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