ClonebotsClonebots University of California, …2008.igem.org/files/poster/UC_Berkeley.pdfTerry...
Transcript of ClonebotsClonebots University of California, …2008.igem.org/files/poster/UC_Berkeley.pdfTerry...
ClonebotsClonebotsOutsourcing Manufacturing to E. coliOutsourcing Manufacturing to E. coli
Undergraduates: Undergraduates: Molly Allen, Christie Brown, Aron Lau, Marlee Tichenor, Madhvi Venkatesh, Bing Xia Molly Allen, Christie Brown, Aron Lau, Marlee Tichenor, Madhvi Venkatesh, Bing Xia High School:High School: Cici Chen, Sherine Cheung Cici Chen, Sherine Cheung Teacher: Teacher: Dirk VandePol Dirk VandePol
Instructors: Instructors: Jin Huh, Terry Johnson, Megan Dueck, J. Christopher AndersonJin Huh, Terry Johnson, Megan Dueck, J. Christopher Anderson
University of California, BerkeleyUniversity of California, Berkeley iGEM08iGEM08
E xcis ionase Integrase Integration H ost Factor
xis int ihfa ihfb
attR1attL1 attL2 attR2 attB1attP1 attP2 attB2
Gateway Device
ccdA
Transform Entry Vector
AmpRCmR
Kill Device Gateway DeviceKill Device Gateway Device
ccdB xis intPts
Entry
ccdA
Ampicillin and Chloramphenicol Sensitive
Ampicillin and Chloramphenicol Sensitive
Contains Lethal ccdB
Survives
Transform selective cellsand Plate on
Cm/Amp
[H ]2 − (Ω(ΩH − ΩAHAH +Φ)[Φ)[H ]− ΦΩΦΩH = 0
Transfer function at Steady State Transfer function at Steady State
ΩH =kH PoPSHγγ mRNA,HA,H Hc
Holin ProductionHolin Production
ΩAHAH =kAHAH PoPSAHAHγγ mRNA,AHA,AH Hc
Antiholin ProductionAntiholin Production
Φ = ku + γkcHc
Holin/Antiholin InteractionHolin/Antiholin Interaction
Modeling shows that antiholin buffers against premature lysis
ΩAH = 300
ΩAH = 3
0.20 .40 .60 .81
1.21 .41 .61 .82
10 100 1000 10000
Φ = 400
H/H
criti
cal Critical Holin Concentration
ΩH
ΩAH = 300
ΩAH = 3
0.20 .40 .60 .81
1.21 .41 .61 .82
Φ = 40
H/H
criti
cal Critical Holin Concentration
R S Rz
PBAD Pcon
Lysozym e H olin A ntiholin
PoPS LysisLysis
Device
Induced with Arabinose
Without Arabinose
Lysozyme
Holin & AntiholinDimer
Lambda lysis device responses to arabinose
Desired product Co-transformation
0
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OD
■ Purpose: to give form and forum to questions surrounding synthetic biology ■ Methodology: • Giving larger context to synthetic biology research • Raising question of what defines the good life • Calling for collaboration between stakeholders ■ Production:
In an effort to optimize the manufacturing of parts, we have designed - a collection of devices and strains that aid in the synthesis and analysis of new parts. Building engineered biological systems requires cumber-some laboratory protocols that significantly impede the advancement of our field. However, there are some unit operations that can be cost effectively auto-mated at scale in the laboratory such as small volume liquid transfers, fluores-cence measurements, and heating/cooling steps. If we can reduce all synthesis and analysis methodology to these simple operations, we can readily automate many aspects of synthetic biology research - a cost-effective, BioCAD-friendly approach to large-scale projects. This project is an effort to solve these basic technical problems of synthetic biology in vivo. We successfully constructed two devices designed to automate sythetic biology: a Gateway cloning device and a genetic self-lysis device.
Miniprep
Transformation
Biochemical Manipulation
Gateway Device
Self-Lysis Device
Phagemid Device
Layered Standard Assembly
Two-Antibiotic Assembly
Robots ■ Precise & accurate liquid handling ■ High-throughput
■ Reagent-free ■ Highly efficient enzymatic reactions
Robots and working together
Plasmid-Based GatewayPlasmid-Based Gateway
Self Lysis-Based GatewaySelf Lysis-Based Gateway Phagemid-Based Gateway Phagemid-Based Gateway
ccdA
Kill Device Gateway Device
ccdB xis intR S Rz
Self-Lysis Device
CmR
Transform Entry Vector
ccdA
AdvisorsMegan DueckJin HuhTerry Johnson
Human Practices AdvisorsGaymon BennettPaul RabinowAnthony Stavrianakis
Logo Artwork & DesignKarin Wu
SupportKevin CostaKate SpohrInvitrogen
Ars Synthetica CollaboratorsElizabeth HaNoah WittmanAdrian Van Allen
Berkeley iGEM Advisory GroupChris AndersonAdam ArkinJohn DueberJay KeaslingSusan Marqusee
Slideshow InterfaceVuvox.com
Acknowledgements
1. Gateway device successfully replaced biochemical manipulation in vivo 2. Lysis device further replaced minipreps when it was installed on the plasmid with the gateway device3. Further study will replace both minipreps and transformations with phagemid-based gateway
coi repL pacApacPBAD
Phagemid DeviceWill DeLoache
PoPS Phage ParticlePhagemid
Device ‡
‡ Constructed by Will DeLoache
coi repL pacAPBAD
xis intPtspac ~~
P1 Lysogen Device
Entry
P1 Lysogen Device
Entry
P1 Lysogen Device
Phagemid Device Gateway Device
UC Berkeley iGEM 2008Clonebots
We Thank the Generous Support of:
F O U N D A T I O N
SynBERC
Restriction/ligationless part/vector matching in vivo
Replacing biochemical manipulation
Procedure
Outgrow in Cm/Amp/Spec
Co-transformation is eliminated by diluting the intermediate minipreps
Miniprep
Lysis Device is installed along with gateway device
Replacing biochemical manipulations and minipreps
Successful gateway and lysis without co-transformation
Procedure
Holin multimers allows lysozyme to reach periplasm
Phagemid Device is installed along with gateway device
Replacing biochemicals, minipreps, and transformation
Procedure
1 2 3 4 5 6
A
B
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1 2 3 4 5 6
Colonies spotted on Cm/Amp
Desired product Co-transformation
A
B
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D
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1 2 3 4 5 6 1 2 3 4 5 6Desired product Co-transformation
Colonies spotted on Spec
Induce Lysis
Outgrow at 37°Infect
Induce phage formation
Re-Infect cells of choice
Lysozyme degrades peptidoglycan layer
Holin pore
AbstractAbstract
Clonebots
Clonebots
ConclusionConclusion
Human PracticesHuman Practices
GatewayReaction
Concentrated 1/20 Dilution
Outer Membrane
Periplasm
Inner Membrane
1 hour after induction at mid-log Lysis device induced at mid-log
Arabinose concentration (micromolar)
ClonebotsConventional
Clonebots
SpecR
Colonies spotted on Cm/Amp Colonies spotted on Spec
Mayco-transform
Colonies spotted on Cm/Amp Colonies spotted on Spec
Ampicillin and Chloramphenicol Sensitive
Ampicillin and Chloramphenicol Sensitive
Contains Lethal ccdB
Survives
Mayco-transform
Entry
SpecR
Outgrow in Cm/Amp/Spec
AmpRTransform
selective cells (GenR)
and Plate on Cm/Amp/Gen
Holin production
Gen sensitive
SpecR
AmpR
KanR
CamR