Difference between revisions of "Part:BBa K1992002"
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PctA-Tar
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__NOTOC__
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<!-- ######################## TITLE ######################### -->
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<partinfo>BBa_K1992002 short</partinfo>
  
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Introduction
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<!-- ######################## SUB TITLE ######################### -->
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Novel Histamine-Tar chemoreceptor in E.coli
  
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<!-- ######################## SEQUENCE ######################### -->
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<span class='h3bb'>Sequence and Features</span>
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<partinfo>BBa_K1992002 SequenceAndFeatures</partinfo>
  
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<!-- ######################## INTRODUCTION ######################### -->
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==Introduction==
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Tar is a chemoreceptor found in the bacterium E-coli which mediates chemotaxis towards Aspartic acid and away from Nickel and Cobalt (1). Using designs from bioinformatic tool, a novel chemoreceptor which mediates chemotaxis towards Histamine, was formed by inducing directed point mutations to the Tar ligand-binding domain (LBD) (part <partinfo>BBa_K777000</partinfo>). This novel receptor is a part of the S.Tar platform.
  
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<!-- ######################## Usage and Biology ######################### -->
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Methods
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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).
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==Usage and Biology==
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Histamine is a derivative of Histidine, which is also an amino acid as the native Tar ligand. The motivation to mediate chemotaxis towards Histamine is due to it’s presence in food poison, especially in rotten fish.
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The new chemoreceptor enables chemotactic attractant response to Histamine, althougth Histamin chemoreceptor could be found  in human cells, this is the first time this receptor is conducted in bacterial cell.
  
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<!-- ######################## Design considerations ######################### -->
  
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Figure 1: (a) Amino acids sequence of PctA-Tar chimera (1) (b) PctA-Tar chimera crystallography
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==Design considerations==
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[[File:T--Technion_Israel--Scheme_Histamine.png|300px|thumb|right|Figure 1: Design process schema for designing novel Histamine-Tar chemoreceptor with Rosetta software.]]
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<p align="justify">
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The point mutations deisgn was made by the Rosetta software. Rosetta is a powerfull bioniformatics tool for macromolecular modeling and design. To redesign the Tar ligand-binding domain (LBD), we followed a protocol from the literature(2). The output of the protocol is a library of about 870 variants, this fact means that filtering the results is an extremely crucial part of the process. Rosetta is able to predict which protein designs are likely to have improved protein activity, these predeictions enable to filter out the results and get a final library of 11 variants.The analysis of the variants in the library illustrates two main regions of point mutations, one around amino acid number 34 in the LBD sequence and the second around the 115th amino acid.
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Those results led us to design and perform a two-step cloning assay, in each step we insert the mutations with single PCR reaction.
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All variants went under microscope experiment, but only one variant (Histamine_9) succeed to show chemotaxis ability.
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</p>
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[[File:Histamine_alignment.png|450px|thumb|center|Figure 2: Alignment of Tar ligand binding domain (LBD). The alignment presents the 11 variants in the library with the native Tar (wild type). Variant Histamine_9 enable to show chemotaxis ability under microscope experiment.]]
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<br style="clear: both" />
  
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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.
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<!-- ######################## Experiments ######################### -->
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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.
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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.
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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.
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Results
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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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==Experiments==
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===GFP Based Migration Test===
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<p>
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E.coli native chemoreceptors cluster in the cell poles. This property is critical for signal amplification and adaptation of the cell, since it is crucial for additional proteins, such as kinases and adaptors to interact with the receptor, once it is situated in its proper location in the membran. Although little is known about the mechanism of localization, it is important to retain this property with our newly designed Histaine-Tar receptor - to ensure a functional and sensitive chemotactic response.
  
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To prove the correct localization, GFP was fused to the C-terminus of Histamine-Tar chemoreceptor, with a short linker sequence (<partinfo>BBa_E0040</partinfo>).The results of these tests as seen in figure 3, prove our assumption of correct localizations.
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Figur 2: Swarming assay for attractant response of the PctA-Tar chimera. From left to right: PctA chimera, UU1250, ∆Z.
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</p>
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[[File:T--Technion_Israel--Histamine-Tar-GFP.png|450px|thumb|center|Figure 3: Results of GFP fusion.
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<br><b>a.</b> Positive control- <i>E. Coli</i> strain expressing GFP protein.
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<br><b>b.</b> Negative control- <i>E. Coli</i> UU1250 strain expressing Tar chemoreceptor.
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<br><b>c.</b> <i>E. Coli</i> UU1250 strain expressing Tar-GFP chemoreceptor.
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<br><b>d.</b> <i>E. Coli</i> UU1250 strain expressing Histamine-Tar-GFP Chimera, fluorescence (490nm excitation).]]
  
