Difference between revisions of "Part:BBa K1992002"

Revision as of 20:58, 15 October 2016

Histamin-Tar receptor

Novel Histamine-Tar chemoreceptor in E.coli

Sequence and Features

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
INCOMPATIBLE WITH RFC[12]
Illegal NheI site found at 7
Illegal NheI site found at 30
21
COMPATIBLE WITH RFC[21]
23
COMPATIBLE WITH RFC[23]
25
INCOMPATIBLE WITH RFC[25]
Illegal AgeI site found at 1355
1000
INCOMPATIBLE WITH RFC[1000]
Illegal SapI.rc site found at 184

Introduction

Tar is a chemoreceptor found in the bacterium E-coli which mediates chemotaxis toward Aspartic acid and away from Nickel and Cobalt (1). Using designs from bioinformatic tool, a noval chemoreceptor, which mediates chemotaxis toward Histamine, was formed by induce directed point mutations to the Tar ligand-binding domain (LBD) (part BBa_K777000). This noval 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 mediates chemotaxis toward Histamine is duo to it’s presence in food poison, especially in rotten fish. The new chemoreceptor enable chemotactic attractant response to Histamine, althogth Histamin chemoreceptor could be found in human cells, this is the first time this receptor conducted in bacterium cell.

Design considerations

Figure 1: Alignment of Tar ligand binding domain (LBD). The alignment presents the 11 variants in the library with the native Tar (wild type). Variant His_9 enable to show chemotaxis ability under microscope experiment

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 900 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, this predeictions enable to filter out the results and get a final library of 11 varaints. Analyzing the variants in the library illustrating 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 go under microscope experiment with only one variant succeed to show chemotaxis ability

Experiments

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

@@ Line 21: / Line 21: @@
 <!-- ######################## Design considerations ######################### -->
 ==Design considerations==
-[[File:Histamine_alignment.png|450px|thumb|right|Figure 1: Alignment of Tar ligand binding domain (LBD). The alignment presents the 11 variants in the library with the native Tar (wild type). Variant His_9 is the one who showed chemotaxis ability ]]
+[[File:Histamine_alignment.png|450px|thumb|right|Figure 1: Alignment of Tar ligand binding domain (LBD). The alignment presents the 11 variants in the library with the native Tar (wild type). Variant His_9 enable to show chemotaxis ability under microscope experiment]]
 <p align="justify">
 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 900 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, this predeictions enable to filter out the results and get a final library of 11 varaints. Analyzing the variants in the library illustrating two main regions of point mutations, one around amino acid number 34 in the LBD sequence and the second around the 115th amino acid.