Team:Bielefeld-Germany/Project/Theory

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     <li><a href="/Team:Bielefeld-Germany/Project/Theory">Theory</a></li>
     <li><a href="/Team:Bielefeld-Germany/Project/Theory">Theory</a></li>
     <li><a href="/Team:Bielefeld-Germany/Project/Protocols">Protocols</a></li>
     <li><a href="/Team:Bielefeld-Germany/Project/Protocols">Protocols</a></li>
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    <li><a href="/Team:Bielefeld-Germany/Project/Results">Results</a></li>
 
     <li><a href="/Team:Bielefeld-Germany/Project/Model">Model</a></li>
     <li><a href="/Team:Bielefeld-Germany/Project/Model">Model</a></li>
     <li><a href="/Team:Bielefeld-Germany/Project/Outlook">Outlook</a></li>
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= Introduction =
= Introduction =
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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.
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In our iGEM project we tried to create an ''Escherichia coli'' cell which is capable to sense capsaicin in a complex sample and report the concentration with a luciferase light signal. We combined a native receptor system of ''Agrobacterium tumefaciens'' with the readout system of the firefly luciferase. The native receptor senses the phenolic compound acetosyringone. For this reason we used directed evolution to modify the binding region of the native receptor to generate a new capsaicin receptor because of the chemical similarities of acetosyringone and capsaicin. 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 derived from different organisms were assembled. The readout system taken from firefly, luciferase, the receptor sensing system derived from ''A. tumefaciens'' and sensitivity tuners consist of phage DNA.
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= ''Agrobacterium tumefaciens'' =
= ''Agrobacterium tumefaciens'' =
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[[Image:Bielefeld_Agrobacterium_tumefaciens_attached_to_a_plant_cell_Image_by_Martha_Hawe.jpg|500px|right|thumb|right|Image by Martha Hawe]]  
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[[Image:Bielefeld_Agrobacterium_tumefaciens_attached_to_a_plant_cell_Image_by_Martha_Hawe.jpg|500px|right|thumb|right| '''Figure 1: Image by Martha Hawe''']]  
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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( DeCleene M, DeLay J, 1976). 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 (Palmer AG. ''et al.''2004),(Brencic A, Winans SC, 2005). 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.
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The model organism ''A. tumefaciens'' is a soil bacterium and can be found at nearly every place in the world. ''Agrobacteria'' became known as a phyto-pathogen leading to the crown gall disease in dicotyledonous species ([http://www.springerlink.com/content/hm17520m287ht766/ DeCleene M and DeLay J, 1976]). The infection is caused by a gene transfer system located on an extrachromosomal element, the Ti-plasmid. Furthermore, the infection can be divided into several steps: The first step is the localisation of the hurt plant by the bacteria. Predominantely ''A. tumefaciens'' senses phenolic compunds from hurt plants, but also aldose monosaccharides, low pH and low phosphate ([http://www.annualreviews.org/doi/abs/10.1146/annurev.phyto.41.052002.095701?journalCode=phyto Palmer AG. ''et al.''2004]; [http://mmbr.asm.org/cgi/content/abstract/69/1/155 Brencic A and Winans SC, 2005]). When ''A. tumefaciens'' recognizes phenols with the VirA receptor, a signal transduction cascade is initiated leading 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.
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For biotechnological purposes the Ti-plasmid was disharmed. Instead of 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 ([http://people.uleth.ca/~alicja.ziemienowicz/extra/Ziemienowicz_ABP2001.pdf Ziemienowicz A, 2001]).
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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 (Ziemienowicz A, 2001).
 
= Native receptor =
= Native receptor =
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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
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''A. tumefaciens'' needs a precise recognition system for potential hosts to gain an evolutionary advance. The native sensing system is a two-component phospho-relay system in which VirA is a transmembrane-bound sensor while VirG is the intracellular response regulator ([http://www.biomedcentral.com/content/pdf/gb-2002-3-10-reviews3013.pdf Wolanin PM ''et al.'', 2002]). The two genes for the sensing system are ''virA'' and ''virG'' ([http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1166964/ Stachel SE and Nester EW, 1986]) which are constitutively expressed at a basal level. VirA is a histidine kinase. An autophosphorylation occurs at the His-474 residue, after sensing the phenol 3,5-dimethoxyacetophenoneacetosyringone ([http://www.ncbi.nlm.nih.gov/pmc/articles/PMC208549/?pageindex=1&tool=pmcentrez Huang Y ''et al.'', 1990] ; [http://jb.asm.org/cgi/content/abstract/172/2/531 Jin SG ''et al.'', 1990a]). In the next step in the signal transduction cascade, the phosphorylated VirA leads to the transfer of the phosphate to Asp-52 residue of VirG ([http://jb.asm.org/cgi/content/abstract/172/2/531 Jin SG ''et al.'', 1990a] ; [http://jb.asm.org/cgi/content/abstract/172/9/4945 Jin SG ''et al.'', 1990b] ; [http://nar.oxfordjournals.org/content/18/23/6909.short Pazour GJ and Das A, 1990]). VirG is the response regulator of the two-component system [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1082791/ (Brencic A, Winans SC, 2005)] and acts as a transcription factor. Hence it binds to the virulence box (vir Box) containing promoters, for example the ''virB'' promoter ([http://jb.asm.org/cgi/content/abstract/172/2/531 Jin SG ''et al.'', 1990a] ; [http://jb.asm.org/cgi/content/abstract/172/3/1241 Pazour GJ and Das A, 1990]).
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system in which VirA is an transmembrane-bound sensor while VirG is the intracellular response regulator (Wolanin PM ''et al.'', 2002). The two genes for the sensing system are virA and virG (Stachel SE, Nester EW, 1986) which are constitutively expressed at a basal level. VirA is a hitstidine kinase and after sensing the phenol 3,5-DimethoxyacetophenoneAcetosyringone an autophosphorylation occurs at the His-474 residue (Huang Y ''et al.'', 1990),(Jin S ''et al.'', 1990). Later in the signal transduction cascade, the phosphorylated VirA leads to the transfer of the phosphate to Asp-52 residue of VirG (Jin SG, Prusti RK ''et al.'', 1990), (Jin SG, Roitsch T ''et al.'', 1990), (Pazour GJ, Das A, 1990). VirG is the response regulator of the two-component system (Brencic A, Winans SC, 2005). VirG acts as a transcription factor and binds to virulence box (vir Box) containing promotors, for example virB (] Jin SG, Roitsch T ''et al.'', 1990), (Pazour GJ, Das A, 1990)
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== VirA receptor structure ==
== VirA receptor structure ==
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The VirA receptor consists of 829 amino acids and is a transmembrane protein in the inner menbrane of ''A. tumefaciens'' ([http://www.ncbi.nlm.nih.gov/pmc/articles/PMC401051/ Melchers LS, 1989]). VirA spans the inner membrane, with two transmembrane domains, a large periplasmic region, and a large C-terminal cytoplasmic domain ([http://jb.asm.org/cgi/content/abstract/176/11/3242 Banta LM, 1994]). VirA directly senses the phenolic compounds for vir activation ([http://linkinghub.elsevier.com/retrieve/pii/S0378111996003289 Lee YW ''et al.'', 1996]). Therefore the linker domain is essential for induction by phenolic compounds ([http://jb.asm.org/cgi/content/abstract/174/21/7033 Chang CH and Winans SC., 1992]). The linker region is located in the cytosolic site at position 280 to 414 ([http://linkinghub.elsevier.com/retrieve/pii/S0378111996003289 Lee YW ''et al.'', 1996]). This region between the amino acids 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 ([http://www.springerlink.com/index/G548R7745685341P.pdf Turk SC ''et al.'', 1994]).
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The virA receptor consists of 829 amino acids and is a transmembrane protein in the inner menbrane of A.tumefaciens (Melchers LS, 1989). VirA spans the inner membrane, with two transmembrane domains, a large periplasmic region, and a large C-terminal cytoplasmic domain (Banta LM, 1994). VirA directly senses the phenolic compounds for vir activation (Lee YW, Jin S ''et al.'', 1996) Therefore the linker domain is essential for induction by phenolic compounds (Chang CH, Winans SC., 1992). The linker region is located in the cytosolic site at position 280 to 414 (Lee YW, Jin S ''et al.'', 1996). 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 (Turk SC ''et al.'', 1994).
 
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[[Image:Bielefeld_Melcher_1989_VirA_structure.jpg|300px|left|thumb|right| Melchers et al., 1989 ]]
 
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[[Image:Bielefeld_VirA_structure_Lee_1996.jpg|300px|center|thumb|right|Lee et al., 1996 ]]  
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[[Image:Bielefeld_Melcher_1989_VirA_structure.jpg|300px|left|thumb|right| '''Figure 2: The structur of virA (Melchers et al., 1989)''' ]]  
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[[Image:Bielefeld_VirA_structure_Lee_1996.jpg|300px|center|thumb|right| '''Figure 3: The funtional parts of the virA receptor (Lee et al., 1996)''' ]]
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Chang and Winnans revealed in their studies, the parts of the VirA receptor essential for the signal transduction (Chang CH, Winans SC., 1992). A structured model for different inducing conditions are shown in the figure below.
 