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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.
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===Microscope Activity Test===
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Use of a microscope provided us with a relatively simple way to track bacterial chemotaxis in real time and with clear results. In order to perform the experiment we used an inverted microscope with the ability to record the data as a movie or as a time lapse. The purpose of this assay is to test the bacterial chemotactic response towards attractants.
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In this assay, a microfluidic chip was filled with a suspension of bacteria in motility buffer and placed under the microscope to ascertain the bacteria’s condition (alive and swimming)
  
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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.
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<!-- Center Figure -->
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[[File:His_9_microscope.png|450px|thumb|center|Figure 4: Experimental results from the microscope assay with bacteria engineered to detect Histamine. a) Immediately after Histamine addition b) 20 minutes after Histamine addition
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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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]]
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conclution
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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
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<br style="clear: both" />
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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.
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1)Reyes-Darias, J.A., Yang, Y., Sourjik, V., and Krell, T. (2015). Correlation between signal input and output in PctA and
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  PctB amino acid chemoreceptor of Pseudomonas aeruginosa. Mol. Microbiol. 96, 513–525.
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====References====
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2) https://eu.idtdna.com/site/order/gblockentry
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(1) Bi, Shuangyu, and Luhua Lai. "Bacterial chemoreceptors and chemoeffectors." Cellular and Molecular Life Sciences 72.4 (2015): 691-708. <br>
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3) http://www.pseudomonas.com/feature/show?id=111456
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(2) 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

Latest revision as of 23:00, 21 October 2016

Histamin-Tar receptor

Novel Histamine-Tar chemoreceptor in E.coli

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 1282
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI.rc site found at 111

Introduction

Tar is a chemoreceptor found in the bacterium E-coli which mediates chemotaxis towards Aspartic acid and away from Nickel and Cobalt (1). Using designs from bioinformatic tool, a novel chemoreceptor which mediates chemotaxis towards Histamine, was formed by inducing directed point mutations to the Tar ligand-binding domain (LBD) (part BBa_K777000). This novel receptor is a part of the S.Tar platform.


Usage and Biology

Histamine is a derivative of Histidine, which is also an amino acid as the native Tar ligand. The motivation to mediate chemotaxis towards Histamine is due to it’s presence in food poison, especially in rotten fish. The new chemoreceptor enables chemotactic attractant response to Histamine, althougth Histamin chemoreceptor could be found in human cells, this is the first time this receptor is conducted in bacterial cell.


Design considerations

Figure 1: Design process schema for designing novel Histamine-Tar chemoreceptor with Rosetta software.

The point mutations deisgn was made by the Rosetta software. Rosetta is a powerfull bioniformatics tool for macromolecular modeling and design. To redesign the Tar ligand-binding domain (LBD), we followed a protocol from the literature(2). The output of the protocol is a library of about 870 variants, this fact means that filtering the results is an extremely crucial part of the process. Rosetta is able to predict which protein designs are likely to have improved protein activity, these predeictions enable to filter out the results and get a final library of 11 variants.The analysis of the variants in the library illustrates two main regions of point mutations, one around amino acid number 34 in the LBD sequence and the second around the 115th amino acid. Those results led us to design and perform a two-step cloning assay, in each step we insert the mutations with single PCR reaction. All variants went under microscope experiment, but only one variant (Histamine_9) succeed to show chemotaxis ability.

Figure 2: Alignment of Tar ligand binding domain (LBD). The alignment presents the 11 variants in the library with the native Tar (wild type). Variant Histamine_9 enable to show chemotaxis ability under microscope experiment.



Experiments

GFP Based Migration Test

E.coli native chemoreceptors cluster in the cell poles. This property is critical for signal amplification and adaptation of the cell, since it is crucial for additional proteins, such as kinases and adaptors to interact with the receptor, once it is situated in its proper location in the membran. Although little is known about the mechanism of localization, it is important to retain this property with our newly designed Histaine-Tar receptor - to ensure a functional and sensitive chemotactic response. To prove the correct localization, GFP was fused to the C-terminus of Histamine-Tar chemoreceptor, with a short linker sequence (BBa_E0040).The results of these tests as seen in figure 3, prove our assumption of correct localizations.

Figure 3: Results of GFP fusion.
a. Positive control- E. Coli strain expressing GFP protein.
b. Negative control- E. Coli UU1250 strain expressing Tar chemoreceptor.
c. E. Coli UU1250 strain expressing Tar-GFP chemoreceptor.
d. E. Coli UU1250 strain expressing Histamine-Tar-GFP Chimera, fluorescence (490nm excitation).

Microscope Activity Test

Use of a microscope provided us with a relatively simple way to track bacterial chemotaxis in real time and with clear results. In order to perform the experiment we used an inverted microscope with the ability to record the data as a movie or as a time lapse. The purpose of this assay is to test the bacterial chemotactic response towards attractants. In this assay, a microfluidic chip was filled with a suspension of bacteria in motility buffer and placed under the microscope to ascertain the bacteria’s condition (alive and swimming)

Figure 4: Experimental results from the microscope assay with bacteria engineered to detect Histamine. a) Immediately after Histamine addition b) 20 minutes after Histamine addition




References

(1) Bi, Shuangyu, and Luhua Lai. "Bacterial chemoreceptors and chemoeffectors." Cellular and Molecular Life Sciences 72.4 (2015): 691-708.
(2) 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