the university of arizona. the a team students patrick hollinger brian heinze josh kittleson tim...
TRANSCRIPT
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The University of Arizona
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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
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The Watercolor Palette
Goal: Create a color image utilizing bacterial fluorescence
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Pointilism/image/piesky
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Water color/Paintbrush
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Vision
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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.
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2 Seconds of Operon Regulation
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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
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Design: Induction Details
Regulator Type Controlled By
LuxR Positive Homoserine Lactone (HSL)
TetR Negative Anhydrotetracycline (AHT)
LacI Negative Isopropyl β-D-1-
thiogalactopyranoside
(IPTG)
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Some Assembly Required
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Parts Created
Name Type Description LengthBBa_J21000 Composite LuxR mCherry, TetR ECFP, LacI EYFP 2916BBa_J21001 Reporter LuxR controlled RFP 932BBa_J21002 Regulatory Promoter + LuxR 998BBa_J21003 Regulatory Promoter + TetR 904BBa_J21004 Regulatory Promoter + LacL 1372BBa_J21005 Reporter LxR,LcY,TC 2829BBa_J21006 Regulatory LuxR, TetR Generator 1910BBa_J21007 Regulatory LuxR,TetR,LacL Generator 3290BBa_J21008 Composite WaterColorDesign 6127
Arizona iGEM 2006 Parts Sandbox
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Inducer Application Methods
Pipette application Grown in inducers Inkjet Printer PDMS Stamp / Rapid Prototyping Mold
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Characterization
Cells Grown and induced Fluorescent microscopy Bright Field Microscopy
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Inducer added to lawn
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Cultures were grown on LB + amp in inducer concentrations ranging from no inducer to 10,000x Km
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Images 1
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Images 2
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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.
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A New Plan
Revisit intermediate constructs• Two strains of cells
• Constitutive expression
• One for each ‘color’
Place cells with inkjet printer
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Inkjet Printer
Replace ink with cells Print onto transfer paper and ‘stamp’
media
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Results
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Rapid Prototyping Stamp
Machine generated ‘negative’ image PDMS stamp is made [insert picture of PDMS]
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RP Results
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Conclusions
This Week
Mucho Printo
Last day to print Weds
Imaging
Thurs
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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