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[[Image:Bielefeld_Chang_1992.jpg|300px|center|thumb|right|Chang, Winans, 1992 ]]
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Chang and Winnans (1992) revealed in their studies the parts of the VirA receptor which are essential for the signal transduction ([http://jb.asm.org/cgi/content/abstract/174/21/7033 Chang CH and Winans SC., 1992]). A structured model for different inducing conditions are shown in figure 4.
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[[Image:Bielefeld_Chang_1992.jpg|300px|center|thumb|right| '''Figure 4 : The different binding conditions (Chang and  Winans, 1992)''' ]]
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<html><div style="font-size:16px; font-weight:bold;"><a href="/Team:Bielefeld-Germany/Project/Approach#The_Approach">For information about modulation strategy click here</a></div></html>
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== Phenolic Compounds ==  
== Phenolic Compounds ==  
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The Ligand receptor interaction between Acetosyringone and VirA is based on the interaction of several chemical groups. First of all, the hydroxylated aromat 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. The potential capacity of the group para to the phenolic hydroxyl group is associated with higher activities and chirality at this carbon center is critical for inducing activity (Yi-Han Lin ''et al.'', 2007), (McCullen CA., Binns AN. , 2006), (Winans SC., 1992,).
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The ligand receptor interaction between acetosyringone and VirA is based on the interaction of several chemical groups. First of all the hydroxylated aromat is essential. Methoxy groups in the ''ortho'' position of the phenol play a crucial role in the signaling as well. It should be mentioned that dimethoxy compounds have a higher activity than monomethoxy compounds. The acetyl and alkyl groups in ''para'' position enhance the binding affinity. VirA activating compounds must have two methoxy groups in ''ortho'' position and an additional carbonyl group on the R3 chain. The potential capacity of the group ''para'' to the phenolic hydroxyl group is associated with higher activities. Moreover the chirality at this carbon center is critical for the inducing activity ([http://www.ncbi.nlm.nih.gov/pmc/articles/PMC372852/?pageindex=1&tool=pmcentrez Winans SC, 1992]).
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Regarding to the proton transfer model of Hess et al 1996 (Hess KM ''et al.'', 1991) the VirA activator transfers a proton to the basic area receptor binding site. The allosteric change leads to the phosphotransfer and the signaltransduction (Yi Han Linn ''et al.'', 2008), (Kyunghee Lee, 1996).
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Regarding to the proton transfer model of Hess ''et al.'' (1996) the VirA activator transfers a proton to the basic area receptor binding site. The allosteric change leads to the phosphotransfer and the signaltransduction ([http://www.ncbi.nlm.nih.gov/pmc/articles/PMC52402/?pageindex=1&tool=pmcentrez Hess KM ''et al.'', 1991]).
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[[Image:Bielefeld_benzol_structure.jpg|400px|left|thumb|right| '''Figure 5: Chemical structure of phenol ''']]
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[[Image:Bielefeld_proton_transfer_model.jpg|400px|center|thumb|right| '''Figure 6: The proton transfer model according to Hess et al., 1991''']]
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[[Image:Bielefeld_Winans_1992.jpg|400px|center|thumb|right| '''Figure 7: Compounds with a structural similarity, which induce virA Winans, 1992''']]
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<html><div style="font-size:16px; font-weight:bold;"><a href="/Team:Bielefeld-Germany/Project/Approach#The_Approach">See more possible compounds by clicking here</a></div></html>
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[[Image:Bielefeld_benzol_structure.jpg|400px|left|thumb|right|Chemical structure of phenol]]
 
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[[Image:Bielefeld_proton_transfer_model.jpg|400px|center|thumb|right|Hess et al., 1991]]
 
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[[Image:Bielefeld_Winans_1992.jpg|400px|center|thumb|right|Winans, 1992]]
 
== Inducing enhancers ==
== Inducing enhancers ==
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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 (Shimoda N ''et al.'', 1993)
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The sensitivity of this system is highly enhanced when additional aldose monosacchardic suggars occur in the environment of ''Agrobacterium''. The sugar binding protein ChvE interacts with the VirA receptor, leading to a much stronger ''vir'' gene expression ([http://jb.asm.org/cgi/content/abstract/176/11/3242 Banta LM, ''et al.'', 1994]).
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== Subcloning into ''E.coli'' and receptor function in new host ==
 
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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 ( Lohrke SM ''et al.'', 1990) For this reason it was necessary to subclone a modified virG gene that is capaple to be trancribed by the E. coli expression system.
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== Subcloning into ''E. coli'' and receptor function in new host ==
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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 genes ([http://www.ncbi.nlm.nih.gov/pmc/articles/PMC103583/?report=abstract&tool=pmcentrez Lohrke SM ''et al.'', 1990]) For this reason it was necessary to subclone a modified ''virG'' gene that is capable to be trancribed by the ''E. coli'' expression system.
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Yong-Chul ''et al.'' (2004) described VirG mutants that are capable of expressing the ''virB'' promoter-driven genes 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'' ([http://www.springerlink.com/content/wmq06kua5qkma1au/fulltext.pdf Yong-Chul J ''et al.'', 2004]).
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In VirG the amino acid at position 56 is likely to play a key role in the interaction with the RpoA of ''E. coli''.
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Regarding to Yong-Chul J ''et al.'' (2004) 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.
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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 (Yong-Chul J ''et al.'', 2004).
 
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In VirG the amino acid at position 56 is likely to play a key role in the interaction with the RpoA of E. coli.
 
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Regarding to Yong-Chul J ''et al.'', 2004 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.
 
