Team:SJTU-BioX-Shanghai/Project/Application

From 2011.igem.org

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__NOTOC__
__NOTOC__
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==Application==
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==Expansion of Regulating Tools for Synthetic Biology==
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===I. Expansion of Regulating Tools for Synthetic Biology===
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===I. Multi-Level Regulation===
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====1. Multi-Level Regulation====
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[[image:11SJTU_App_05.jpg|frame|''Fig.1'' Multi-Level Regulation]]
[[image:11SJTU_App_05.jpg|frame|''Fig.1'' Multi-Level Regulation]]
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rare tRNA amount: controlled by different strengths of promoters or riboswitch.
rare tRNA amount: controlled by different strengths of promoters or riboswitch.
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====2. Direct and Precise Regulation====
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===II. Direct and Precise Regulation===
As a translational regulatory tool, our device achieves more precise tuning of protein expression when compared with transcriptional tools. The controlling elements we use in this device are codons and tRNAs, the major participants of translation process. Thus manipulating these elements exert direct effects on protein biosynthesis. Here we offer two potential models for how this device can be used in pathway construction.  
As a translational regulatory tool, our device achieves more precise tuning of protein expression when compared with transcriptional tools. The controlling elements we use in this device are codons and tRNAs, the major participants of translation process. Thus manipulating these elements exert direct effects on protein biosynthesis. Here we offer two potential models for how this device can be used in pathway construction.  
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*Many genes are involved in complex metabolic pathways. The expression level of each gene may be different and affecting the production of final product. If a series of genes are under the control of a single promoter, these genes are likely to express on almost the same level. '''(a)'''
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====Model 1====
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Many genes are involved in complex metabolic pathways. The expression level of each gene may be different and affecting the production of final product. If a series of genes are under the control of a single promoter, these genes are likely to express on almost the same level. '''(a)'''
Yet to achieve maximum production, each protein amount should be individually regulated. Traditionally we use different promoters to control different genes. '''(b)'''
Yet to achieve maximum production, each protein amount should be individually regulated. Traditionally we use different promoters to control different genes. '''(b)'''
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[[image:11SJTU_App_01.jpg|center|application model]]
[[image:11SJTU_App_01.jpg|center|application model]]
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*In biosynthetic pathways, feedback can affect protein production greatly. Our device can act as a feedback regulating tool to regulate the biosynthesis of target protein.Target protein and rare tRNA are under the control of the same promoter. Rare tRNA amount can regulate target protein biosynthesis through the rare codons inserted into the gene.  
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Our model can be used in building certain metabolic pathways, such as the violacein synthetic pathway. The relative amount of VioE and VioD will influence violacein production. Based on our model, the two genes can be placed under a single promoter yet different productions of the two enzymes can be achieved when we add different number of AGG codons to different sites on the tag with different copy numbers of AGG tandems.  
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[[image:11SJTU_App_02.jpg|center|application model]]
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[[image:11SJTU-app-vioEvioD.jpg|center]]
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Another factor that influences protein biosynthesis may be the relative amount of two proteins. Our device can serve as a linker to regulate the relative amount of Protein A and Protein B. Rare codons are put into Gene A and Gene B. When rare tRNA is produced, it can influence the amount of both Protein A and Protein B, along with rare codon types and numbers.  
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Different expression levels of the two genes can be achieved which may be used to increase final production.
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[[image:11SJTU_App_03.jpg|center|application model]]
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====Model 2====
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===II. A New Method to Incorporate Point Mutation into Protein===
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=====Feedback=====
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aaRS Modulator can be used as a tool to incorporate point mutation into protein.  
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In biosynthetic pathways, feedback can affect protein production greatly. Our device can act as a feedback regulating tool to regulate the biosynthesis of target protein.Target protein and rare tRNA are under the control of the same promoter. Rare tRNA amount can regulate target protein biosynthesis through the rare codons inserted into the gene.  
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In our project for instance, we have modified tRNA<sup>Asp</sup>’s anticodon to base pair with AGG, which is originally the codon for Arg. Then we have modified the AspRS. This modified AspRS can charge Asp to tRNA<sup>Asp</sup>-AGG. For the protein, codon AGG is originally decoded as Arg yet now it is translated into Asp, thus incorporating point mutation.  
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[[image:11SJTU_App_02.jpg|center|application model]]
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This method is fit for any other codon and any other tRNAs.
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We have simplified the process of constructing positive feedback in the metabolic pathways. We simply add AGG codons before the target gene and insert a 74bp tRNA<sup>Arg</sup> gene in the same operon.  
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[[image:11SJTU_App_04.jpg|center|incorporate point mutation]]
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We inserted the tDNA<sup>Arg</sup> and luciferase gene into T7 operon so that both genes can be transcribed. tRNA<sup>Arg</sup> can be correctly processed and become matured. Luciferase mRNA will be further translated into protein. The induced tRNA<sup>Arg</sup> can facilitate the translation of luciferase-nAGG, achieving positive feedback.  
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===III. A New Method to Study the Important Domains of a Protein===
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[[image:11SJTU_FB2468.jpg|frame|center|''Fig.1'']]
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Initial-Codon Switch can be used as a tool to study the important domains of a protein. With Initial-Codon Switch, we can start translation at any given site, selectively expressing part of an ORF by introducing a new start codon, a new tRNA<sup>fmet</sup>, a new aminoacyl-tRNA synthetase and a new 16S rRNA.
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The rest of the working curves are shown here:
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We replace the first codon of the selected part of the peptide with a rare codon. Here, the rare codon is used as the start codon (Reporter).
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<gallery caption="Different Number of Rare Codons with tRNA<sup>Arg</sup>">
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image:11SJTU_FB2.jpg|''Fig.2'' 2AGG-tRNA<sup>Arg</sup>
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image:11SJTU_FB4.jpg|''Fig.3'' 4AGG-tRNA<sup>Arg</sup>
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image:11SJTU_FB6.jpg|''Fig.4'' 6AGG-tRNA<sup>Arg</sup>
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image:11SJTU_FB8.jpg|''Fig.5'' 8AGG-tRNA<sup>Arg</sup>
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</gallery>
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'''Note''':Click to see large figures.
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Concurrently, an altered tRNA<sup>met</sup> with anticodon base pairing the rare codon and its corresponding aminoacyl-tRNA synthetase are introduced into the cell (Initial-Codon Switch).  
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According to these working curves we proposed that the positive feedback can make the working curves linear instead of titration curves.  
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In order to guarantee the recognition of ribosome to mRNA, the base pairing between RBS of mRNA and MBS of 16SrRNA should be assured. The 5 nucleotides which are 7 nucleotides upstream of start (rare) codon is selected as the RBS sequence. The gene of an MBS base-pairing the selected RBS sequence is introduced into the 16s rRNA gene, replacing the original MBS. Thus the cell has a new type of ribosome which can recognize the selected RBS. This procedure mimics the original base pairing between RBS and MBS by changing the conserved RBS according to the sequence of the gene.
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=====Linkage=====
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Another factor that influences protein biosynthesis may be the relative amount of two proteins. Our device can serve as a linker to regulate the relative amount of Protein A and Protein B. Rare codons are put into Gene A and Gene B. When rare tRNA is produced, it can influence the amount of both Protein A and Protein B, along with rare codon types and numbers.  
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[[image:11SJTU_App_03.jpg|center|application model]]
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With the design above, we can achieve the selective expression of part of an ORF.
 
