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Team:Bielefeld-Germany/Project/Theory
From 2010.igem.org
Contents |
Introduction
Our iGEM project is to create an E. coli cell which is capable to sense Capsaicin in a complex sample and report the concentration with a luciferase light signal. The native receptor senses Acetosyringone. We used directed evolution to modify the binding region of the native receptor to generate a new capsaicin receptor. We established a screening system based on antibiotic concentration gradients to screen the newly generated receptors. In our E. coli Acetosyringone sensing system several parts driving from different organisms were assemlbed. The native receptor was subcloned from Agrobacterium tumefaciens to create a read out system with firefly luciferase.
Agrobacterium tumefaciens
The model organism Agrobacterium tumefaciens is a soil bacterium and can be found at nearly every point of the world Agrobacterium became known as a phyto-pathogen leading to the crown gall disease in dicotyledonous species [1]. The infection is actually caused by a gene transfer system located on an extrachromosomal element, the Ti plasmid. The infection can be divided into several steps. Predominantely A. tumefaciens senses phenolic compunds from hurted plants, but also aldose monosaccharides, low pH and low phosphate [2],[3]. When A. tumefaciens recognizes phenols with the virA receptor, a signal transduction cascade is initiated leadig to the expression of virulence genes. The next step is a physical interaction with the host plant. A type three secretion system is responsible for the DNA transfer of the Ti-plasmid from the bacterium into the host. The DNA is translocated to the nucleus, leading to the gene expression and the production of opin. A. tumefaciens uses the reprogrammed plant cells for metabolite production and therefore as a nutrient supplier.
For biotechnology purpose the Ti-Plasmid was disharmed. Instead the native transfer region (T-region) and a gene of interest could be easily introduced into the Ti plasmid. Agrobacterium-mediated DNA transfer is one of the most commonly used techniques of plant transformation [6].
Native receptor
To gain an evolutionary advance, A. tumefaciens needs a precise recognition system for potential hosts. The native sensing system is a two-component phospho-relay system in which VirA is an transmembrane-bound sensor while VirG is the intracellular response regulator [4]. The two genes for the sensing system are virA and virG [5]which are constitutively expressed at a basal level. VirA is a hitstidine kinase and after sensing the phenol 3,5-Dimethoxyacetophenone, Acetosyringone an autophosphorylation occurs at the His-474 residue [9],[10]. Later in the signal transduction cascade, the phosphorylated VirA leads to the transfer of the phosphate to Asp-52 residue of VirG [11],[12],[13]. VirG is the response regulator of the two-component system[3]. VirG acts as a transcription factor and binds to virulence box (vir Box) containing promotors, for example virB [7], [8]
VirA receptor structure
The virA receptor consists of 829 amino acids and is a transmembrane protein in the inner menbrane of A.tumefaciens [14]. VirA spans the inner membrane, with two transmembrane domains, a large periplasmic region, and a large C-terminal cytoplasmic domain. [16]. VirA directly senses the phenolic compounds for vir activation [15]. Therefore the linker domain is essential for induction by phenolic compounds [17]. The linker region is located in the cytosolic site at position 280 to 414 [15]. This region between aa 283 and 304 was highly conserved in four different strains of Agrobacterium, and therefore likely to serve as the receptor region for the phenolic inducers which are common to all four strains [18].
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Chang and Winnans revealed in their studies, the parts of the VirA receptor essential for the signal transduction [17]. A structured model for different inducing conditions are shown in the figure below.
Phenolic Compounds
The Ligand receptor interaction between Acetosyringone and VirA is based on the interaction of several chemical groups. First of all, the aromatic hydroxyl is essential. Metoxy groups in ortho position of the phenol play also crucial role in the signaling. Dimetoxy compounds have a higher activity than monometoxy compounds. The acetyl and alkyl groups in para position enhancing the binding affinity. VirA activating compounds must have two met-oxy groups in the ortho position and an additional carbonyl group on the R3 chain[22], [21], [20]. Regarding to the proton transfer model of Hess et al 1996 [26], the VirA activator transfers a proton to the basic area receptor binding site. The allosteric change leads to the phosphotransfer and the signaltransduction [24], [25].
Inducing enhancers
The sensitivity of this system is highly enhanced when additional aldose monosacchardic suggars occur in the environment of Agrobacterium. The sugar binding protein ChvE interact with the VirA receptor, leading to a much stronger vir gene expression [27].
Subcloning into E.coli and receptor function in new host
In our project we decided to work with E coli instead of A. tumefaciens. The transcription procedure in E. coli is very similar to A. tumefaciens but not complete homolog. In E. coli the rpoA gene - encoding the α-subunit of RNA polymerase in A. tumefaciens is not present but essential for the transcription of a virB promoter driven genen [29]. For this reason it was necessary to subclone a modified virG gene that is capaple to be trancribed by the E. coli expression system.
Yong-Chul described VirG conderived mutants that are capable of expressing the virB promoter-driven gene in E. coli without the requirement for the RpoA from A. tumefaciens, suggesting that the virG mutants are able to interact with the transcription system of E.coli [28]. In VirG the amino acid at position 56 is likely to play a key role in the interaction with the RpoA of E. coli. Regarding to [28 we used virG mutants, with amino acid substitutions of G56V and I77V that are capable of activating vir genes in E. coli in response to inducer acetosyringone in a virA-dependent manner.
