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

0.2

0.4

0.6

0.8

1

1.2

0 1 10 100 1000

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

C

D

E

F

G

H

1 2 3 4 5 6

Colonies spotted on Cm/Amp

Desired product Co-transformation

A

B

C

D

E

F

G

H

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