Engineering bacteria to solve the Burnt Pancake Problem

1

EngineeringbacteriatosolvetheBurntPancakeProblem

KarmellaAHaynes

1*

,MarianLBroderick

4

,AdamDBrown

3

,TrevorLButner

3

,JamesODickson

2

,LanceHarden

2

,LaneHHeard

3,6

,EricLJessen

3

,KellyJMalloy

3

,BradJOgden

2

,SabriyaRosemond

1,5

,SamanthaSimpson

1

,ErinZwack

1

,MalcolmCampbell

1

,ToddTEckdahl

3

,LaurieJHeyer

2

,JeffreyLPoet

4



1

DavidsonCollege,DepartmentofBiology,Davidson,NC28036,USA

2

DavidsonCollege,DepartmentofMathematics,Davidson,NC28036,USA

3

MissouriWesternStateUniversity,DepartmentofBiology,St.Joseph,MO64507,USA

4

MissouriWesternStateUniversity,DepartmentofComputerScience,MathandPhysics,St.Joseph,MO64507,USA

5

HamptonUniversity,BiologyDepartment,Hampton,VA23668,USA

6

CentralHighSchool,St.Joseph,MO64506,USA*CorrespondingAuthor:kahaynes@davidson.edu,orkarmella@gmail.comKAH–kahaynes@davidson.eduMLB–mlb8172@missouriwestern.eduADB–adb1207@missouriwestern.eduTLB–tlb3238@missouriwestern.eduJOD–jidickson@davidson.eduWLH–laharden@davidson.eduLHH–axenmoon@hotmail.comELJ–elj0152@missouriwestern.eduKJM–kjm6938@stowers-institute.orgBJO–bjo3000@missouriwestern.eduSR–sabriyarosemond@yahoo.comSS–sasimpson@davidson.eduEZ–erzwack@davidson.eduAMC–macampbell@davidson.eduTTE–eckdahl@missouriwestern.eduLJH–laheyer@davidson.eduJLP–poet@missouriwestern.edu

2

Abstract



Background:

WeinvestigatedthepossibilityofexecutingDNA-basedcomputationinlivingcellsbyengineering

Escherichiacoli

toaddressaclassicmathematicalpuzzlecalledtheBurntPancakeProblem(BPP).TheBPPissolvedbysortingastackofdistinctobjects(pancakes)intoproperorderandorientationusingtheminimumnumberofmanipulations.Eachmanipulationreversestheorderandorientationofoneormoreadjacentobjectsinthestack.Wehavedesignedasystemthatusessite-specificDNArecombinationtomediateinversionsofgeneticelementsthatrepresentpancakeswithinplasmidDNA.

Results:

Inversions(or“flips”)oftheDNAfragmentpancakesaredrivenbythe

Salmonellatyphimurium

Hin/

hix

DNArecombinasesystemthatwereconstitutedasacollectionofmodulargeneticelementsforusein

E.coli

.OursystemsortsDNAsegmentsbyinversionstoproducedifferentpermutationsofapromoterandatetracyclineresistancecodingregion;

E.coli

cellsbecomeantibioticresistantwhenthesegmentsareproperlysorted.HinrecombinasecanmediateallpossibleinversionoperationsonadjacentflippableDNAfragments.Mathematicalmodelingpredictsthatthesystemreachesequilibriumafterveryfewflips,whereequalnumbersofpermutationsarerandomlysortedandunsorted.SemiquantitativePCRanalysisof

invivo

flippingsuggeststhatinversionproductsaccumulateonatimescaleofhoursordaysratherthanminutes.

Conclusions:

TheHin/

hix

systemisaproof-of-conceptdemonstrationof

invivo

computationwiththepotentialtobescaleduptoaccommodatelargerandmorechallengingproblems.Hin/

hix

mayprovideaflexiblenewtoolformanipulatingtransgenicDNA

invivo

.

3

Background

ThetremendousinformationstoragecapacityofDNAandtheremarkableefficiencyofbiomolecularself-assemblyhaveinspiredresearcherstodesignbiologicalcomputers.Previousworkhascreatedproof-of-conceptbiologicalcomputersbasedon

invitro

self-assemblyofDNA[1]andprotein-DNAinteractions[2-4].Thusfar,biologicalcomputingislimitedtononlivingdevicesthathavenotutilizedtheparallelprocessingpoweraffordedbyDNAreplicationandcellulardivision.Inordertodemonstratethefeasibilityof

invivo

computing,weprogrammed

Escherichiacoli

toaddressaclassicmathematicalchallengecalledtheBurntPancakeProblem(BPP)[5].TheBPPcanbevisualizedasastackofdifferentsizedpancakes,eachhavingoneburntsideandonegoldenside,arrangedinanarbitraryorder.Thestackmustbesortedbyflippingindividualpancakesorsubsetsofadjacentpancakesuntilthepancakesareorderedfromsmallesttolargestwitheachpancakeorientedgoldensideup(seeexampleinFig.1).TheBPPisalsoknownassortingbyreversalssinceboththeorderandorientationofthepancakesarechangedwhentheyareflipped.TheBPPisasubjectofinterestinbasicmathematicalandcomputationalresearch(

e.g.

,[5]).OfparticularinteresttobiologistsistheapplicationoftheBPPtocomparativegenomics.Theevolutionarydistancebetweensyntenicgenomesoftwoorganismsisdeterminedbytheminimumnumberofreversalsrequiredtosortregionsofgenesinoneorganismtomatchtheorderandorientationoforthologousgenesintheotherorganism[6-8].Thetotalnumberofpossiblearrangementsof

n

objects(

i.e.

,pancakesorgenes)is2

n

(

n

!),anexponentialincreaseinarrangementsasthestackofobjects(pancakesorgenes)becomeslarger.PlasmidDNAreplicationandexponentialcellgrowthinbacteriaareinexpensive,occupymuchlessspacethancomputerhardware,andmaintainparitywiththeexponentialincreaseinBPParrangements.



Therefore,solvingtheBPPinlivingcellsoffersuniqueadvantagesoverusingcomputerhardware.