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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.