AQUARIUM:ProgrammableWetlabTileli Amimeur,Yaoyu Yang,DavidYounger,MilesGander,ChrisTakahashi,AnthonyLaMarca,

JeffScofield,DieterFox,JeffBilmes,EricKlavinsUniversityofWashington,Seattle,WA

ReproducibilityIssuesinWetlabs

AquariumApproach

ProtocolsWith‘Krill’ WorkflowOptimization

Metacols With‘Oyster’

Training

CurrentandFutureDirections

Astudyin2012foundthatonlysixoffifty-threeselected‘landmark’papersincancerbiologywerereproducible.

Anotherfoundthatretractions,duetoerrorsorotherwise,areincreasinglyfrequent.Engineersattemptingtobuildonscientificresultsinthelifesciencesinordertoexplorethestudyofengineeringlifeitselfarefindingthattheseresultsdonothold.

Aquariumhastwoseparateserversforprotocoldevelopmentandin-labprotocolexecution.TheRehearsalserverisusedfortestingofprotocols/metacols only.TheProductionserverhoststheversionofAquariumthatgetsruninthelabbytechniciansandholdsanaccuraterepresentationofthelab’sinventory.

InordertoensurereproducibilityandconfidenceinAquariumlabtechnicians,Aquariumitselfprovidesstandardtrainingforallincomingtechnicians.Alibraryofprotocolsexistsfornewtechlaborientationandtrainingandprovideslabmanagerswithawayofmonitoringandmanagingtechnicians’skills.

CurrentlywehaveaworkingbuildpipelinelibraryforE.coli andYeast,andareintheprocessofoptimizingasmanyoftheseprotocolsaspossible.

WearecollaboratingwiththeIntelCenterforPervasiveComputingtoexpandAquariumtoadd:

•Toolsforprotocoldevelopment•Objecttracking– knowingtheexactlocationofallobjectsinthelabatalltimes•Automatedlabassistance– protocolprogresstrackinganderrorchecking,locationassistance,physicalroboticassistant

FutureGoals include:

•ExecutablecodeasMaterials/Methods:Reproducibilitystandard•Networkedcloudlab•Self-bootinglab

Researcher

LabTechnicians LogDatabase Analysis

Interpreter(touchscreen)

InventoryManager

(foranykindofsample)

Squid

Manta

LabequipmentanddevicesaremanagedbytheSquiddevicemanager.

Mantahandlesvideorecordingandother

sensors.

ProtocolsandMetacols arewritteninstandard languagesandstoredin

versioncontrolledGithubrepositories. Imagesarekept inthe

Anglerimageserver.

Researcher Design

Build / Test

Formal• Ruby-based protocol specification• Control, branching, etc.• Inventory interactions• Transfers of liquids between containers• Interactions with lab equipment

Informal• Textual descriptions of steps• Photos/cartoons/drawings

KrillProtocolLanguage

LabTechnicians

LogDatabase

ProductionServer

RehearsalServer

Aquarium istheKlavinslabapproachtoresolvingreproducibilityissuesinthewetlab.ItaimstoseparatetheDesignportionofsyntheticbiologyfromtheBuildandTestportions.Aquariumcomeswithalanguageforsemi-formalprotocoldescriptions(Krill)andaformalprocesscontrollanguage(Oyster)fordirectingtheflowofprotocols.ThiswasusedtobuildaversioncontrolledGithub libraryofprotocolsforstrainbuilding/testing.

TheresearcherusesAquariumtoselectdesiredprotocols/metacols fromtheGithub repoandschedulesthemtobeexecutedbyatrainedlabtechnician.Atexecutiontime,theInterpreterdisplaystheprotocolsasaseriesofstepsonatouchscreenforthetechniciantorunthrough.Dataisloggedintoadatabaseaccessiblebytheresearcher.

error

!error

primer_ids

plate_id

• AMetacol specifiesprocesscontrolandisawiringofprotocolsandtheirtransitionsintheformofPetriNets

• Places arejobsassociatedwith• aprotocol• parameters• ausergroup• adesiredstart/stoptime

• Transitions haveconditions• errors/cancelations• inventorystatus• useravailability

• Inputs andoutputs canbewired

argumentgels : sample(“Gel slice”) arrayfragments : sample(“Fragment”) array

end

place purify_gelprotocol : ”purify_gel.pl"argument

gel : gelendgroup : aquarium_usermarked : truestart : now()window : minutes(30)

end

wire (purify_gel,”fragment_stock_ids") => (gibson_prepare,"fragments")

# forward transitionstransition [purify_gel] => [dpnI_digest] when !error(0) && completed(0) endtransition [dpnI_digest] => [pcr_purification] when !error(0) && completed(0) endtransition [pcr_purification] => [gibson_prepare] when !error(0) && completed(0) endtransition [gibson_prepare] => [stock_fragment] when !error(0) && completed(0) endtransition [stock_fragment] => [] when !error(0) && completed(0) end

OysterMetacol Language

PCR/Gels PurifyFragments Gibson Transform Miniprep Sequence

PipelineforBuildingaNewStrain

Transform

Plate

Incubate

Image

ElectrocompetentcellsLiquidMedia

SolidMediaPlate

InventorySamples

InventoryObjects

AssociatedWatchersInordertokeepthepipelineflowing,allnecessaryinputparametersneedtobeavailable(dependenciesneedtoberesolved).Wehavewhatiscalledawatcherthatmonitorskeyinventorysamplesandobjects andfiresrestockingprotocolswhenanitemrunslow.

QualityChecks

Optimization

Buildinganewlogicfunctionintoacellinvolvessteppingthroughacomplexsetofhighlyvariablewetlab cloningprotocols.Amainportionofthebuildpipelineisshownabove.OneplaceforhighvariabilityistheTransform step.Transformationinvolveselectroshockingthecellsenoughtotemporarilyweakentheircellwallstoallowacceptanceofthenewplasmid,whichhasthedesiredlogicfunctionencodedontoit.Thesetofprotocolsnecessaryforexecutingandtestingthetransformationareshownhereontheright.

Inordertominimizevariabilityinthebuildprocess,weintroducequalitycheckstotestcrucialpointsinthepipeline.

Therearenumerousplacesinthebuildprotocolswhereparameterscanbeoptimized.Forexample,thetemperatureatwhichcertainchemicalsareadded,thelengthoftimeatwhichthey’remixed,theconcentrationofplasmidmixturetogrowup.Wecanteaseouttheidealvaluesfortheseparametersbyrunningvariationsofprotocolsandextractingthevaluesthatgiveusthebestresults.Inaddition,withAquarium’sdatacollectionmethods,wecanconceivablyrevealexperimentalrelationshipsbetweenparametersthatwedidn’tknowexistedbefore.

Waitforlowcompcellstock

Makecompcells

QC:Testcompcells

Discardbatch

Electrocompetentcells

PassFail

Inadditiontotechtraining,we’veusedAquariumtoteachsyntheticbiologytograduatestudentsfromallbackgrounds.Infiveweeksstudentslearnedbasiclabskills,molecularcloning,Gibsonassembly,andgeneknockoutswithverylittleexternalassistanceoutsideofAquarium.

Protocol/ObjectTracking

RoboticAssistance

Evenwhenaresultisvalid,themethodsarepoorlyexplained,purposefullynotexplained,orsimplyburiedinsomeresearcher’snotes.Sciencesuffersfromagrowingneedtobeabletospecify,aspreciselyaspossible,themeansforobtainingaresult.Astandardlanguageforreproduciblemethods.