bactricity

MFCs can be thought of as bacteria-powered renewable batteries. They use bacteria as a catalyst to convert chemical energy into electrical energy. Typically, this chemical energy comes in the form of degradable organic matter. Under anaerobic conditions,

some bacteria break down this matter, releasing electrons in a process called an oxidation reaction. In a fuel cell, removal of oxygen from the cells’ environment means that electron receptors are separated. The electrons must be transmitted from the bacteria to an anode, from which they move to a separate compartmentwhere they can complete the reaction by combining with protons and oxygen to make water, known as reduction. The net result is an electrical current.

We use MFCs to monitor the electrical current output of S. oneidensis. This organism’s ability to produce current is directly affected by its gene expression. By coupling environmental stimuli to gene expression, we have devised not only an innovative biosensor, but also a unique readout of molecular processes.

We were able to create a chemically inducible system in which LacI represses the current production of mtrB in knock-out S. oneidensis. This system can be induced with IPTG, an analogue of lactose.

We also created a new thermoinducible cI system with a GFP reporter.

We tested both the Lac and cI systems in E. coli with a GFP reporter.

The current production of mtrB knockout S. oneidensis is approximately 20-25% of that of wildtype S. oneidensis. This is consistent with published data1

In this experiment, S. Oneidensis ΔmtrB mutants with plasmid lac-inducible mtrB were tested with or without the addition of IPTG. The resulting exponential growth in current production for cells receiving the induction signal seems to indicate successful production of the protein mtrB. Although IPTG typically turns on lacI systems in shorter time frames, the nutrient poor environment of a microbial fuel cell may contribute to the lengthy span between receiving an induction signal and producing the target protein.

The graph shows that the wt E.coli and wt S.oneidensis can work together to produce current using lactose, relative to the Shewanella-lactate control. The delay can be attributed to the time it takes for E. coli to break down lactose into lactate. Current production by the ecology relies on both members and can be manipulated in either.

Induction of GFP expression was observed at both 2 and 4 hours after moving samples to 40 ºC. We hypothesize that elevated temperature affects the GFP expression (e.g. by disrupting protein folding).

GFP levels increased in response to IPTG induction after 2 and 4 hours. This shows that the lac QPI was successfully induced.

Our parts

cOntrOlling current

testing inducible systems

bacterial biosensors with electrical outputThilini Ariyawansa, Joy Ding, Dan Gong, Meng Xiao He, Amy Li, Erica Lin, Lauren Schumacher, Anna Marie Wagner, Sam Workman

cI system

lac systemwildtype vs. ΔmtrB

IPTG-inducible strains

engineered ecologies E. coli co-cultures

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Thermoinduction of Temperature Sensitive cI Systems

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GFP - BBa_K098981(negative control)

GFP + BBa_K098991(positive control)

ThermoinduciblecI System (high)BBa_K098988

Thermoinducible cI system (low)BBa_K098987

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IPTG Inducible systemBBa_K098982

• SuccessfullyclonedmtrB,agenetoxictoE. coli

• Implemented and designed hardware and softwareutilities for microbial fuel cells

• Transducedchemicalsignalintoelectricaloutput

• Demonstrated, through co-culture experiments, thepossibility of combining induction in a wide range of E. coli with electrical output from S. oneidensis

• Createdmeanstocoupleelectricaloutputwithexistingbiosensors such as:

- Mercury

- Light

- Arsenic

• BiobrickedanewthermoinducibleQPI

Our work in creating a system of inducible electrical output in S. oneidensis has laid the foundations for different methods of implementing a bacteria-computer interface that combines the sensitivity and versatility of bacteria with the speed and analytical abilities of electricity and computers.

cOnclusiOns

1.Bretschger,O.,Obraztsove,A.,Sturm,C.,Chang,I.,Gorby,Y.,Reed,S.,Culley,D.,Reardon,C.,Barua,S.,Romine,M.,Zhou,J.,Beliaev,A.,Bouhenni,R.,Saffrini,D.,Mansfeld,F.,Kim,B.,Fredrickson,J.,andNealson,K.(2007).CurrentProductionandMetalOxideReductionby Shewanella oneidensis MR-1 Wild Type and Mutants. Appl. Environ. Microbiol. 73,7003-7012.

2.Saffrini,Daad.(2008)Personalcorrespondence.

AlainViel,OriannaBretschger,DaadSaffarini,HelenWhite,RemyChait,NatalieFarny,ChristinaAgapakis,JasonLohmueller,KimdeMora,ColleenHansel,PeterGirguis,ChristopherMarx,GeorgeChurch,JageshV.Shah,PamSilver,TamaraBrenner,DianneNewman,HarvardBiolabs,LauraCroal,MargaretRomine,MayraMollinedo,SamanthaReed,JizhongZhou,HaichunChung,RenaHill,JeffTabor,RandyRettberg,JimFredrickson,ChadSaltikov,HowardHughesMedicalInstitute.

references

acknOwledgements

cymA mtrAe-

e-e-e-

mtrCOmcA

mtrB

Shewanellaoneidensisisametabolicallyversatilebacterium.Whengrownanaerobically,S. oneidensis can metabolize lactate and use the anode of a microbial fuel cell as a terminal electron acceptor, thereby producing an electrical current. We sought to engineer S. oneidensis to report variations in environmental conditions through changes in current production. A previous study has shown that S. oneidensis mutants deficient in the mtrB gene produce less current than the wildtype strain, and that current production in these mutants can be restored by the addition of exogenous mtrB . 1 We attempted to control current production in mtrB knockouts by introducing mtrB on lactose, tetracycline, and heat-inducible BioBrick based plasmids. These novel biosensors integrate directly with electrical circuits, paving the way for the development of automated, biological measurement and reporter systems.

intrOductiOn

mfcs

microbial fuel cells

lac inducible systemheat inducible cI QPI

summary of results

implications

StrongPromoter(BBa J23114)

Terminator(BBa B0014)

cI Promoter(BBa B0014)

RBS(Part of BBa E0240)

Terminator(Part of BBa E0240)

cIts Repressor GFP Part of BBa E0240

RBS(BBa B0034)

StrongPromoter(BBa J23114)

Terminators(BBa B0010) (BBa B0012)

lacI RegulatedPromoter(BBa R0011)

RBS(BBa B0034)

DoubleTerminator

(BBa B0014)

lacI + LVA

lac QPI (BBa Q04121)

mtrB

RBS(BBa B0034)

Xbal

EcoRI

SpeI

PstI

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