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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
Recombinant gene technologies revolutionized biological sciences
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Promoters drive the expression of genes
Promoter Protein (Gene)
Transcription Factor Binds
RNA Polymerase Recruited
Gene Expression
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
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
More Robust Expression with ABS2
Black Bars: ABS Grey Bars :ABS2
ABS4 Does Not Induce Strongly
Cu (II) Ion, μM
Nanomoles(x10-9)
Micromoles (x10-6)
Cu (II) Ion Exclusively Activates ABS2
ABS2 for Tobacco?
From cited by 110 publications
Constitutively Express ACE (Transcription factor) in Tobacco
Transform ACE-Tobacco with ABS2-Reporter Construct
Assay Reporter Activity
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
•Determine ACE1 Expression in Tobacco▫RNA▫Protein
•Secondary Transformations of Tobacco with ABS2:β-galactosidase•β-galactosidase assays
Future Work
Conclusions
•In Yeasts, ABS2 outperforms ABS▫Tighter Control▫Higher Expression•Potential for ABS2 to work in Tobacco
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
Translating a Synthetic, High Expressing, Copper-Inducible Promoter System from Yeast into Tobacco
for Controlled Expression of Recombinant Genes
Alex LouieApril 17, 2009