1. The University of Arizona

2. 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

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

4. Pointilism/image/piesky

5. Water color/Paintbrush

6. Vision

7. 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.

8. 2 Seconds of Operon Regulation

9. 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

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

11. Some Assembly Required

12. Parts Created

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

14. Characterization Cells Grown and induced Fluorescent microscopy Bright Field Microscopy

15. Inducer added to lawn

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

17. Images 1

18. Images 2

19. 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.

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

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

22. Results

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

24. RP Results

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

26. 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