= Read out system =
= Read out system =
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== Firefly Luciferase==
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We are using luciferase as the read out system. The luciferase was originally extracted from the firefly ''Photinus pyralis''. It is one of the best studied and characterized read out systems. The luciferase enzyme catalyses the chemical reaction from its substrate luceferin to oxyluciferin and light ([http://mcb.asm.org/cgi/reprint/7/2/725 De Wet ''et al.'' 1986]). Figure 8 shows the reaction in detail.
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We used luciferase as the read out system because it causes only slight negligible noise, hence the signal to noise ration is excellent.
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[[Image:Bielefeld Luct.jpg|300px|left|thumb| '''Figure 8: Firefly luciferase reaction''' (De Wet ''et al.''1986)]]
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==Output-signal amplification by Sensitivity Tuner implementation==
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Using a standard, inducible promoter with reporter system often results in weak reporter expression. So difficulties in quantification can occur. The quantification can be enhanced by an amplification of the transcription rate of the desired reporter genes. Such an amplification can be realized by using a so called ''sensitivity tuner'' device. This takes place as promoter induction upregulates a phage activator, which binds to a phage promoter upstream of a reporter gene. As result, a PoPs input (Inducer) generates a PoPs output at a higher signal. PoPs is equivalent to the flow of RNA polymerase molecules along DNA ([http://jb.asm.org/cgi/content/abstract/178/19/5668 Julien and Calendar, 1996] ; [https://2007.igem.org/Cambridge/Amplifier_project iGEM Team Cambridge, 2007]).
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[[Image:Bielefeld readout.png|600px|center|thumb| '''Figure 9: Gene sequence of final test construct including Sensitivity Tuner elements.''']]
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'''Purpose of Sensitivity Tuner application'''
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We presumed weak expression rates of our reporter luciferase indicated by pretesting the native system <partinfo>K389015</partinfo>. For having a broader range of quantification for our prototype test system, an amplification device was implemented.
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For amplifying the output signal of luciferase induced by acetosyringone, three sensitivity tuners, distinguished by the amplification factor, were combined with our detection system. To modify the sensitivity tuner for our purpose we took BioBricks with amplification factors from 10 (<partinfo>I746380</partinfo>) to 35 (<partinfo>I746390</partinfo>), removed the pBAD/araC promoter (<partinfo>I0500</partinfo>) and GFP read out (<partinfo>E0040</partinfo>) and replaced it by the reporter gene luciferase <partinfo>K389004</partinfo>(Figure 9). This enhanced luciferase BioBrick was assembled to the VirA/G signaling system BioBrick. The benefits of luciferase as reporter gene instead of GFP are a broader range of measurement, higher sensitivity and low half-live making cinetic tests possible ([http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6W9V-4F031H9-30&_user=10&_coverDate=01%2F31%2F1989&_rdoc=1&_fmt=high&_orig=search&_origin=search&_sort=d&_docanchor=&view=c&_searchStrId=1514624813&_rerunOrigin=google&_acct=C000050221&_version=1&_urlVersion=0&_userid=10&md5=ee400628b119490fcdc44ccdd856c4e8&searchtype=a Williams ''et al.''1989]).
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For test results click [https://2010.igem.org/Team:Bielefeld-Germany/Results/Tests#BBa_K389421.2C_BBa_K389422.2C_BBa_K389423:_Sensitivity_Tuner_amplified_Vir-test_system Sensitivity Tuner amplified Vir-test system]
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= Receptor modification strategy =
= Receptor modification strategy =
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The smartest way of receptor-modification is initiated by a silico approach based on a 3D structure of the native receptor. Followed by primer mutagenesis of the computantional gained results a precise adapted peptide emerges. This concept has been proven by [http://www.nature.com/nature/journal/v423/n6936/abs/nature01556.html Looger ''et al.'', 2003].
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Because of time limitations within the iGEM competition and a lack of biological data in literature - no x-ray cristalography structure data for VirA linker region available- this strategy was not applicable.
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Therefore we developed two different stragies in our MARSS project:
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The first strategy was an <u> error prone PCR </u> approach by building a mutant data base for selection. The second strategy was a <u> primer mutagenesis based </u> approach.
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== Random mutagenesis by error-prone PCR (EP-PCR)==
== Random mutagenesis by error-prone PCR (EP-PCR)==
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In order to detect novel substances (e.g. capsaicin) with the virA receptor, the first step was to create a mutagenised library of virA variants, which could subsequently be screened for new binding characteristics. Plenty of different strategies for mutagenesis of DNA are known, including the use nucleotide analogues, bacteria containing mutator genes, the mutagenesis with UV light or chemicals and inaccurate PCR ([http://genome.cshlp.org/content/2/1/28.short Cadwell RC and Joyce GF, 1992]).
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In order to detect novel substances (''e.g.'' capsaicin) with the VirA receptor, the first step was to create a mutagenised library of ''virA'' variants, which could subsequently be screened for new binding characteristics. Plenty of different strategies for mutagenesis of DNA are known, including the use of nucleotide analogues, bacteria containing mutator genes, the mutagenesis with UV light or chemicals and inaccurate PCR ([http://genome.cshlp.org/content/2/1/28.short Cadwell RC and Joyce GF, 1992]).
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When designing our strategy we rejected the use of bacteria strains with high mutation rates, since the changes in base sequence would occur all over the transformed plasmids. Thereby some mutations would also take place in the backbone of the plasmid, our might even been found in the standardized BB prefix and suffix. We also excluded the possibility of using UV light or mutagenic chemicals, due to reasons of safety and minimizing the exposure toxic substances.
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When designing our strategy we rejected the use of bacteria strains with high mutation rates, since the changes in base sequence would occur all over the transformed plasmids. Thereby some mutations would also take place in the backbone of the plasmid, or might even been found in the standardized BioBrick prefix and suffix. We also excluded the possibility of using UV light or mutagenic chemicals, due to reasons of safety and minimizing the exposure of toxic substances.
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In our experiment we wanted to alter only the part of the plasmid coding for the virA receptor, while using a not harmful and thereby safe technique. Thus, our method of choice was inaccurate PCR that allows the exclusive variations of a distinct region of a plasmid, which is defined by the location of the upstream and downstream primers. This mutagenic method of PCR, called error-prone PCR (EP-PCR) has been described and improved a lot in scientific community ([http://www.springerlink.com/content/r62q360t82764508/#section=746282&page=1 McCullum EO ''et al.'', 2010]).
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In our experiment we wanted to alter only the part of the plasmid coding for the VirA receptor, while using a not harmful and thereby safe technique. Thus, our method of choice was inaccurate PCR that allows the exclusive variations of a distinct region of a plasmid, which is defined by the location of the upstream and downstream primers. This mutagenic method of PCR, called error-prone PCR (EP-PCR) has been described and improved a lot in scientific community ([http://www.springerlink.com/content/r62q360t82764508/#section=746282&page=1 McCullum EO ''et al.'', 2010]).
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The basic principle of this technique uses the natural high infidelity of the ''taq'' DNA polymerase, which can even be increased by special changes in buffer conditions compared to standard PCR. These alterations may include the unequal distribution of dNTPs (5 mM purines, 25 mM pyrimidines) as well as an increased amount of MgCl<sub>2</sub> and the addition of MnCl<sub>2</sub>. The total rate of base exchange can be adjusted by the number of PCR cycles, since mutations will accumulate during the exponential amplification of the sequence ([http://onlinelibrary.wiley.com/doi/10.1002/0471142727.mb0803s51/full Wilson DS and Keefe AD, 2000]). The experimental conditions of the performed error-prone PCR are described in the section “protocols”.
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== Directed mutagenesis ==
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After the identification of the 100 amino acids linker region responsible for VirA ligand binding ([[Team:Bielefeld-Germany/Project/Theory#VirA_receptor_structure|Compare VirA-Receptor]]) we compared this region with well characterized capsaicin receptors derived from animal model organisms (TRPV1). The conserved receptor region is shown in the figure beneath.
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The species-specific sensitivity of TRPV1 can be ascribed to about eight amino acids in the vicinity of TM3 ([http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6WSN-4C5H6M5-F&_user=2459438&_coverDate=02%2F08%2F2002&_rdoc=1&_fmt=high&_orig=search&_origin=search&_sort=d&_docanchor=&view=c&_acct=C000057302&_version=1&_urlVersion=0&_userid=2459438&md5=93a9e63f48d785a0b400131a058ee269&searchtype=a Jordt an Julius 2002]).
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[[Image:Bielefeld_VirA_conserved_sequence.jpg|600px|center|thumb|''' Figure 10: Molecular determinants of species-specific vanilloid sensitivity. Sequence alignment of rat (top), human (middle) and chicken (bottom) VR1 within the TM3-4 region is shown. Conserved residues are indicated by black background. The chimera V3/C contains a minimal segment of rat VR1 that is sufficient to confer vanilloid sensitivity ([http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6WSN-4C5H6M5-F&_user=2459438&_coverDate=02%2F08%2F2002&_rdoc=1&_fmt=high&_orig=search&_origin=search&_sort=d&_docanchor=&view=c&_acct=C000057302&_version=1&_urlVersion=0&_userid=2459438&md5=93a9e63f48d785a0b400131a058ee269&searchtype=a Jordt and Julius, 2002])'''.]]
 +
 
 +
We further aligned the TM3 region of the TRPV1 receptor to the ligand binding region of the native VirA receptor from ''Agrobacterium'' by the use of the tool '''MU'''ltiple '''S'''equence '''C'''omparison by Log-Expectation ([http://www.ebi.ac.uk/Tools/muscle/index.html Muscle]).
 +
 
 +
[[Team:Bielefeld-Germany/Project/Approach#Directed_mutagenesis| '''For detailed approach of directed mutagenesis for VirA_mut1 and VirA_mut2 click here''']]
-
The basic principle of this technique uses the natural high infidelity of the Taq DNA polymerase, which can even be increased by special changes in buffer conditions compared to standard PCR. These alterations may include the unequal distribution of dNTPs (5 mM purines, 25 mM pyrimidines) as well as an increased amount of MgCl<sub>2</sub> and the addition of MnCl<sub>2</sub>. The total rate of base exchange can be adjusted by the number of PCR cycles, since mutations will accumulate during the exponential amplification of the sequence ([http://onlinelibrary.wiley.com/doi/10.1002/0471142727.mb0803s51/full Wilson DS and Keefe AD, 2000]). The experimental conditions of the performed error-prone PCR are described in the section “protocols”.
 
= Screening system =
= Screening system =
-
The screening of randomly mutagenised genes for a desired function or application is always very time-consuming procedure ([http://www.ncbi.nlm.nih.gov/pubmed/1496376 Beaudry and Joyce, 1992]). It requires a huge amount of material and might takes several months or even years to result in a promising new version of gene ([http://mbe.oxfordjournals.org/content/17/7/1050.long Hanczyc and Dorit, 2000]). As we faced the challenge to modify the virA receptor in only few weeks we designed a strategy for a fast high-throughput screening, by using subsequent steps of different read out systems and a strategy with two different plasmids.
+
==Development of a high-troughput screening==
-
In the first step after mutagenesis of virA it was necessary to separate thousands of transformants with minor or unwanted changes in the virA gene, from few bacteria that included interesting virA variants. Thus, we constructed our system to lead in the expression of a kanamycin resistance after the induction of the virA receptor, enabling the quick exclusion of all unwanted virA variant.
+
 
 +
The screening of randomly mutagenised genes for a desired function or application is always a very time-consuming procedure ([http://www.ncbi.nlm.nih.gov/pubmed/1496376 Beaudry and Joyce, 1992]). It requires a huge amount of material and might takes several months or even years to result in a promising new version of a gene ([http://mbe.oxfordjournals.org/content/17/7/1050.long Hanczyc and Dorit, 2000]). As we faced the challenge to modify the VirA receptor in only a few weeks we designed a strategy for a fast high-throughput screening by using subsequent steps of different read out systems and a strategy with two different plasmids.
 +
 
 +
In the first step after mutagenesis of ''virA'' it is necessary to separate thousands of transformants with minor or unwanted changes in the ''virA'' gene, from few bacteria that included interesting ''virA'' variants. Thus, we wanted to construct our system to lead in the expression of a kanamycin resistance after the induction of the VirA receptor, enabling the quick exclusion of all unwanted ''virA'' variants.
 +
 
