?id=111456