Part:BBa_K1992011

Histamin-Tar expression system(promoter+RBS+coding+terminator)

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

The point mutations design 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, 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 (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