 +
For that purpose a kanamycin resistance cassette should be set under control of the ''virB'' promoter, leading in the expression of aminoglycoside phosphotransferase (APH) that can inactivate kanamycin. The mode of inactivation is the transfer of the y-phosphate from ATP to the hydroxyl group at C3 of the antibiotic ([http://www.bioscience.org/1999/v4/d/wright/wright.pdf Wright and Thompson, 1999]). This phosphorylation results in the loss of binding capacity of the aminoglycoside to the 30S subunit of bacterial ribosomes, which would lead to inhibition of protein synthesis without the presence of the APH ([http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1365070/pdf/brjclinpharm00008-0014.pdf Begg EJ and Barclay ML, 1995]).
 +
 
 +
As indicated by the first results, the reporter genes under control of the ''virB'' promoter showed a slight but measureable expression without any induction of VirA with acetosyringone. This basal transcription resulted in the growth of bacteria without induction of VirA at normal working concentrations of kanamycin of 25 to 50 µg mL<sup>-1</sup> ([http://books.google.de/books?hl=de&lr=&id=9mO2Fx0CuEYC&oi=fnd&pg=PA4&dq=Molecular+Cloning:+A+Laboratory+Manual,+Vol+1.+&ots=Ctw-SlcSLm&sig=FvweaKb_qiUxClNNiRfdwpmbqdo#v=onepage&q&f=false Sambrook J and Russell DW, 2001]).
 +
 
 +
Concludingly, prior to the screening experiments it was necessary to adjust the concentration of kanamycin, which inhibits the growth of uninduced bacteria, while allowing bacteria to grow when an appropriate inductor was present and able to activate VirA. This analysis was performed using the method of determination of minimal inhibitory concentrations (MIC) as described below.
 +
 
 +
 
 +
==Determination of minimal inhibitory concentration (MIC) of kanamycin==
 +
 
 +
There are several ways to investigate the susceptibility of bacteria to inhibiting drugs like antibiotics. Nevertheless, the result of all these tests is the amount of an assayed substance that inhibits visible growth of the bacteria, called the minimal inhibitory concentration (MIC). The most common way of determination is to grow bacteria in liquids with several concentrations of the inhibiting drug. This procedure is chosen mostly, since many different conditions can be measured at the same time by using microtiterplates ([http://www.nature.com/nprot/journal/v3/n2/abs/nprot.2007.521.html Wiegand I ''et al.'', 2008]).
 +
 
 +
As we planned not to use the kanamycin in liquid culture but in LB-Agar, we chose to determine the MIC at the same conditions as the desired experiment. Therefore, we planned to construct ''E. coli'' inhabiting the native VirA/G signaling system and a kanamycin resistance read out and plated a small volume in different dilutions on LB-Agar without kanamycin. The grown colonies could then be transferred to agar plates with rising concentrations of kanamycin using replica plating. By counting the colonies it should be possible to calculate the percentage of colonies that could withstand each kanamycin concentration. This experiment should be carried out with and without acetosyringone to determine a kanamycin concentration induced ''E. coli'' could withstand, while the same population of bacteria dies without the presence of acetosyringone.
 +
 
 +
 
 +
==Primary selection of ''virA'' variants with novel binding properties==
 +
 
 +
After the determination of the MIC the screening for ''virA'' variants with new binding properties could be started. The aim of this screening is to find versions of ''virA'' that can be induced by one of the tested substances capsaicin, homovanillic acid, dopamine and 3-O methyldopamine.
 +
 
 +
For that purpose one should transform the mutagenised variants of ''virA'' to ''E. coli'' and plate the bacteria on LB-agar with the determined kanamycin concentration. At the same time a mixture of all mentioned substances should be present in the agar. All bacteria including a ''virA'' variant that is activated by at least one of the substances will grow on the selective agar, since it expresses the kanamycin resistance. With this step it is thereby possible to select thousands of bacteria with unwanted versions of ''virA'' from few individuals with wanted binding properties.
 +
 
 +
At this point it must be mentioned that some grown colonies might still be non-induced and false positive results. As the ''virA'' gene has been randomly changed before, it is possible that some variants occur where the receptor is always active. This would lead to a constitutive expression of the reporter gene and thereby a high level of kanamycin resistance.
 +
To exclude those false positive clones, the bacteria should be tested whether they are only resistance to the MIC of kanamycin when one of the tested substances is present. Every colony that can withstand the high kanamycin concentration without any inductor includes a constitutive version of virA and should be discarded in further analysis.
 +
 
 +
 
 +
==Quantitative analysis of ''virA'' variants after induction with novel substances==
 +
 
 +
Should we find some bacteria that respond to the presence of the tested substances (capsaicin, homovanillic acid, dopamine and 3-O methyldopamine) by growing on the MIC of kanamycin, it is desirable to quantify the induction. For that purpose it is appropriate to change the read out system from kanamycin resistence to luciferase expression. This complex and time consuming task can easily be achieved without any cloning step, when using the advantage of our two plasmid system.
 +
 
 +
After primary selection each bacteria includes two plasmids with different origins of replication (oris). The plasmid with the ''virA'' is in a common <partinfo>pSB1AT3</partinfo> backbone with a ColE1 ori. Contrary to that the read out plasmid with KanR has a special ori, named R6K, which can only amplify in ''E. coli'' strains that express the gene ''pir'' to produce the so called Pir protein. Most of the strains used in laboratory are pir<sup>-</sup> but few (''e.g'' EC100D) are pir<sup>+</sup> ([http://www.ncbi.nlm.nih.gov/pubmed/17383678 Bowers et al., 2007]).
 +
 
 +
The setup of the different oris was chosen to separate both plamids at this experimental stage. To change the read out system from KanR to luciferase, one just needs to perform two transformations and isolations of plasmids. In the first step plasmids are isolated from colonies with positive binding properties to one of the tested substances. This mixture of plasmids with ColE1 and R6K oris is then transformed to a pir<sup>-</sup> strain (''e.g.'' TOP10). In the following only the plasmid with ColE1 ori will be amplified during the growth of the transformants, leading to pure plasmids with ''virA'' when plasmids are isolated for a second time. In the last step this isolated DNA can be transformed to bacteria including another read out plasmid (''e.g.'' with luciferase).
 +
 
