E. coli Traffic Light: An Analog Biosensor2009 UBC iGEM Team

Alex Ng, Amelia Hardjasa, Calvin Chan, Charles Howes, Eric J. L. Ma, Hank Yu, Heather Kempthorne, Janny Ke, Karen Cheshire, Mark Ling, Paul Jaschke, Eric T. Lagally, Joanne A. Fox, Dave Ng

Results(1) PBAD variants were fabricated

by site-directed mutagenesis

Figure 1. Site-directed mutagenesis was performed on BBa_I13453 to induce mutations in PBAD (indicated in red). Mutated wild type coordinates are indicated in green.

5’...ATGCCATAGCATTTTTATCCATAAGAT...3’Wild Type

5’...ATGCCATAGCTTTTTTATCCATAAGAT...3’Weak

5’...ATGCCATAGCAAGATAGTCCATAAGAT...3’Strong

(3) PBAD variants respond specifically to arabinose

0

75

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ribose xylose lyxose rhamnose arabinose

RFP

Pro

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Sugar

PBAD weak

PBAD wt

PBAD strong

Control

Figure 4. RFP under control of PBAD is expressed only when induced with arabinose and none of the other four pentoses tested. Sugar concentrations were at 0.05% (w/v) except for lyxose at 0.01% (w/v).

(2) PBAD Strong, WT and Weak exhibit differential responses to arabinose

Figure 2. RFP induction becomes visible in culture at 2.33 µM in PBAD strong and 4.30 µM in PBAD wild type and weak.

Stro

ngW

TW

eak

00.751.322.354.307.50Arabinose concentration (µM)

Ara

bino

se P

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oter

Arabinose Concentration (µM)

RFP

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)

PBAD Strong

PBAD Wild TypePBAD Weak

0

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0 2.5 5.0 7.5 10.0

Figure 3. Transfer function for arabinose induction of RFP confirms the relative strengths of each promoter (strong > wild type > weak) at varying concentrations of arabinose. Representative data points of flow cytometry measurements are shown; fitted curves are solid lines. RFP production represented as the mean of 585 nm histogram values.

Introduction• Biosensing is an important foundational tool

for multiple applications including medical diagnostics and environmental protection.

• Whole-cell biosensors potentially offer greater range and specificity towards biological targets than mechanical & electrical sensors.

• Analog biosensors are able to respond to inputs in a dose-dependent fashion, enabling the development of thresholds for reporter expression.

• RNA-level regulation can be used to execute threshold-dependent outputs.

• We have constructed and characterized two parts classes for a prototype arabinose threshold-dependent whole cell biosensor.

Gold SponsorsTeaching and Learning Enhancement Fund

The Traffic Light

Conclusions• We have successfully fabricated and

characterized a strong and weak PBAD that are respectively more and less sensitive to arabinose than wild type PBAD.

• PBAD variants are highly specific for arabinose.• The Jammer is an effective tool for specific

gene knockdown.

ReferencesHutchinson CA et. al. (1978). Mutagenesis at a specific position in a DNA sequence. J. Biol. Chem. 253(18):6551-6560.

Isaacs FJ et .al. (2004). Engineered riboregulators enable post-transcriptional control of gene expression. Nat. Biotech. 22(7):841-847.

Niland P, Hühne R, Müller-Hill B. (1996). How AraC Interacts Specifically with its Target DNAs. J. Mol. Biol. 264:667-674.

O’Connor CD & Timmis KN (1987). Highly repressible expression system for cloning genes that specify potentially toxic proteins. J Bact. 169(10):4457-4462.

Win MN & Smolke CD. (2007). A modular and extensible RNA-based gene-regulatory platform for engineering cellular function. Proc. Natl. Acad. Sci. USA. 104(36):14283-8.

(4) RFP Expression occurs 9 hours post-induction

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2.25

4.50

6.75

9.00

0 15 30 45 60Hours Post Induction

RFP

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D60

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PBAD StrongPBAD Wild TypePBAD WeakControl

Figure 5. RFP induction occurs at 9 hours. RFP production values are means of flow cytometry measurements normalized against optical density of the culture measured at 600 nm (OD600).

(5) Jammer knocks down reporter levels

Figure 6. Following induction by 33 mM arabinose, the Jammer expression causes reduction in GFP levels.