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Transcript of The University of Arizona. The A Team Students Patrick Hollinger Brian Heinze Josh Kittleson Tim...
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
Regulator Type Controlled By
LuxR Positive Homoserine Lactone (HSL)
TetR Negative Anhydrotetracycline (AHT)
LacI Negative Isopropyl β-D-1-
thiogalactopyranoside
(IPTG)
Some Assembly Required
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
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