ABSTRACTAlternative Oxidase (AOX) is known to uncouple the oxidative phosphorylationpathway in thermogenic plants and transfer electrons to exothermic redoxreactions. The AOX gene of the Sacred Lotus plant was cloned into E. Coli, downstream from an inducible cold shock promoter, and directed to the periplasm via an OmpA targeting sequence. Analytical models were developed to predict the thermogenic behavior in different growth media, and faster heating as well as elevated steady state temperatures were observed over controls.

Inducing a Thermogenic Response to Cold Shock in E. coliMitesh Agrawal, Margo Clark, Robert Fee, Christina Graves, Atta Hassan, Scott Holmes, Monica Huynh, Gita Mahmoudabadi, Christian Mandrycky,

Debika Mitra, Amy Schwartz, Shadeah Suleiman and Siddharth Tantia

Georgia Institute of Technology, Atlanta, GA, USA

The synthesis of ATP during normal respiration is coupled with the flow of electrons through the electron transport chain to a terminal electron acceptor. In addition to this pathway, higher plants contain a cyanide resistant pathway involving alternative oxidase, which transfers electrons from the cytochrome pathway at ubiquinone. Whereas the free energy from the cytochrome pathway is used to generate ATP, free energy in alternative oxidasereactions is lost as heat.

Nelumbo nucifera (Sacred Lotus) is one plant species known to briefly maintain temperatures as high as 15 °C above ambient to volatilize insect attractants [1]. Two classes of AOX genes have been demonstrated to lead to increase in alternative pathway capacity and this is believed to be regulated by the changes in AOX mRNA levels [2]. We predict AOX over-expression and localization in the bacterial inner membrane will lead to increases in heat production.

DISCUSSION

[1]Grant, N et al. (2009). Two Cys or Not Two Cys? That is the Question: Alternative Oxidase in the Thermogenic Plant Sacred Lotus. Plant Physiology, 150: 987-995

[2]Maxwell, D, Yong, W, & McIntosh, L. (1999). The alternative oxidase lowers mitochondrial reactive oxygen production in plant cells. Proceedings of the National Academy of Sciences, 96, 8271-8276

Kumar, A., & Soll, Dieter. (1992). Arabidopsis alternative oxidase sustains escherichia coli respiration. Proc. Nati. Acad. Sci., 89.

Ingledew, W. John, & Poole, Robert. (1984). The Respiratory Chains of Escherichia Coli.Microbiological Reviews, 48(3), 222-271.

Albury, M, Elliott, C, & Moore, A. (2009). Towards a structural elucidation of the alternative oxidase in plants. PhysiologiaPlantarum, 137(4), 316-327

REFERENCES

ACKNOWLEDGEMENTSWe would like to thank our advisors Dr. Eric Gaucher, Dr. Joshua Weitz, Dr. Mark Styczynski, and our advisors Megan Cole, Richard In-Ho Joh, and Ryan Randall

RESULTSMODELING

• A. Liquid Culture Model• 800 mV electric potential drop of 4 electrons

generates 5.12 x 10-19 Joules

• 70% of electrons enter AOX pathway

• Power generated per cell is 1.6 x 10-13 Watt

• Temperature of system can be raised by 1 K in

40-400 min.

• B. Colony Based Model

General Assumptions

• Petri dish is insulated and kept at 288 K• Ambient temperature is 288 K• Media, constant convective coefficient for air• Aspect ratio : width of colony >> height of colony

B1. Analytical

METHODOLOGY

Biobrick submitted to the registry: BBA_K18000

Heat CharacterizationLiquid cultures were grown overnight at 37 °C, cold-shocked at 10 °C for one hour, acclimated to temperatures ranging from 15 °C to 25 °C, and then measured by a thermistor thermometer at regular intervals for 1-8 hours.

BACKGROUND

4 C – 25 C: hybB is active

37 C: hybB is inactive

hybB OmpA AOX1a

B2. Computational

288.000

288.030

288.060

288.065

Conduction in E. coli

Conduction in agarose

BC2: Conductive Flux

BC3: Continuity

BC1: Thermal Insulation

X=0

X=d

X=f

288.102288.100288.098288.096

288.150288.100288.050288.000

Temperature Profile In E. coli (K)

Temperature Profile in Agarose (K)

1.288])288[(2

)( 2. +−+

−= ecoliE T

khx

kQxT

288)(58.21)( +−−= fxxT Agarose

hybB mRFPOmpA

TARGETING SIGNAL VERIFICATION

Fig 1: hybB-RFP Fig 2: hybB-OmpA-RFP

Fig 1: Non-targeted expression of mRFP in E. coli.

Fig 2: OmpA targeted localization of mRFP in the periplasm

• Growth conditions and cold shock activity in E. Coli containing AOX could be explored and further optimized to increase heat generation• The post-translational metabolic regulation of AOX in thermogenic plants could be studied to improve understanding of how heating could be maintained for extended periods

FUTURE WORK

• The AOX1a gene was successfully cloned into E. Coli, expressed at low temperatures via the HybB cold shock promoter, and routed to the bacterial periplasm using a OmpA targeting sequence • Repeated experiments demonstrated faster rates of heating and higher steady state temperatures of 0.2 K over control strains. These effects were more pronounced in colder environments• Increases in temperature agreed closely with derived analytical models for heat output and heat transfer in liquid and solid media

E. Coli expressing RFP with the HybB cold shock promoter release significant amounts of heat during normal cell growth and respiration. E. Coli containing Bba_K410000 with heat generating AOX1a exhibited statistically significant increases in heating rate and higher steady state temperatures than controls. These results were more pronounced at lower temperatures.

Targeting signal verificationThe construct HybB-OmpA-RFP was cloned to verify OmpA targeting via fluorescence microscope.

Thermal Imaging Separate E. Coli cultures with RFP and AOX constructs were prepared and grown on plates, and imaged using a Ti40 IR Armaflex thermal camera over a period of 2 hours.

Measurement by an IR camera revealed a small thermal gradient produced by AOX1a expressing E. Coli (bottom right) in comparison to RFP E. Coli (top right) and agar media (left).

Plate 1 IR 11/2/2010 6:58:14 PM

Plate 1 IR 11/2/2010 7:21:24 PM

OD measurements at ‘on’ and ‘off’ temperatures suggested that local heating of the bacterial microenvironment could have an impact on cell vitality and growth rates.

A. Liquid Culture Model

B. Colony Based Model

1. Analytical

Solutions

2. Computational

Solution

Solving for Boundary Conditions

1. Temperature and flux at E. coli –agarose boundary are equal

2. Convective flux and conductive flux at the E. coli – Air boundary are equal

A 0.1K rise in temperature is predicted

T(x)Ecoli =−Q2k

x 2 + C1x + C2

T(x)Agarose = C3x + C4

A two dimensional temperature profile was created in COMSOL. Very similar to analytical solution, a 0.06K rise in temperature is expected.