translating a synthetic, high expressing, copper-inducible promoter system from yeast into tobacco...

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