 +
 
 +
= References =
-
===Primary selection of virA variants with novel binding properties===
+
*Banta LM, Joerger RD, Howitz VR, Campbell AM, Binns AN., (1994) ''Glu-255 outside the predicted ChvE binding site in VirA is crucial for sugar enhancement of acetosyringone perception by Agrobacterium tumefaciens'', J Bacteriol. 176(11):,3242-9.
-
For that purpose a kanamycin resistance cassette was set under control of the virB promotor, leading in the expression of aminoglycoside phosphotransferase (APH) that can inactivate kanamycin. The mode of inactivation is the transfer of the y-phosphate from ATP to the hydroxyl group at C3 of the antibiotic ([http://www.bioscience.org/1999/v4/d/wright/wright.pdf Wright and Thompson, 1999]). This phosphorylation results in the loss binding capacity of the aminoglycoside to the 30S subunit of bacterial ribosomes, which would lead to inhibition of protein synthesis without the presence of the APH ([http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1365070/pdf/brjclinpharm00008-0014.pdf Begg EJ and Barclay ML, 1995]).
+
*Beaudry AA, Joyce GF (1992) ''Directed evolution of an RNA enzyme'', Science, 257(5070):, 635-41.
-
As indicated by the first results, the reporter genes under control of the virB promoter showed a slight but measureable expression without any induction of virA with acetosyringone. This basal transcription resulted in the growth of bacteria without induction of virA at normal working concentrations of kanamycin of 25 to 50 µg mL<sup>-1</sup>) ([http://books.google.de/books?hl=de&lr=&id=9mO2Fx0CuEYC&oi=fnd&pg=PA4&dq=Molecular+Cloning:+A+Laboratory+Manual,+Vol+1.+&ots=Ctw-SlcSLm&sig=FvweaKb_qiUxClNNiRfdwpmbqdo#v=onepage&q&f=false Sambrook J and Russell DW, 2001]).
+
-
Concludingly, prior to the screening experiments one needs to adjust the concentration of kanamycin, which inhibits the growth of uninduced bacteria, while allowing bacteria to grow when an appropriate inductor was present and able to activate virA. This analysis was performed using the method of determination of minimal inhibitory concentrations (MIC) as described below.
+
-
====Determination of minimal inhibitory concentration (MIC) of kanamycin====
+
*Begg EJ, Barclay ML (1995) ''Aminoglycosides – 50 years on'', Br J clin Pharmac, 39:, 597-603.
-
= Modeling =
+
*Bowers LM, Krüger R, Filutowicz M (2007), ''Mechanism of origin activation by monomers of R6K-encoded pi protein'', J Mol Biol, 368(4):, 928-938.
-
= Weblinks =
+
*Brencic A, Winans SC (2005), ''Detection of and response to signals involved in host-microbe interactions by plant-associated bacteria'', Microbiol Mol Biol, Rev 69:, 155–194.
-
*Banta LM, Joerger RD, Howitz VR, Campbell AM, Binns AN., 1994, Glu-255 outside the predicted ChvE binding site in VirA is crucial for sugar enhancement of acetosyringone perception by Agrobacterium tumefaciens., J Bacteriol. 176(11):3242-9.  
+
*Cadwell RC and Joyce GF (1992), ''Randomization of genes by PCR mutagenesis'', Genome Res., 28-33.
-
*Brencic A, Winans SC, 2005, Detection of and response to signals involved in host-microbe interactions by plant-associated bacteria., Microbiol Mol Biol Rev 69: 155–194.  
+
*Chang CH, Winans SC., (1992) ''Functional roles assigned to the periplasmic, linker, and receiver domains of the Agrobacterium tumefaciens VirA protein'', J Bacteriol., 174(21):, 7033-9.  
-
*Chang CH, Winans SC., 1992, Functional roles assigned to the periplasmic, linker, and receiver domains of the Agrobacterium tumefaciens VirA protein., J Bacteriol. 174(21):7033-9.
+
*DeCleene M, DeLay J (1976), ''The host range of crown gall'', Bot Rev 42, 389–466
-
*DeCleene M, DeLay J, 1976, The host range of crown gall, Bot Rev 42: 389–466
+
*De Wet JR, Wood KV, De Luca M, Helinski DR and Subramani S (1987), ''Firefly Luciferase Gene: Structure and Expression in Mammalian Cells'', Molecular and Cell Biolog, Feb 1987, 725-737
-
*Hess KM, Dudley MW, Lynn DG, Joerger RD, Binns AN., 1991, Mechanism of phenolic activation of Agrobacterium virulence genes: development of a specific inhibitor of bacterial sensor/response systems., Proc. Natl. Acad. Sci. USA 88:7854–58.
+
*Hanczyz MM, Dortit RL (2000), ''Replicability and Recurrence in the Experimental Evolution of a Group I Ribozyme'', Mol Biol Evol, 17 (7):, 1050-1060
-
*Huang Y, Morel P, Powell B, Cado CI, 1990, VirA, a corregulator of Ti-specific virulence genes, is phosphorylated in vitro., J Bacteriol 172:1142–1144.
+
*Hess KM, Dudley MW, Lynn DG, Joerger RD, Binns AN., (1991) ''Mechanism of phenolic activation of Agrobacterium virulence genes: development of a specific inhibitor of bacterial sensor/response systems'', Proc. Natl. Acad. Sci., USA 88:, 7854–58
-
*Jin S, Roitsch T, Ankenbauer RG, Gordon MP, Nester EW, 1990, The VirA protein of Agrobacterium tumefaciens is autophosphorylated and is essential for vir gene regulation., J Bacteriol 172: 525–530.
+
*Huang Y, Morel P, Powell B, Cado CI (1990), ''VirA, a corregulator of Ti-specific virulence genes, is phosphorylated in vitro'', J Bacteriol 172:, 1142–1144
-
*Jin SG, Prusti RK, Roitsch T, Ankenbauer RG, Nester EW, 1990, Phosphorylation of the VirG protein of Agrobacterium tumefaciens by the autophosphorylated VirA protein: Essential role in biological activity of VirG., J Bacteriol 172:4945–4950.
+
*Jin S, Roitsch T, Ankenbauer RG, Gordon MP, Nester EW (1990), ''The VirA protein of Agrobacterium tumefaciens is autophosphorylated and is essential for vir gene regulation'', J Bacteriol 172:, 525–530
-
*Jin SG, Roitsch T, Christie PJ, Nester EW, 1990, The regulatory VirG protein specifically binds to a cis-acting regulatory sequence involved in transcriptional activation of Agrobacterium tumefaciens virulence genes., J Bacteriol 172:531–537
+
*Jin SG, Prusti RK, Roitsch T, Ankenbauer RG, Nester EW (1990a), ''Phosphorylation of the VirG protein of Agrobacterium tumefaciens by the autophosphorylated VirA protein: Essential role in biological activity of VirG'', J Bacteriol, 172:, 4945–4950
-
*Jin SG, Roitsch T, Christie PJ, Nester EW, 1990, The regulatory VirG protein specifically binds to a cis-acting regulatory sequence involved in transcriptional activation of Agrobacterium tumefaciens virulence genes., J Bacteriol 172:531–537.
+
*Jin SG, Roitsch T, Christie PJ, Nester EW (1990b), ''The regulatory VirG protein specifically binds to a cis-acting regulatory sequence involved in transcriptional activation of Agrobacterium tumefaciens virulence genes'', J Bacteriol, 172:, 531–537  
-
*Kyunghee Lee, 1996, A structure-based activation model of phenol-receptor protein interactions.
+
*Jordt SE, Julius D (2002), ''Molecular Basis for Species-Specific Sensitivity to „Hot“ Chili Peppers'', Cell, 108:, 421-430
-
Lee YW, Jin S, Sim WS, Nester EW, 1996, The sensing of plant signal molecules by Agrobacterium: genetic evidence for direct recognition of phenolic inducers by the VirA protein., Gene. 179(1):83-8.
+
-
*Lohrke SM, Nechaev S, Yang H, Severinov K, Jin SJ, 1999, Transcriptional activation of Agrobacterium tumefaciens virulence gene promoters in Escherichia coli requires the A. tumefaciens RpoA gene, encoding the alpha subunit of RNA polymerase., J Bacteriol 181:4533–4539.  
+
*Kyunghee Lee (1996), ''A structure-based activation model of phenol-receptor protein interactions.  
-
*McCullen CA., Binns AN. , 2006, Agrobacterium tumefaciens and Plant Cell Interactions and Activities Required for Interkingdom Macromolecular Transfer, Annu. Rev. Cell Dev. Biol. 22:101-127
+
*Lee YW, Jin S, Sim WS, Nester EW (1996), ''The sensing of plant signal molecules by Agrobacterium: genetic evidence for direct recognition of phenolic inducers by the VirA protein'', Gene, 179(1):, 83-8.
-
*Melchers LS, 1989, Membrane topology and functional analysis of the sensory protein VirA of Agrobacterium tumefaciens., The EMBO Journal vol.8 no.7 pp.1919- 1925.  
+
*Lin, Y, Gao R, Binns A, Lynn D (2008), ''Bacterial signal transduction: networks and drug targets'' Adv Exp Med Biol., 161–177.
-
*Palmer AG, Gao R, Maresh J, Erbil WK, Lynn DG, 2004 Chemical biology of multi-host/pathogen interactions: chemical perception and metabolic complementation, Annu Rev Phytopathol 42: 439–464.
+
*Lohrke SM, Nechaev S, Yang H, Severinov K, Jin SJ (1999), ''Transcriptional activation of Agrobacterium tumefaciens virulence gene promoters in Escherichia coli requires the A. tumefaciens RpoA gene, encoding the alpha subunit of RNA polymerase'', J Bacteriol, 181:, 4533–4539
-
*Pazour GJ, Das A, 1990, Characterization of the VirG binding site of Agrobacterium tumefaciens., Nucleic Acids Res 18:6909–6913.
+
*Looger L, Dwyer MA, Smith JJ and Hellinga HW  (2003), ''Computational design of receptor and sensor proteins with novel functions'', Nature, 423:, 185-190
-
*Pazour GJ, Das A, 1990, virG, an Agrobacterium tumefaciens transcriptional activator, initiates translation at a UUG codon and is a sequence-specific DNA-binding protein., J Bacteriol 172:1241– 1249.
+
*McCullen CA, Binns AN  (2006), ''Agrobacterium tumefaciens and Plant Cell Interactions and Activities Required for Interkingdom Macromolecular Transfer'', Annu. Rev. Cell Dev. Biol., 22:, 101-127
-
*Scott M., Lohrke, 2001, Reconstitution of Acetosyringone-Mediated Agrobacterium tumefaciens Virulence Gene Expression in the Heterologous Host Escherichia coli ., J. of Bacteriology, p. 3704–3711 Vol. 183, No. 12.  
+
*McCullum EO, Willliams BAR, Zhang J, Chaput JC (2010) ''Random Mutagenesis by Error-Prone PCR, In vitro mutagenesis protocols'', Methods in Molecular Biology, Vol 634:, 103-109.
-
*Shimoda N,Toyoda-Yamamoto A, Shinsuke S, Machica Y., 1993, Genetic evidence for an interaction between the VirA sensor protein and the ChvE sugar-binding protein of Agrobacterium. J. Biol. Chem. 268:26552–58
+
*Melchers LS, (1989), ''Membrane topology and functional analysis of the sensory protein VirA of Agrobacterium tumefaciens'', The EMBO Journal, 8(7):, 1919-1925
-
*Stachel SE, Nester EW, 1986, The genetic and transcriptional organization of the vir region of the A6 Ti plasmid of Agrobacterium tumefaciens., EMBO J 5: 1445–1454.  
+
*MUltiple Sequence Comparison by Log-Expectation. MUSCLE http://www.ebi.ac.uk/Tools/muscle/index.html
-
*Turk SC, van Lange RP, Regensburg-Tuïnk TJ, Hooykaas PJ., 1994, Localization of the VirA domain involved in acetosyringone-mediated vir gene induction in Agrobacterium tumefaciens., Plant Mol Biol. 25(5):899-907.  
+
*Palmer AG, Gao R, Maresh J, Erbil WK, Lynn DG (2004), ''Chemical biology of multi-host/pathogen interactions: chemical perception and metabolic complementation'', Annu Rev Phytopathol, 42:, 439–464.  
-
*Winans SC., 1992, Two-way chemical signaling in Agrobacterium-plant interactions., Microbiol Rev. 56(1):12-31.
+
*Pazour GJ, Das A (1990a), ''Characterization of the VirG binding site of Agrobacterium tumefaciens'', Nucleic Acids Res, 18:, 6909–6913
-
*Wolanin PM, Thomason PA, Stock J.B., 2002, Histidine protein kinases: key signal transducers outside the animal kingdom., Genome Biol 3: REVIEWS3013.  
+
*Pazour GJ, Das A (1990b), ''virG, an Agrobacterium tumefaciens transcriptional activator, initiates translation at a UUG codon and is a sequence-specific DNA-binding protein'', J Bacteriol, 172:, 1241– 1249.  
-
*Yi Han Linn et al., 2008, Capturing the VirA/VirG TCS of Agrobacterium tumefaciens., Adv Exp Med Biol. 631:161-77.
+
*Sambrook J, Russel DW (2001), ''Molecular Cloning – A Laboratory Manual'', Cold Spring Harbor Laboratory Press, 1:
-
*Yi-Han Lin et al., 2007, The initial steps in Agrobacterium tumefaciens pathogenesis: chemical biology of host recognition, Agrobacterium: From Biology to Biotechnology,2008, pp. 221-241.
+
*Scott M, Lohrke SM (2001), ''Reconstitution of Acetosyringone-Mediated Agrobacterium tumefaciens Virulence Gene Expression in the Heterologous Host Escherichia coli'', J. of Bacteriology, 183(12):,3704–3711 
-
*Yong-Chul J., Yunrong G., Donghai W., Shouguang J., 2004, Mutants of Agrobacterium tumefaciens virG Gene That Activate Transcription of vir Promoter in Escherichia coli, Current Microbiology Vol. 49, pp. 334–340.  
+
*Shimoda N, Toyoda-Yamamoto A, Shinsuke S, Machica Y (1993), ''Genetic evidence for an interaction between the VirA sensor protein and the ChvE sugar-binding protein of Agrobacterium'' J. Biol. Chem., 268:,26552–58
-
*Ziemienowicz A., 2001, Odyssey of agrobacterium T-DNA,. Acta Biochim Pol. 2001;48(3):623-35.  
+
*Stachel SE, Nester EW, (1986) ''The genetic and transcriptional organization of the vir region of the A6 Ti plasmid of Agrobacterium tumefaciens'', EMBO J, 5:, 1445–1454.  
 +
*Turk SC, van Lange RP, Regensburg-Tuïnk TJ, Hooykaas PJ., 1994, ''Localization of the VirA domain involved in acetosyringone-mediated vir gene induction in Agrobacterium tumefaciens'', Plant Mol Biol. 25(5):,899-907
 +
*Wiegand I, Hilpert K, Hancok REW (2008) ''Agar and broth dilution methods to determine the minimal inhibitory concentration (MIC) of antimicrobial substances'', Nature Protocols, 3:, 163-175.
-
=== Literature of error-prone PCR and screening ===
+
*Williams TM, Burlein JE, Ogden S, Kricka LJ and Kant JA, 1989 ''Advantages of firefly luciferase as a reporter gene: Application to the interleukin-2 gene promoter'', Analytical Biochemistry, 176:, 28-32
-
*McCullum EO, Willliams BAR, Zhang J, Chaput JC (2010) ''Random Mutagenesis by Error-Prone PCR, In vitro mutagenesis protocols'', Methods in Molecular Biology, Vol 634, 103-109.
+
*Winans SC., 1992, Two-way chemical signaling in Agrobacterium-plant interactions., Microbiol Rev. 56(1):,12-31.
-
*Cadwell RC and Joyce GF (1992) ''Randomization of genes by PCR mutagenesis'', Genome Res., 28-33.
+
*Wilson DS, Keefe AD (2000) ''Random Mutagenesis by PCR'', Current Protocols in Molecular Biology, 8.3.1-8.3.9.  
-
*Wilson DS, Keefe AD (2000) ''Random Mutagenesis by PCR'', Current Protocols in Molecular Biology, 8.3.1-8.3.9.
+
*Wolanin PM, Thomason PA, Stock J.B. (2002), ''Histidine protein kinases: key signal transducers outside the animal kingdom'', Genome Biol 3: REVIEWS3013.  
*Wright GD, Thompson PR (1999) ''Aminoglycoside phosphotransferases: Proteins, Structure and Mechanism'', Frontiers in Bioscience 4, d9-21.
*Wright GD, Thompson PR (1999) ''Aminoglycoside phosphotransferases: Proteins, Structure and Mechanism'', Frontiers in Bioscience 4, d9-21.
-
*Beaudry AA, Joyce GF (1992) ''Directed evolution of an RNA enzyme'', Science, 257(5070):635-41.
+
*Yi Han Linn, Goa R, Bins AN, Lynn DG (2008), ''Capturing the VirA/VirG TCS of Agrobacterium tumefaciens'', Adv Exp Med Biol. 631:, 161-77
-
*Hanczyz MM, Dortit RL (2000) ''Replicability and Recurrence in the Experimental Evolution of a Group I Ribozyme'', Mol Biol Evol, 17 (7), 1050-1060.
+
*Yi Han Linn, Goa R, Bins AN, Lynn DG (2007), ''The initial steps in Agrobacterium tumefaciens pathogenesis: chemical biology of host recognition, Agrobacterium: From Biology to Biotechnology'', 2008, 221-241
-
*Begg EJ, Barclay ML (1995) ''Aminoglycosides – 50 years on'', Br J clin Pharmac, 39, 597-603.
+
*Yong-Chul J, Yunrong G, Donghai W, Shouguang J (2004), ''Mutants of Agrobacterium tumefaciens virG Gene That Activate Transcription of vir Promoter in Escherichia coli'', Current Microbiology, 49, 334–340
-
*Sambrook J, Russel DW (2001) ''Molecular Cloning – A Laboratory Manual'', Cold Spring Harbor Laboratory Press, Vol 1.
+
*Ziemienowicz A (2001), Odyssey of agrobacterium T-DNA,. Acta Biochim Pol. 2001, 48(3):, 623-35.

