Difference between revisions of "Part:BBa K3009039"
(The tyrosyl-tRNA-synthetase from M.jannashii and cognate tRNA pair are acting orthogonally to the endogenous translational machinery of E.coli. By binding and loading non-canonical amino acids onto the tRNA, the tyrosyl-tRNA-synthetase enables their incor) |
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| + | Composite Part :SPD5 with MCP, synthetase BBa_K3009039 | ||
| + | Group: Freiburg | ||
| + | Author: Alisa | ||
| + | Summary: The tyrosyl-tRNA-synthetase from M.jannashii and cognate tRNA pair are acting orthogonally to the endogenous translational machinery of E.coli. By binding and loading non-canonical amino acids onto the tRNA, the tyrosyl-tRNA-synthetase enables their incorporation into proteins. The tRNA is cognate towards the amber stop codon, which is suppressed by the non-canonical amino acid. [1] MS2 Capsid Protein(MCP), binds strongly to specific stem-loop structures derived from the MS2 phage genome. Proteins fused | ||
| + | to MCP can be brought in close proximity to mRNA tagged with stem-loops.pindle-deficient protein 5 derived from C.elegans is able to assemble into condensates that built a separate phase from the surronding cytosol, therefore emulating a reaction compartment. Fusion proteins will also be confined to this organelle. | ||
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| − | + | Documentation: | |
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| + | Usage | ||
| + | SPD5 proteins assemble into dynamic droplet structures along with their fusion proteins. [1]. By fusing an othogonal aminoacyl tRNA Synthetase to the assembler proteins, the droplets are confined to a seprated phase inside the droplet and do not impede endogenous processes in the cytosol. [2] MS2 Capsid Proteins bind stem-loops which can be used to tag mRNA. [3] An mRNA of interest can therefore be recruited into the artificial organelle. The synthetase then incorporates a noncanonical amino acid inside the translated protein sequence. | ||
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| + | Biology | ||
| + | In C. elegans, SPD5 , forms condensates that aid the organization of microtubule arrays in the centrosome. [1] | ||
| + | The MS2 Capsid Protein performs regulatory functions in the early stages of an MS2 bacteriophage infection of ... It forms a protein-RNA complex with a specific RNA stem-loop structure derived from the phage genome. The original function of the MS2 Capsid Protein is to stabilize RNA to prevent replicase synthesis and enable the encapsidation of the genome. [4] | ||
| + | In nature, the tyrosyl-tRNA-synthetase catalyzes the bond between the tyrosyl-tRNA and tyrosine in the archaea Methanocaldococcus jannaschii. The tRNA can then bind to the complementary codon of a translated mRNA sequence and enable the incorporation of a tyrosine into the protein. [5] | ||
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| − | + | Characterization | |
| − | + | By creating a trimolecular fusion of SPD5:MCP:TyrRS, we aimed to confine an orthogonal translation system to a membraneless organelle inside a cell, so that background and unspecific incorporation could be minimized. Other components necessary for incorporation should be able to move freely into the organelle by diffusion. | |
| − | + | [[File:sp.png|200px|thumb|center|Fig.1:Fluorescence intensity quantification of cells expressing bi- and trimolecular fusions of TyrRS with sfGFP variants containing amber stop codons. n = 50 cells, error bars show standard error.]] | |
| + | Fig.1:Fluorescence intensity quantification of cells expressing bi- and trimolecular fusions of TyrRS with sfGFP variants containing amber stop codons. n = 50 cells, error bars show standard error. | ||
| + | Fluorescence intensity quantification of C321.delta A E.coli cells expressing an SPD5:MCP:TyrRS trimolecular fusion and sfGFP variants with amber stop codons at different sites was performed. Incorporation of D-Phenylalanine in stead of the amber stop codon is considerably higher for cells expressing the trimolecular fusion compared to the bimolecular fusion of MCP:TyrRS . | ||
| + | References | ||
| + | [1]Woodruff et. al (2017): The Centrosome Is a Selective Condensate that Nucleates Microtubules by Concentrating Tubulin. In: Cell 169 (6), 1066-1077.e10. DOI: 10.1016/j.cell.2017.05.028. | ||
| + | [2]Reinkemeier et. al (2019): Designer membraneless organelles enable codon reassignment of selected mRNAs in eukaryotes. In: Science (New York, N.Y.) 363 (6434 | ||
