Translating a Synthetic, High Expressing, Copper-Inducible Promoter System from Yeast into Tobacco...
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Transcript of Translating a Synthetic, High Expressing, Copper-Inducible Promoter System from Yeast into Tobacco...
![Page 1: Translating a Synthetic, High Expressing, Copper-Inducible Promoter System from Yeast into Tobacco for Controlled Expression of Recombinant Genes Alex.](https://reader035.fdocuments.net/reader035/viewer/2022062422/56649f285503460f94c4155f/html5/thumbnails/1.jpg)
Translating a Synthetic, High Expressing, Copper-Inducible Promoter System from Yeast into Tobacco
for Controlled Expression of Recombinant Genes
Alex LouieApril 17, 2009
![Page 2: Translating a Synthetic, High Expressing, Copper-Inducible Promoter System from Yeast into Tobacco for Controlled Expression of Recombinant Genes Alex.](https://reader035.fdocuments.net/reader035/viewer/2022062422/56649f285503460f94c4155f/html5/thumbnails/2.jpg)
Recombinant gene technologies revolutionized biological sciences
http://genetik.fu-berlin.de/institut/en_GFP_fly3.jpg
http://www.biotech.ucdavis.edu/TBCWebsites/TBC07/Agricultural&IndustrialApplications/EdwardsHayes&Overhouse-ChristianBros/Derek%20Matt%20Emily%20Website/rice.jpghttp://confocal.owu.edu/gallery/GFP27CELLS_DSP.jpg
http://www.edinformatics.com/inventions_inventors/Insulincrystals.jpg
http://www.sanger.ac.uk/Info/Press/gfx/060907_knockout-mice_300.jpg
http://www.cepolina.com/freephoto/f/nature.fruits.food/banana.jpg
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Promoters drive the expression of genes
Promoter Protein (Gene)
Transcription Factor Binds
RNA Polymerase Recruited
Gene Expression
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ABS Expression System
• Yeast Metallothionein Gene
• Controlled by Transcription Factor: Activating copper metallothionein expression (ACE1)
• Promoter Contains ACE1-Binding Sites (ABS)
• Copper Ion Inducible Expression
Metallothionein Expression
Copper Ions
ACE1
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Tandem Repeats?
• Graduate Student (K. Trujillo) wanted to use ABS
• ABS did not induce strongly
• More is better?
Promoter
ABS
ABS
ABS2
ABS4
Β-galactosidase reporter gene
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More Robust Expression with ABS2
Black Bars: ABS Grey Bars :ABS2
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ABS4 Does Not Induce Strongly
Cu (II) Ion, μM
Nanomoles(x10-9)
Micromoles (x10-6)
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Cu (II) Ion Exclusively Activates ABS2
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ABS2 for Tobacco?
From cited by 110 publications
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Constitutively Express ACE (Transcription factor) in Tobacco
Transform ACE-Tobacco with ABS2-Reporter Construct
Assay Reporter Activity
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ACE Transformed Tobacco1 2 3 4 5 6 7L
L: Ladder1: ABS2-βgal-Yeast2: ACE-Tobacco #53: ACE-Tobacco #64. ACE-Tobacco #85. ACE-Tobacco #96. ACE-Tobacco #107. Wild Type Tobacco
1 2 3 4 5 6 7LL: Ladder1: Wild Type Tobacco2: ABS2-βgal-Yeast3: ACE-Tobacco #14. ACE-Tobacco #25. ACE-Tobacco #36. ACE-Tobacco #47. ACE-Tobacco #7
To constitutively express ACE in Tobacco, Cauliflower Mosaic Virus 35S RNA promoter (CaMV) drives ACE expression
Polymerase Chain Reactions (PCR) with ACE Primers
Polymerase Chain Reactions (PCR) with ACE Primers
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•Determine ACE1 Expression in Tobacco▫RNA▫Protein
•Secondary Transformations of Tobacco with ABS2:β-galactosidase•β-galactosidase assays
Future Work
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Conclusions
•In Yeasts, ABS2 outperforms ABS▫Tighter Control▫Higher Expression•Potential for ABS2 to work in Tobacco
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Acknowledgements
•Dr. Glenn Kuehn• Dr. Kristina Trujillo
•Norah Gonzalez • Maria Ordaz• Murphy Hollie• Bernice Carrillo• Antonia Dominguez• Earlphia Sells• Don Benn• Hiranya Roychowdhury• Research Supported by MARC NIH GMO7667-31 and
GM008136-34 SCORE SC3 grant
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Translating a Synthetic, High Expressing, Copper-Inducible Promoter System from Yeast into Tobacco
for Controlled Expression of Recombinant Genes
Alex LouieApril 17, 2009