Difference between revisions of "Part:BBa K1992002"
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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) | 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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INBAL | INBAL | ||
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===GFP Based Migration Test=== | ===GFP Based Migration Test=== | ||
Revision as of 20:25, 15 October 2016
Histamin-Tar receptor
Novel Histamine-Tar chemoreceptor in E.coli
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 7
Illegal NheI site found at 30 - 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 1355
- 1000INCOMPATIBLE 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) (link to K777000 part). 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
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.
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)
INBAL