Latest revision as of 22:39, 27 October 2010

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Contents

Introduction

In our iGEM project we tried to create an Escherichia coli cell which is capable to sense capsaicin in a complex sample and report the concentration with a luciferase light signal. We combined a native receptor system of Agrobacterium tumefaciens with the readout system of the firefly luciferase. The native receptor senses the phenolic compound acetosyringone. For this reason we used directed evolution to modify the binding region of the native receptor to generate a new capsaicin receptor because of the chemical similarities of acetosyringone and capsaicin. 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 derived from different organisms were assembled. The readout system taken from firefly, luciferase, the receptor sensing system derived from A. tumefaciens and sensitivity tuners consist of phage DNA.


Agrobacterium tumefaciens

Figure 1: Image by Martha Hawe


The model organism A. tumefaciens is a soil bacterium and can be found at nearly every place in the world. Agrobacteria became known as a phyto-pathogen leading to the crown gall disease in dicotyledonous species ([http://www.springerlink.com/content/hm17520m287ht766/ DeCleene M and DeLay J, 1976]). The infection is caused by a gene transfer system located on an extrachromosomal element, the Ti-plasmid. Furthermore, the infection can be divided into several steps: The first step is the localisation of the hurt plant by the bacteria. Predominantely A. tumefaciens senses phenolic compunds from hurt plants, but also aldose monosaccharides, low pH and low phosphate ([http://www.annualreviews.org/doi/abs/10.1146/annurev.phyto.41.052002.095701?journalCode=phyto Palmer AG. et al.2004]; [http://mmbr.asm.org/cgi/content/abstract/69/1/155 Brencic A and Winans SC, 2005]). When A. tumefaciens recognizes phenols with the VirA receptor, a signal transduction cascade is initiated leading 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 biotechnological purposes the Ti-plasmid was disharmed. Instead of 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 ([http://people.uleth.ca/~alicja.ziemienowicz/extra/Ziemienowicz_ABP2001.pdf Ziemienowicz A, 2001]).


Native receptor

A. tumefaciens needs a precise recognition system for potential hosts to gain an evolutionary advance. The native sensing system is a two-component phospho-relay system in which VirA is a transmembrane-bound sensor while VirG is the intracellular response regulator ([http://www.biomedcentral.com/content/pdf/gb-2002-3-10-reviews3013.pdf Wolanin PM et al., 2002]). The two genes for the sensing system are virA and virG ([http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1166964/ Stachel SE and Nester EW, 1986]) which are constitutively expressed at a basal level. VirA is a histidine kinase. An autophosphorylation occurs at the His-474 residue, after sensing the phenol 3,5-dimethoxyacetophenone, acetosyringone ([http://www.ncbi.nlm.nih.gov/pmc/articles/PMC208549/?pageindex=1&tool=pmcentrez Huang Y et al., 1990] ; [http://jb.asm.org/cgi/content/abstract/172/2/531 Jin SG et al., 1990a]). In the next step in the signal transduction cascade, the phosphorylated VirA leads to the transfer of the phosphate to Asp-52 residue of VirG ([http://jb.asm.org/cgi/content/abstract/172/2/531 Jin SG et al., 1990a] ; [http://jb.asm.org/cgi/content/abstract/172/9/4945 Jin SG et al., 1990b] ; [http://nar.oxfordjournals.org/content/18/23/6909.short Pazour GJ and Das A, 1990]). VirG is the response regulator of the two-component system [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1082791/ (Brencic A, Winans SC, 2005)] and acts as a transcription factor. Hence it binds to the virulence box (vir Box) containing promoters, for example the virB promoter ([http://jb.asm.org/cgi/content/abstract/172/2/531 Jin SG et al., 1990a] ; [http://jb.asm.org/cgi/content/abstract/172/3/1241 Pazour GJ and Das A, 1990]).


