Molly Allen Christie Brown Cici Chen Sherine Cheung Aron Lau - - PowerPoint PPT Presentation

Molly Allen  Christie Brown  Cici Chen  Sherine Cheung  Aron Lau

 Marlee Tichenor  Madhvi Venkatesh  Bing Xia  Dirk VandePol

Limitations of Current Methods of Parts Manufacture

Improve Efficiency by Using Robots and in vivo Reactions

An Automatable Scheme with Reactions that can be done in vivo

Layered Standard Assembly

Layered Standard Assembly

Two-Antibiotic Assembly

Gateway Reaction

SpecR AmpR KanR CamR

Gateway Reaction: Transfer of Sequences Flanked by att Sites

attR1 attL1 attL2 attR2 attB1 attP1 attP2 attB2

Biologically Encoding Unit Operations for Transferring Parts

Miniprep Transformation Biochemical Manipulation

Gateway Device Self-Lysis Device Phagemid Device

Gateway Device: Performing LR Gateway in vivo

Excisionase Integrase Integration Host Factor Temperature Sensitive Promoter

attL attR attB attP Gateway Device

xis int ihfa ihfb

Pts

Gateway Device Replaces Biochemical Manipulation

ccdA

Chloramphicol Resistance Ampicillin Resistance Kill Device Gateway Device

ccdB xis int

Pts

Gateway Device Replaces Biochemical Manipulation

ccdA

Ampicillin and Chloramphenicol Sensitive Contains Lethal ccdB Ampicillin and Chloramphenicol Sensitive Survives

Gateway Device Replaces Biochemical Manipulation

Plasmid Based Gateway in vivo is very efficient

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 Colonies spotted on Spec

Concentrated

1.4% ± 0.8% co-transformation 0.8% ± 0.7% co-transformation

A B C D E F G H 1 2 3 4 5 6 1 2 3 4 5 6

Desired product Co-transformation

1/20 Dilution

Colonies spotted on Cm/Amp

Lysis Device: Alternative to Miniprep PoPS Lysis Lysis Device vs.

Natural Lambda Phage Lysis Pathway at Work

Inner Membrane Outer Membrane Periplasm Lysozyme Holin Antiholin

Lambda Phage Lysis: Governing Equations

Lambda Phage Lysis: Steady-State Solution & Parameters

Holin Production

Anti-Holin Production Holin / Anti-Holin Interaction

Lambda Phage Lysis: Reaching the Critical Holin Concentration

Critical Holin Concentration

Lambda Phage Lysis: Reaching the Critical Holin Concentration

Critical Holin Concentration

Lysis Device: Alternative to Miniprep PoPS Lysis Lysis Device

R S Rz

PBAD Pcon

Lysozyme Holin Antiholin Inducible Promoter Constitutive Promoter

Arabinose-Induced Lysis Device at Work

Induced with Arabinose Without Arabinose 1 hour after induction at mid-log

Lysis Device Results Show Critical Arabinose Concentration

Lysis Device Replaces Miniprep in Plasmid Based Gateway

ccdA

Chloramphicol Resistance Ampicillin Resistance Kill Device Gateway Device

ccdB xis int R S Rz

Self-Lysis Device

ccdA

Lysis Device Replaces Miniprep in Plasmid Based Gateway

Survives

Lysis Device Replaces Miniprep in Plasmid Based Gateway

Successful use of Lysis Device for Gateway in vivo

Gateway Device + Lysis Device in Assembly Vector Cam/Amp Spec

PoPS P1 Particles Phagemid Device* vs.

* Phagemid Device built by Will Deloache

Phagemid Device: Alternative to Miniprep and Transformation

Phagemid Device Replaces Minipreps and Transformation

Phagemid Device Gateway Device

Phagemid Device Replaces Minipreps and Transformation

Conclusions

• Simplified biochemical manipulations using

Gateway device

• Replaced miniprep in Gateway protocol with

lysis device

• Further study will replace both minipreps

and transformations with phagemid-based gateway

• Our devices simplified unit operations used

in the Gateway protocol

• Easy to automate
• BioCAD friendly
• Less room for error

Human Practices on Berkeley’s 2008 iGEM Team

What Can This New Form Look Like?

Links

• www.ars-synthetica.net
• blogs.coe.berkeley.edu/igem

Advisors Megan Dueck Jin Huh Terry Johnson

Acknowledgements

Berkeley iGEM Advisory Group Chris Anderson Adam Arkin John Dueber Jay Keasling Susan Marqusee Support Kevin Costa Kate Spohr Invitrogen, Inc. Logo Artwork & Design Karin Wu Human Practices Advisors Gaymon Bennett Paul Rabinow Anthony Stavrianakis Ars Synthetica Collaborators Elizabeth Ha Noah Wittman Adrian Van Allen Slideshow Interface Vuvox.com