Team:PKU Beijing/Project/AND Gate 2 Design
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{{PKU_Beijing/Sidebar_Project}} | {{PKU_Beijing/Sidebar_Project}} | ||
{{PKU_Beijing/Header2}} | {{PKU_Beijing/Header2}} | ||
- | + | [[Team:PKU_Beijing/Project|Project]] > [[Team:PKU_Beijing/Project/AND_Gate_2|AND Gate 2]] > [[Team:PKU_Beijing/Project/AND_Gate_2_Design|Design]] | |
==='''One of the Second AND Gate Design'''=== | ==='''One of the Second AND Gate Design'''=== | ||
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- | In order to creat a amber mutation inside of the PhiR73 delta/T3pol coding sequence, we carried out site directed mutagenesis with primers introduced with TAG amber mutation at the original Ser site. One problem that comes to us is how to decide which serine codon to mutate. In order to avoid that the half translated protein has function, we picked first few serine of the whole sequence of either PhiR73 delta activator and T3 polymerase. For PhiR73 delta it is on the first and second serine. (For PhiR73 delta sequence click [http://partsregistry.org/Part:BBa_I746352 Here]) | + | In order to creat a amber mutation inside of the PhiR73 delta/T3pol coding sequence, we carried out site directed mutagenesis with primers introduced with TAG amber mutation at the original Ser site. One problem that comes to us is how to decide which serine codon to mutate. In order to avoid that the half translated protein has function, we picked first few serine of the whole sequence of either PhiR73 delta activator and T3 polymerase. For PhiR73 delta it is on the first and second serine. (For PhiR73 delta sequence click [http://partsregistry.org/Part:BBa_I746352 Here]). In T3 polymerase, we picked four sites, they are on the 37th, 91th, 130th, 283th nucleotide of its coding sequence. |
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[[Image:PKU_Point_Mutation.png|500px|center|thumb|fi2. Creating the Amber mutation]] | [[Image:PKU_Point_Mutation.png|500px|center|thumb|fi2. Creating the Amber mutation]] | ||
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+ | In order to test the second AND Gate we place the mutated PhiR73 delta downstream of the salicylate sensor, so that salicylate can induce the expression of the mutated PhiR73 delta. Because our earlier work is very robust, there are many constructed SupD indicible expression set. We picked Arabinose sensor-SupD set for its well performance in the construction of the first AND Gate. The supD PhiR73 delta expression set is on the plasmid pSB4K5, and the PO promoter-GFP is on the pSB1A2 plasmid. | ||
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==='''Another Design of the Second AND Gate'''=== | ==='''Another Design of the Second AND Gate'''=== | ||
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First, we should figure out the -35 region and the -10 region of the salicylate inducible promoter. Then, the region between -35 and -10 is replaced with a CI434 binding site. Another CI434 binding site is placed downstream of -10 region. In this way, the promoter is silenced when CI434 is bound. After CI434 is removed from the system, this hybrid promoter still needs salicylate to activate. | First, we should figure out the -35 region and the -10 region of the salicylate inducible promoter. Then, the region between -35 and -10 is replaced with a CI434 binding site. Another CI434 binding site is placed downstream of -10 region. In this way, the promoter is silenced when CI434 is bound. After CI434 is removed from the system, this hybrid promoter still needs salicylate to activate. | ||
- | [[Image:PKU_Second_AND_Gate_Hybrid_Promoter.png| | + | [[Image:PKU_Second_AND_Gate_Hybrid_Promoter.png|600px|center|thumb|fig3. Another Design of the Second AND Gate]] |
{{PKU_Beijing/Foot}} | {{PKU_Beijing/Foot}} | ||
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Latest revision as of 23:44, 17 October 2009
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