VirA receptor structure

The VirA receptor consists of 829 amino acids and is a transmembrane protein in the inner menbrane of A. tumefaciens ([http://www.ncbi.nlm.nih.gov/pmc/articles/PMC401051/ Melchers LS, 1989]). VirA spans the inner membrane, with two transmembrane domains, a large periplasmic region, and a large C-terminal cytoplasmic domain ([http://jb.asm.org/cgi/content/abstract/176/11/3242 Banta LM, 1994]). VirA directly senses the phenolic compounds for vir activation ([http://linkinghub.elsevier.com/retrieve/pii/S0378111996003289 Lee YW et al., 1996]). Therefore the linker domain is essential for induction by phenolic compounds ([http://jb.asm.org/cgi/content/abstract/174/21/7033 Chang CH and Winans SC., 1992]). The linker region is located in the cytosolic site at position 280 to 414 ([http://linkinghub.elsevier.com/retrieve/pii/S0378111996003289 Lee YW et al., 1996]). This region between the amino acids 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 ([http://www.springerlink.com/index/G548R7745685341P.pdf Turk SC et al., 1994]).


Figure 2: The structur of virA (Melchers et al., 1989)
Figure 3: The funtional parts of the virA receptor (Lee et al., 1996)


Chang and Winnans (1992) revealed in their studies the parts of the VirA receptor which are essential for the signal transduction ([http://jb.asm.org/cgi/content/abstract/174/21/7033 Chang CH and Winans SC., 1992]). A structured model for different inducing conditions are shown in figure 4.

Figure 4 : The different binding conditions (Chang and Winans, 1992)

For information about modulation strategy click here


Phenolic Compounds

The ligand receptor interaction between acetosyringone and VirA is based on the interaction of several chemical groups. First of all the hydroxylated aromat is essential. Methoxy groups in the ortho position of the phenol play a crucial role in the signaling as well. It should be mentioned that dimethoxy compounds have a higher activity than monomethoxy compounds. The acetyl and alkyl groups in para position enhance the binding affinity. VirA activating compounds must have two methoxy groups in ortho position and an additional carbonyl group on the R3 chain. The potential capacity of the group para to the phenolic hydroxyl group is associated with higher activities. Moreover the chirality at this carbon center is critical for the inducing activity ([http://www.ncbi.nlm.nih.gov/pmc/articles/PMC372852/?pageindex=1&tool=pmcentrez Winans SC, 1992]). Regarding to the proton transfer model of Hess et al. (1996) the VirA activator transfers a proton to the basic area receptor binding site. The allosteric change leads to the phosphotransfer and the signaltransduction ([http://www.ncbi.nlm.nih.gov/pmc/articles/PMC52402/?pageindex=1&tool=pmcentrez Hess KM et al., 1991]).

Figure 5: Chemical structure of phenol
Figure 6: The proton transfer model according to Hess et al., 1991
Figure 7: Compounds with a structural similarity, which induce virA Winans, 1992

See more possible compounds by clicking here


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 interacts with the VirA receptor, leading to a much stronger vir gene expression ([http://jb.asm.org/cgi/content/abstract/176/11/3242 Banta LM, et al., 1994]).


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 genes ([http://www.ncbi.nlm.nih.gov/pmc/articles/PMC103583/?report=abstract&tool=pmcentrez Lohrke SM et al., 1990]) For this reason it was necessary to subclone a modified virG gene that is capable to be trancribed by the E. coli expression system.

Yong-Chul et al. (2004) described VirG mutants that are capable of expressing the virB promoter-driven genes 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 ([http://www.springerlink.com/content/wmq06kua5qkma1au/fulltext.pdf Yong-Chul J et al., 2004]). 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 Yong-Chul J et al. (2004) 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.


Read out system

Firefly Luciferase

We are using luciferase as the read out system. The luciferase was originally extracted from the firefly Photinus pyralis. It is one of the best studied and characterized read out systems. The luciferase enzyme catalyses the chemical reaction from its substrate luceferin to oxyluciferin and light ([http://mcb.asm.org/cgi/reprint/7/2/725 De Wet et al. 1986]). Figure 8 shows the reaction in detail.

We used luciferase as the read out system because it causes only slight negligible noise, hence the signal to noise ration is excellent.

Figure 8: Firefly luciferase reaction (De Wet et al.1986)






Output-signal amplification by Sensitivity Tuner implementation

Using a standard, inducible promoter with reporter system often results in weak reporter expression. So difficulties in quantification can occur. The quantification can be enhanced by an amplification of the transcription rate of the desired reporter genes. Such an amplification can be realized by using a so called sensitivity tuner device. This takes place as promoter induction upregulates a phage activator, which binds to a phage promoter upstream of a reporter gene. As result, a PoPs input (Inducer) generates a PoPs output at a higher signal. PoPs is equivalent to the flow of RNA polymerase molecules along DNA ([http://jb.asm.org/cgi/content/abstract/178/19/5668 Julien and Calendar, 1996] ; iGEM Team Cambridge, 2007).

Figure 9: Gene sequence of final test construct including Sensitivity Tuner elements.



Purpose of Sensitivity Tuner application

We presumed weak expression rates of our reporter luciferase indicated by pretesting the native system <partinfo>K389015</partinfo>. For having a broader range of quantification for our prototype test system, an amplification device was implemented. For amplifying the output signal of luciferase induced by acetosyringone, three sensitivity tuners, distinguished by the amplification factor, were combined with our detection system. To modify the sensitivity tuner for our purpose we took BioBricks with amplification factors from 10 (<partinfo>I746380</partinfo>) to 35 (<partinfo>I746390</partinfo>), removed the pBAD/araC promoter (<partinfo>I0500</partinfo>) and GFP read out (<partinfo>E0040</partinfo>) and replaced it by the reporter gene luciferase <partinfo>K389004</partinfo>(Figure 9). This enhanced luciferase BioBrick was assembled to the VirA/G signaling system BioBrick. The benefits of luciferase as reporter gene instead of GFP are a broader range of measurement, higher sensitivity and low half-live making cinetic tests possible ([http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6W9V-4F031H9-30&_user=10&_coverDate=01%2F31%2F1989&_rdoc=1&_fmt=high&_orig=search&_origin=search&_sort=d&_docanchor=&view=c&_searchStrId=1514624813&_rerunOrigin=google&_acct=C000050221&_version=1&_urlVersion=0&_userid=10&md5=ee400628b119490fcdc44ccdd856c4e8&searchtype=a Williams et al.1989]).



For test results click Sensitivity Tuner amplified Vir-test system


Receptor modification strategy

The smartest way of receptor-modification is initiated by a silico approach based on a 3D structure of the native receptor. Followed by primer mutagenesis of the computantional gained results a precise adapted peptide emerges. This concept has been proven by [http://www.nature.com/nature/journal/v423/n6936/abs/nature01556.html Looger et al., 2003].

Because of time limitations within the iGEM competition and a lack of biological data in literature - no x-ray cristalography structure data for VirA linker region available- this strategy was not applicable. Therefore we developed two different stragies in our MARSS project:

The first strategy was an error prone PCR approach by building a mutant data base for selection. The second strategy was a primer mutagenesis based approach.


Random mutagenesis by error-prone PCR (EP-PCR)

In order to detect novel substances (e.g. capsaicin) with the VirA receptor, the first step was to create a mutagenised library of virA variants, which could subsequently be screened for new binding characteristics. Plenty of different strategies for mutagenesis of DNA are known, including the use of nucleotide analogues, bacteria containing mutator genes, the mutagenesis with UV light or chemicals and inaccurate PCR ([http://genome.cshlp.org/content/2/1/28.short Cadwell RC and Joyce GF, 1992]).

When designing our strategy we rejected the use of bacteria strains with high mutation rates, since the changes in base sequence would occur all over the transformed plasmids. Thereby some mutations would also take place in the backbone of the plasmid, or might even been found in the standardized BioBrick prefix and suffix. We also excluded the possibility of using UV light or mutagenic chemicals, due to reasons of safety and minimizing the exposure of toxic substances.

In our experiment we wanted to alter only the part of the plasmid coding for the VirA receptor, while using a not harmful and thereby safe technique. Thus, our method of choice was inaccurate PCR that allows the exclusive variations of a distinct region of a plasmid, which is defined by the location of the upstream and downstream primers. This mutagenic method of PCR, called error-prone PCR (EP-PCR) has been described and improved a lot in scientific community ([http://www.springerlink.com/content/r62q360t82764508/#section=746282&page=1 McCullum EO et al., 2010]).

