The University of Arizona. The A Team Students Patrick Hollinger Brian Heinze Josh Kittleson Tim Spriggs Tyler Brown Dan Reavis Kevin MacDow Carlos Chang.

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Presentation transcript:

The University of Arizona

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

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

Pointilism/image/piesky

Water color/Paintbrush

Vision

Implementation Overview 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

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

Design: Induction Details RegulatorTypeControlled By LuxRPositiveHomoserine Lactone (HSL) TetRNegativeAnhydrotetracycline (AHT) LacINegativeIsopropyl β-D-1- thiogalactopyranoside (IPTG)

Some Assembly Required

Parts Created

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

Characterization Cells Grown and induced Fluorescent microscopy Bright Field Microscopy

Inducer added to lawn

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

Images 1

Images 2

Results 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 Revisit intermediate constructs Two strains of cells Constitutive expression One for each ‘color’ Place cells with inkjet printer

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

Results

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

RP Results

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

Acknowledgements 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 McGinnis and Lyuda XYZ Dr. Jay Hoying

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