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Team:Harvard/Project/Expression Plasmids
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The zinc finger expression plasmid was assembled through two-piece Gibson assembly, and contains the ColE1 origin of replication in addition to a spectinomycin resistance cassette. The zinc finger array is conjugated to the omega subunit of RNA polymerase, providing the basis for a one-hybrid system such that zinc finger target binding recruits RNA polymerase to transcribe downstream genes. In order to visualize and confirm zinc finger expression, GFP has been added to the end of the mRNA transcript, and is translated through the Shine-Dalgarno sequence AGGAGG that prefaces GFP by 7 base pairs. However, it must be noted that this only confirms expression of zinc finger mRNA without confirmation on the protein level. Zinc finger expression is controlled through the lac operator. Intrinsic activation of the lac operator has been found to be sufficient to induce zinc finger expression in significant amounts. | The zinc finger expression plasmid was assembled through two-piece Gibson assembly, and contains the ColE1 origin of replication in addition to a spectinomycin resistance cassette. The zinc finger array is conjugated to the omega subunit of RNA polymerase, providing the basis for a one-hybrid system such that zinc finger target binding recruits RNA polymerase to transcribe downstream genes. In order to visualize and confirm zinc finger expression, GFP has been added to the end of the mRNA transcript, and is translated through the Shine-Dalgarno sequence AGGAGG that prefaces GFP by 7 base pairs. However, it must be noted that this only confirms expression of zinc finger mRNA without confirmation on the protein level. Zinc finger expression is controlled through the lac operator. Intrinsic activation of the lac operator has been found to be sufficient to induce zinc finger expression in significant amounts. | ||
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==ZF Expression Plasmid== | ==ZF Expression Plasmid== | ||
| - | [[File:HARVZFP_with_caption_for_web.jpg|frameless|915px]] | + | [[File:HARVZFP_with_caption_for_web.jpg|frameless|center|915px]] |
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Latest revision as of 17:30, 26 September 2011
Overview | Design | Synthesize | Test | Zinc Finger Background | Protocols
Zinc Finger Expression Plasmids
Expression Plasmid Schematic
The zinc finger expression plasmid was assembled through two-piece Gibson assembly, and contains the ColE1 origin of replication in addition to a spectinomycin resistance cassette. The zinc finger array is conjugated to the omega subunit of RNA polymerase, providing the basis for a one-hybrid system such that zinc finger target binding recruits RNA polymerase to transcribe downstream genes. In order to visualize and confirm zinc finger expression, GFP has been added to the end of the mRNA transcript, and is translated through the Shine-Dalgarno sequence AGGAGG that prefaces GFP by 7 base pairs. However, it must be noted that this only confirms expression of zinc finger mRNA without confirmation on the protein level. Zinc finger expression is controlled through the lac operator. Intrinsic activation of the lac operator has been found to be sufficient to induce zinc finger expression in significant amounts.
ZF Expression Plasmid
