Difference between revisions of "Team:BostonU/Composite Part"
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| − | To create the Gemini parts library we made use of two “classes” of plasmid: guide RNA expressing vectors and guide RNA operator reporter vectors. When these two plasmids are co-transfected with a dCas9-VPR plasmid into a mammalian cell line, a gene of interest is expressed. <b>Our work for iGEM dealt with fluorescent proteins; specifically, GFP for the parts submitted to the registry.<b> </p> | + | To create the Gemini parts library we made use of two “classes” of plasmid: guide RNA expressing vectors and guide RNA operator reporter vectors. When these two plasmids are co-transfected with a dCas9-VPR plasmid into a mammalian cell line, a gene of interest is expressed. <b>Our work for iGEM dealt with fluorescent proteins; specifically, GFP for the parts submitted to the registry.</b> </p> |
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| − | The guide RNA operator vector can be considered a composite part. It is constructed by combining the following basic parts in order: BBa_K1875009 (20 bp target sequence), BBa_K1875000 (minimal CMV promoter), BBa_K1875001 (Kozak sequence), BBa_K1875003 (GFP), BBa_K1875002 (Rabbit Beta Globin PolyA terminator). The sources of these parts can be found on their respective registry pages. | + | The guide RNA operator vector can be considered a composite part. It is constructed by combining the following basic parts in order: BBa_K1875009 (20 bp target sequence), BBa_K1875000 (minimal CMV promoter), BBa_K1875001 (Kozak sequence), BBa_K1875003 (GFP), BBa_K1875002 (Rabbit Beta Globin PolyA terminator). The sources of these parts can be found on their respective registry pages. </p> |
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| + | We have submitted <a href = "http://parts.igem.org/Part:BBa_K1875018" style = "color:blue;">BBa_K1875018</a> for best composite part due to both its high expression and consistency in experimentation. This particular composite part has a triple multimerized operator for gRNA 13 (g13), meaning that there are 3 20 bp target sequences for g13 (each separated by 24 random bases) upstream of the minimal CMV promoter. Through our experimentation, we identified that this part, when compared with a full-length CMV promoter, can actually express GFP at a higher level than the strong constitutive promoter. This means that this synthetic composite part enables even higher protein production compared to a natural promoter, and can be used usefully in many protein production applications. Additionally, because its activity is controlled by expression of a specific corresponding gRNA (g13), it can grant temporal control over protein expression. | ||
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Revision as of 23:27, 18 October 2016
To create the Gemini parts library we made use of two “classes” of plasmid: guide RNA expressing vectors and guide RNA operator reporter vectors. When these two plasmids are co-transfected with a dCas9-VPR plasmid into a mammalian cell line, a gene of interest is expressed. Our work for iGEM dealt with fluorescent proteins; specifically, GFP for the parts submitted to the registry.
The guide RNA operator vector can be considered a composite part. It is constructed by combining the following basic parts in order: BBa_K1875009 (20 bp target sequence), BBa_K1875000 (minimal CMV promoter), BBa_K1875001 (Kozak sequence), BBa_K1875003 (GFP), BBa_K1875002 (Rabbit Beta Globin PolyA terminator). The sources of these parts can be found on their respective registry pages.
We have submitted BBa_K1875018 for best composite part due to both its high expression and consistency in experimentation. This particular composite part has a triple multimerized operator for gRNA 13 (g13), meaning that there are 3 20 bp target sequences for g13 (each separated by 24 random bases) upstream of the minimal CMV promoter. Through our experimentation, we identified that this part, when compared with a full-length CMV promoter, can actually express GFP at a higher level than the strong constitutive promoter. This means that this synthetic composite part enables even higher protein production compared to a natural promoter, and can be used usefully in many protein production applications. Additionally, because its activity is controlled by expression of a specific corresponding gRNA (g13), it can grant temporal control over protein expression.