| + | [3]Haimovich et.al (2016): Use of the MS2 aptamer and coat protein for RNA localization in yeast: A response to "MS2 coat proteins bound to yeast mRNAs block 5' to 3' degradation and trap mRNA decay products: implications for the localization of mRNAs by MS2-MCP system". In: RNA (New York, N.Y.) 22 (5), S. 660–666 | ||
| + | [4] Johansson et. al (1997): RNA Recognition by the MS2 Phage Coat Protein. In: Seminars in Virology 8 (3), S. 176–185. | ||
| − | + | [5]Schultz et. al (2006): A genetically encoded infrared probe. In: Journal of the American Chemical Society 128 (43), S. 13984–13985 | |
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Latest revision as of 03:50, 22 October 2019
Composite Part :SPD5 with MCP, synthetase BBa_K3009039 Group: Freiburg Author: Alisa Summary: The tyrosyl-tRNA-synthetase from M.jannashii and cognate tRNA pair are acting orthogonally to the endogenous translational machinery of E.coli. By binding and loading non-canonical amino acids onto the tRNA, the tyrosyl-tRNA-synthetase enables their incorporation into proteins. The tRNA is cognate towards the amber stop codon, which is suppressed by the non-canonical amino acid. [1] MS2 Capsid Protein(MCP), binds strongly to specific stem-loop structures derived from the MS2 phage genome. Proteins fused to MCP can be brought in close proximity to mRNA tagged with stem-loops.pindle-deficient protein 5 derived from C.elegans is able to assemble into condensates that built a separate phase from the surronding cytosol, therefore emulating a reaction compartment. Fusion proteins will also be confined to this organelle.
Documentation:
Usage SPD5 proteins assemble into dynamic droplet structures along with their fusion proteins. [1]. By fusing an othogonal aminoacyl tRNA Synthetase to the assembler proteins, the droplets are confined to a seprated phase inside the droplet and do not impede endogenous processes in the cytosol. [2] MS2 Capsid Proteins bind stem-loops which can be used to tag mRNA. [3] An mRNA of interest can therefore be recruited into the artificial organelle. The synthetase then incorporates a noncanonical amino acid inside the translated protein sequence.
Biology In C. elegans, SPD5 , forms condensates that aid the organization of microtubule arrays in the centrosome. [1] The MS2 Capsid Protein performs regulatory functions in the early stages of an MS2 bacteriophage infection of ... It forms a protein-RNA complex with a specific RNA stem-loop structure derived from the phage genome. The original function of the MS2 Capsid Protein is to stabilize RNA to prevent replicase synthesis and enable the encapsidation of the genome. [4] In nature, the tyrosyl-tRNA-synthetase catalyzes the bond between the tyrosyl-tRNA and tyrosine in the archaea Methanocaldococcus jannaschii. The tRNA can then bind to the complementary codon of a translated mRNA sequence and enable the incorporation of a tyrosine into the protein. [5]
Characterization By creating a trimolecular fusion of SPD5:MCP:TyrRS, we aimed to confine an orthogonal translation system to a membraneless organelle inside a cell, so that background and unspecific incorporation could be minimized. Other components necessary for incorporation should be able to move freely into the organelle by diffusion.
Fig.1:Fluorescence intensity quantification of cells expressing bi- and trimolecular fusions of TyrRS with sfGFP variants containing amber stop codons. n = 50 cells, error bars show standard error.
Fluorescence intensity quantification of C321.delta A E.coli cells expressing an SPD5:MCP:TyrRS trimolecular fusion and sfGFP variants with amber stop codons at different sites was performed. Incorporation of D-Phenylalanine in stead of the amber stop codon is considerably higher for cells expressing the trimolecular fusion compared to the bimolecular fusion of MCP:TyrRS .
References [1]Woodruff et. al (2017): The Centrosome Is a Selective Condensate that Nucleates Microtubules by Concentrating Tubulin. In: Cell 169 (6), 1066-1077.e10. DOI: 10.1016/j.cell.2017.05.028. [2]Reinkemeier et. al (2019): Designer membraneless organelles enable codon reassignment of selected mRNAs in eukaryotes. In: Science (New York, N.Y.) 363 (6434 [3]Haimovich et.al (2016): Use of the MS2 aptamer and coat protein for RNA localization in yeast: A response to "MS2 coat proteins bound to yeast mRNAs block 5' to 3' degradation and trap mRNA decay products: implications for the localization of mRNAs by MS2-MCP system". In: RNA (New York, N.Y.) 22 (5), S. 660–666 [4] Johansson et. al (1997): RNA Recognition by the MS2 Phage Coat Protein. In: Seminars in Virology 8 (3), S. 176–185.
[5]Schultz et. al (2006): A genetically encoded infrared probe. In: Journal of the American Chemical Society 128 (43), S. 13984–13985