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[[image:11SJTU_App_06.jpg|450px|A New Method to Study the Important Domains of a Protein]]
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[[image:11SJTU_arrow_next.jpg|right|Next Page|link=Team:SJTU-BioX-Shanghai/Project/Application-3]]

Latest revision as of 03:53, 29 October 2011

    •



    Expansion of Regulating Tools for Synthetic Biology

    I. Multi-Level Regulation

    Fig.1 Multi-Level Regulation

    Our device has expanded the regulating tools for synthetic biology. We control protein biosynthesis through rare codon types, rare codon numbers and rare tRNA amount. Different combinations of these three elements can bring different outcomes in protein expression levels.

    rare codon types: AGA, AGG, CGA, CUA…

    rare codon numbers: 1, 2,3, 4,5, 6,7, 8,9, 10…

    rare tRNA amount: controlled by different strengths of promoters or riboswitch.

    II. Direct and Precise Regulation

    As a translational regulatory tool, our device achieves more precise tuning of protein expression when compared with transcriptional tools. The controlling elements we use in this device are codons and tRNAs, the major participants of translation process. Thus manipulating these elements exert direct effects on protein biosynthesis. Here we offer two potential models for how this device can be used in pathway construction.

    Model 1

    Many genes are involved in complex metabolic pathways. The expression level of each gene may be different and affecting the production of final product. If a series of genes are under the control of a single promoter, these genes are likely to express on almost the same level. (a)

    Yet to achieve maximum production, each protein amount should be individually regulated. Traditionally we use different promoters to control different genes. (b)

    However with our device, genes can be put under one single promoter yet producing different amount of protein. (c)

    application model

    Our model can be used in building certain metabolic pathways, such as the violacein synthetic pathway. The relative amount of VioE and VioD will influence violacein production. Based on our model, the two genes can be placed under a single promoter yet different productions of the two enzymes can be achieved when we add different number of AGG codons to different sites on the tag with different copy numbers of AGG tandems.

    11SJTU-app-vioEvioD.jpg

    Different expression levels of the two genes can be achieved which may be used to increase final production.

    Model 2

    Feedback

    In biosynthetic pathways, feedback can affect protein production greatly. Our device can act as a feedback regulating tool to regulate the biosynthesis of target protein.Target protein and rare tRNA are under the control of the same promoter. Rare tRNA amount can regulate target protein biosynthesis through the rare codons inserted into the gene.

    application model

    We have simplified the process of constructing positive feedback in the metabolic pathways. We simply add AGG codons before the target gene and insert a 74bp tRNAArg gene in the same operon.

    We inserted the tDNAArg and luciferase gene into T7 operon so that both genes can be transcribed. tRNAArg can be correctly processed and become matured. Luciferase mRNA will be further translated into protein. The induced tRNAArg can facilitate the translation of luciferase-nAGG, achieving positive feedback.

    Fig.1

    The rest of the working curves are shown here:

    Fig.2 2AGG-tRNAArg

    Fig.3 4AGG-tRNAArg

    Fig.4 6AGG-tRNAArg

    Fig.5 8AGG-tRNAArg

    Note:Click to see large figures.

    According to these working curves we proposed that the positive feedback can make the working curves linear instead of titration curves.

    Linkage

    Another factor that influences protein biosynthesis may be the relative amount of two proteins. Our device can serve as a linker to regulate the relative amount of Protein A and Protein B. Rare codons are put into Gene A and Gene B. When rare tRNA is produced, it can influence the amount of both Protein A and Protein B, along with rare codon types and numbers.

    application model


    Next Page
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