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 Gateway Reaction SpecR Two-Antibiotic AmpR Assembly KanR CamR
Gateway Reaction: Transfer of Sequences Flanked by att Sites attR1 attB1 attR2 attB2 attL1 attP1 attL2 attP2
Biologically Encoding Unit Operations for Transferring Parts Biochemical Manipulation Gateway Device Miniprep Self-Lysis Device Transformation Phagemid Device
Gateway Device: Performing LR Gateway in vivo Gateway attL attB Device attR attP Temperature Sensitive Promoter P ts xis int ihfa ihfb Excisionase Integrase Integration Host Factor
Gateway Device Replaces Biochemical Manipulation P ts ccdB xis int Kill Device Gateway Device Chloramphicol Ampicillin Resistance Resistance ccdA
Gateway Device Replaces Biochemical Manipulation ccdA
Gateway Device Replaces Biochemical Manipulation Ampicillin and Ampicillin and Chloramphenicol Contains Chloramphenicol Sensitive Sensitive Lethal ccdB Survives
Plasmid Based Gateway in vivo is very efficient Concentrated 1/20 Dilution Desired product Co-transformation Desired product Co-transformation 1 2 3 4 5 6 1 2 3 4 5 6 1 2 3 4 5 6 1 2 3 4 5 6 A A B B C C D D E E F F G G H H Colonies spotted on Cm/Amp Colonies spotted on Spec Colonies spotted on Cm/Amp 1.4% ± 0.8% co-transformation 0.8% ± 0.7% co-transformation
Lysis Device: Alternative to Miniprep Lysis PoPS Lysis Device vs.
Natural Lambda Phage Lysis Pathway at Work Outer Membrane Periplasm Inner Membrane Lysozyme Holin Antiholin
Lambda Phage Lysis: Governing Equations
Lambda Phage Lysis: Steady-State Solution & Parameters Anti-Holin Production 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 Lysis PoPS Lysis Device Inducible Promoter Constitutive Promoter P BAD P con R S Rz Lysozyme Holin Antiholin
Arabinose-Induced Lysis Device at Work 1 hour after induction at mid-log Induced with Without Arabinose Arabinose
Lysis Device Results Show Critical Arabinose Concentration
Lysis Device Replaces Miniprep in Plasmid Based Gateway R S Rz ccdB xis int Self-Lysis Device Kill Device Gateway Device Chloramphicol Ampicillin Resistance Resistance ccdA
Lysis Device Replaces Miniprep in Plasmid Based Gateway ccdA
Lysis Device Replaces Miniprep in Plasmid Based Gateway Survives
Successful use of Lysis Device for Gateway in vivo Gateway Device + Lysis Device in Assembly Vector Cam/Amp Spec
Phagemid Device: Alternative to Miniprep and Transformation Phagemid PoPS P1 Particles Device* vs. * Phagemid Device built by Will Deloache
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
Acknowledgements Logo Artwork & Human Practices Advisors Advisors Design Gaymon Bennett Megan Dueck Karin Wu Paul Rabinow Jin Huh Anthony Stavrianakis Terry Johnson Support Ars Synthetica Collaborators Slideshow Interface Kevin Costa Elizabeth Ha Vuvox.com Kate Spohr Noah Wittman Invitrogen, Inc. Adrian Van Allen Berkeley iGEM Advisory Group Chris Anderson Adam Arkin John Dueber Jay Keasling Susan Marqusee
Recommend
More recommend