The basic principle of this technique uses the natural high infidelity of the taq DNA polymerase, which can even be increased by special changes in buffer conditions compared to standard PCR. These alterations may include the unequal distribution of dNTPs (5 mM purines, 25 mM pyrimidines) as well as an increased amount of MgCl2 and the addition of MnCl2. The total rate of base exchange can be adjusted by the number of PCR cycles, since mutations will accumulate during the exponential amplification of the sequence ([http://onlinelibrary.wiley.com/doi/10.1002/0471142727.mb0803s51/full Wilson DS and Keefe AD, 2000]). The experimental conditions of the performed error-prone PCR are described in the section “protocols”.


Directed mutagenesis

After the identification of the 100 amino acids linker region responsible for VirA ligand binding (Compare VirA-Receptor) we compared this region with well characterized capsaicin receptors derived from animal model organisms (TRPV1). The conserved receptor region is shown in the figure beneath.

The species-specific sensitivity of TRPV1 can be ascribed to about eight amino acids in the vicinity of TM3 ([http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6WSN-4C5H6M5-F&_user=2459438&_coverDate=02%2F08%2F2002&_rdoc=1&_fmt=high&_orig=search&_origin=search&_sort=d&_docanchor=&view=c&_acct=C000057302&_version=1&_urlVersion=0&_userid=2459438&md5=93a9e63f48d785a0b400131a058ee269&searchtype=a Jordt an Julius 2002]).

Figure 10: Molecular determinants of species-specific vanilloid sensitivity. Sequence alignment of rat (top), human (middle) and chicken (bottom) VR1 within the TM3-4 region is shown. Conserved residues are indicated by black background. The chimera V3/C contains a minimal segment of rat VR1 that is sufficient to confer vanilloid sensitivity ([http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6WSN-4C5H6M5-F&_user=2459438&_coverDate=02%2F08%2F2002&_rdoc=1&_fmt=high&_orig=search&_origin=search&_sort=d&_docanchor=&view=c&_acct=C000057302&_version=1&_urlVersion=0&_userid=2459438&md5=93a9e63f48d785a0b400131a058ee269&searchtype=a Jordt and Julius, 2002]).

We further aligned the TM3 region of the TRPV1 receptor to the ligand binding region of the native VirA receptor from Agrobacterium by the use of the tool MUltiple Sequence Comparison by Log-Expectation ([http://www.ebi.ac.uk/Tools/muscle/index.html Muscle]).

For detailed approach of directed mutagenesis for VirA_mut1 and VirA_mut2 click here


Screening system

Development of a high-troughput screening

The screening of randomly mutagenised genes for a desired function or application is always a very time-consuming procedure ([http://www.ncbi.nlm.nih.gov/pubmed/1496376 Beaudry and Joyce, 1992]). It requires a huge amount of material and might takes several months or even years to result in a promising new version of a gene ([http://mbe.oxfordjournals.org/content/17/7/1050.long Hanczyc and Dorit, 2000]). As we faced the challenge to modify the VirA receptor in only a few weeks we designed a strategy for a fast high-throughput screening by using subsequent steps of different read out systems and a strategy with two different plasmids.

In the first step after mutagenesis of virA it is necessary to separate thousands of transformants with minor or unwanted changes in the virA gene, from few bacteria that included interesting virA variants. Thus, we wanted to construct our system to lead in the expression of a kanamycin resistance after the induction of the VirA receptor, enabling the quick exclusion of all unwanted virA variants.

For that purpose a kanamycin resistance cassette should be set under control of the virB promoter, leading in the expression of aminoglycoside phosphotransferase (APH) that can inactivate kanamycin. The mode of inactivation is the transfer of the y-phosphate from ATP to the hydroxyl group at C3 of the antibiotic ([http://www.bioscience.org/1999/v4/d/wright/wright.pdf Wright and Thompson, 1999]). This phosphorylation results in the loss of binding capacity of the aminoglycoside to the 30S subunit of bacterial ribosomes, which would lead to inhibition of protein synthesis without the presence of the APH ([http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1365070/pdf/brjclinpharm00008-0014.pdf Begg EJ and Barclay ML, 1995]).

As indicated by the first results, the reporter genes under control of the virB promoter showed a slight but measureable expression without any induction of VirA with acetosyringone. This basal transcription resulted in the growth of bacteria without induction of VirA at normal working concentrations of kanamycin of 25 to 50 µg mL-1 ([http://books.google.de/books?hl=de&lr=&id=9mO2Fx0CuEYC&oi=fnd&pg=PA4&dq=Molecular+Cloning:+A+Laboratory+Manual,+Vol+1.+&ots=Ctw-SlcSLm&sig=FvweaKb_qiUxClNNiRfdwpmbqdo#v=onepage&q&f=false Sambrook J and Russell DW, 2001]).

Concludingly, prior to the screening experiments it was necessary to adjust the concentration of kanamycin, which inhibits the growth of uninduced bacteria, while allowing bacteria to grow when an appropriate inductor was present and able to activate VirA. This analysis was performed using the method of determination of minimal inhibitory concentrations (MIC) as described below.


Determination of minimal inhibitory concentration (MIC) of kanamycin

There are several ways to investigate the susceptibility of bacteria to inhibiting drugs like antibiotics. Nevertheless, the result of all these tests is the amount of an assayed substance that inhibits visible growth of the bacteria, called the minimal inhibitory concentration (MIC). The most common way of determination is to grow bacteria in liquids with several concentrations of the inhibiting drug. This procedure is chosen mostly, since many different conditions can be measured at the same time by using microtiterplates ([http://www.nature.com/nprot/journal/v3/n2/abs/nprot.2007.521.html Wiegand I et al., 2008]).

As we planned not to use the kanamycin in liquid culture but in LB-Agar, we chose to determine the MIC at the same conditions as the desired experiment. Therefore, we planned to construct E. coli inhabiting the native VirA/G signaling system and a kanamycin resistance read out and plated a small volume in different dilutions on LB-Agar without kanamycin. The grown colonies could then be transferred to agar plates with rising concentrations of kanamycin using replica plating. By counting the colonies it should be possible to calculate the percentage of colonies that could withstand each kanamycin concentration. This experiment should be carried out with and without acetosyringone to determine a kanamycin concentration induced E. coli could withstand, while the same population of bacteria dies without the presence of acetosyringone.


Primary selection of virA variants with novel binding properties

After the determination of the MIC the screening for virA variants with new binding properties could be started. The aim of this screening is to find versions of virA that can be induced by one of the tested substances capsaicin, homovanillic acid, dopamine and 3-O methyldopamine.

For that purpose one should transform the mutagenised variants of virA to E. coli and plate the bacteria on LB-agar with the determined kanamycin concentration. At the same time a mixture of all mentioned substances should be present in the agar. All bacteria including a virA variant that is activated by at least one of the substances will grow on the selective agar, since it expresses the kanamycin resistance. With this step it is thereby possible to select thousands of bacteria with unwanted versions of virA from few individuals with wanted binding properties.

At this point it must be mentioned that some grown colonies might still be non-induced and false positive results. As the virA gene has been randomly changed before, it is possible that some variants occur where the receptor is always active. This would lead to a constitutive expression of the reporter gene and thereby a high level of kanamycin resistance. To exclude those false positive clones, the bacteria should be tested whether they are only resistance to the MIC of kanamycin when one of the tested substances is present. Every colony that can withstand the high kanamycin concentration without any inductor includes a constitutive version of virA and should be discarded in further analysis.


Quantitative analysis of virA variants after induction with novel substances

Should we find some bacteria that respond to the presence of the tested substances (capsaicin, homovanillic acid, dopamine and 3-O methyldopamine) by growing on the MIC of kanamycin, it is desirable to quantify the induction. For that purpose it is appropriate to change the read out system from kanamycin resistence to luciferase expression. This complex and time consuming task can easily be achieved without any cloning step, when using the advantage of our two plasmid system.

After primary selection each bacteria includes two plasmids with different origins of replication (oris). The plasmid with the virA is in a common <partinfo>pSB1AT3</partinfo> backbone with a ColE1 ori. Contrary to that the read out plasmid with KanR has a special ori, named R6K, which can only amplify in E. coli strains that express the gene pir to produce the so called Pir protein. Most of the strains used in laboratory are pir- but few (e.g EC100D) are pir+ ([http://www.ncbi.nlm.nih.gov/pubmed/17383678 Bowers et al., 2007]).

The setup of the different oris was chosen to separate both plamids at this experimental stage. To change the read out system from KanR to luciferase, one just needs to perform two transformations and isolations of plasmids. In the first step plasmids are isolated from colonies with positive binding properties to one of the tested substances. This mixture of plasmids with ColE1 and R6K oris is then transformed to a pir- strain (e.g. TOP10). In the following only the plasmid with ColE1 ori will be amplified during the growth of the transformants, leading to pure plasmids with virA when plasmids are isolated for a second time. In the last step this isolated DNA can be transformed to bacteria including another read out plasmid (e.g. with luciferase).


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