Скачать презентацию The University of Arizona The A Team Скачать презентацию The University of Arizona The A Team

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The University of Arizona The University of Arizona

The A Team Students Faculty Patrick Hollinger Mark Riley Brian Heinze Joan Curry Josh The A Team Students Faculty Patrick Hollinger Mark Riley Brian Heinze Joan Curry Josh Kittleson Tim Spriggs Tyler Brown Dan Reavis Kevin Mac. Dow Carlos Chang

The Watercolor Palette Goal: Create a color image utilizing bacterial fluorescence The Watercolor Palette Goal: Create a color image utilizing bacterial fluorescence

Pointilism/image/piesky Pointilism/image/piesky

Water color/Paintbrush Water color/Paintbrush

Vision Vision

Implementation Overview l l Select three independent operons to control expression of three different Implementation Overview l l Select three independent operons to control expression of three different fluorescent proteins. Apply inducers via a controlled mechanism to achieve maximum image resolution.

2 Seconds of Operon Regulation 2 Seconds of Operon Regulation

Design: Switches and Lights Three independent operons regulate expression of three fluorescent proteins Lux Design: Switches and Lights Three independent operons regulate expression of three fluorescent proteins Lux Operator red fluorescent protein (m. Cherry) Tet Operator cyan fluorescent protein Lac Operator yellow fluorescent protein

Design: Induction Details Regulator Type Controlled By Lux. R Positive Homoserine Lactone (HSL) Tet. Design: Induction Details Regulator Type Controlled By Lux. R Positive Homoserine Lactone (HSL) Tet. R Negative Anhydrotetracycline (AHT) Lac. I Negative Isopropyl β-D-1 thiogalactopyranoside (IPTG)

Some Assembly Required Some Assembly Required

Parts Created Parts Created

Inducer Application Methods l l Pipette application Grown in inducers Inkjet Printer PDMS Stamp Inducer Application Methods l l Pipette application Grown in inducers Inkjet Printer PDMS Stamp / Rapid Prototyping Mold

Characterization l l l Cells Grown and induced Fluorescent microscopy Bright Field Microscopy Characterization l l l Cells Grown and induced Fluorescent microscopy Bright Field Microscopy

Inducer added to lawn Inducer added to lawn

l Cultures were grown on LB + amp in inducer concentrations ranging from no l Cultures were grown on LB + amp in inducer concentrations ranging from no inducer to 10, 000 x Km

Images 1 Images 1

Images 2 Images 2

Results l l No quantifiable or observable difference between induced and non-induced bacteria Single Results l l No quantifiable or observable difference between induced and non-induced bacteria Single colonies exhibited strong fluorescence in yellow, weak in m-cherry and very little to none in cyan.

A New Plan l Revisit intermediate constructs l Place cells with inkjet printer • A New Plan l Revisit intermediate constructs l Place cells with inkjet printer • Two strains of cells • Constitutive expression • One for each ‘color’

Inkjet Printer l l Replace ink with cells Print onto transfer paper and ‘stamp’ Inkjet Printer l l Replace ink with cells Print onto transfer paper and ‘stamp’ media

Results Results

Rapid Prototyping Stamp l l l Machine generated ‘negative’ image PDMS stamp is made Rapid Prototyping Stamp l l l Machine generated ‘negative’ image PDMS stamp is made [insert picture of PDMS]

RP Results RP Results

Conclusions l This Week Mucho Printo Last day to print Weds Imaging Thurs Conclusions l This Week Mucho Printo Last day to print Weds Imaging Thurs

Acknowledgements l Thanks to our sponsors and supporters: • • Dr. Raina Maier Dr. Acknowledgements l Thanks to our sponsors and supporters: • • Dr. Raina Maier Dr. L. Sandy Pierson (he is in charge of the Koffler lab) Dave Bentley Koffler people (list them) Dr. Vicki Chandler (and her 2 techs who helped train) Karen Mc. Ginnis and Lyuda XYZ Dr. Jay Hoying