Taming Biology as an Engineering Discipline

1. Taming Biology as an Engineering Discipline Tom Knight tk@ginkgobioworks.com DARPA Flexible Microbial Manufacturing Meeting February 23, 2011 Simplicity is the greatest sophistication. -- Leonardo da Vinci

2. Science and Engineering Science & Systems Biology of natural organisms Knowledge & understanding Excellent models Revised knowledge and new techniques Parts Repository De novo DNA synthesis Engineering & Synthetic Biology Metabolic Modules Engineered organisms

3. Key Engineering Ideas • Removal of unnecessary complexity • Simplification with standard reusable parts • Hierarchical design • Modular construction • Standards of construction and measurement • Design rules • Carefully defined interfaces • Modeling at the correct level • Isolation of unrelated parts • Flexibility

4. Challenges to engineering from biology • How do we predict and control the behavior of evolving systems? • How do we ensure safety of systems we build when they inherently self-replicate • How do we construct systems that are as robust to local imperfection and failure as existing living systems

5. Abstraction model catabolism anabolism Constructed complexity Real world complexity Small core of standard parts Design information

6. Design, Build, Test Loop

7. Design, Build, Test Loop

8. Build • Gene synthesis on exponential improvement • Standard parts are understood and available • Biobrick standard partsregistry.org • IGEM competition igem.org • Assembly of standard parts is routine • Biobrick standard assembly (log number of parts) • Rad, Gibson assembly (faster parallel assembly) • Speed and accuracy still need improvement

9. Tecan EVO Ginkgo Fabrication Facility Tecan EVO Biomek FX QiagenBiorobot 8000 Biomek FX Biomek FX Biomicrolab XL-20

10. Test • We can only easily measure a small number of system properties • Need comprehensive measurement of • Protein abundance • Metabolites • Transcripts • Need standard conditions -- turbidostats • Need single cell measurements • Routine, user-friendly, standardized mass-spec and analysis of its results • Tagging of proteins for analysis • Design of organisms to be easy to measure • A pre-condition for good design tools

11. Design • Limited by a lack of good measurements • Limited by the complexity of existing cells • Plan: • Develop a simplified organism with well understood, documented, measured metabolism • Design the organism to be easy to measure • Refactor the genome to standardize parts, re-arrange for modular replacement • Develop metabolic modules for flexible insertion of new capabilities

12. Choosing an organism:Mesoplasma florum • Isolation from the flower of a lemon tree, Florida (McCoy84) • Safe BSL-1 organism -- an insect commensal • Not a human or plant or animal pathogen • No growth at 37C • Fast growing • 40 minute doubling vs. • 16 hour doubling in M. genitalium • Convenient to work with • Facultative anaerobe • Near defined medium • Small genome: • 793,224 bp • 682 coding regions

13. G. Fournier 02/23/04 PTS II System Mfl519, Mfl565 sucrose trehalose xylose beta-glucoside glucose unknown ribose ABC transporter Mfl516, Mfl527, Mfl187 Mfl500 Mfl669 Mfl009, Mfl033, Mfl318, Mfl312 fructose Mfl214, Mfl187 Mfl619, Mfl431, Mfl426 ATP Synthase Complex Mfl181 Mfl497 Mfl515, Mfl526 Mfl499 Mfl317?, Mfl313? Mfl009, Mfl011, Mfl012, Mfl425, Mfl615, Mfl034, Mfl617, Mfl430, Mfl313? ? Mfl109, Mfl110, Mfl111, Mfl112, Mfl113, Mfl114, Mfl115, Mfl116 Mfl666, Mfl667, Mfl668 glucose-6-phosphate chitin degradation Mfl347, Mfl558 ATP ADP sn-glycerol-3-phosphate ABC transporter Pentose-Phosphate Pathway Glycolysis Mfl023, Mfl024, Mfl025, Mfl026 L-lactate, acetate Mfl223, Mfl640, Mfl642, Mfl105, Mfl349 glyceraldehyde-3-phosphate Mfl254, Mfl180, Mfl514, Mfl174, Mfl644, Mfl200, Mfl504, Mfl578, Mfl577, Mfl502, Mfl120, Mfl468, Mfl175, Mfl259 Mfl039, Mfl040, Mfl041, Mfl042, Mfl043, Mfl044, Mfl596, Mfl281 Lipid Synthesis unknown substrate transporters Mfl384, Mfl593, Mfl046, Mfl052 fatty acid/lipid transporter ribose-5-phosphate acetyl-CoA Mfl230, Mfl382, Mfl286, Mfl663, Mfl465, Mfl626 Mfl590, Mfl591 Mfl099, Mfl474,Mfl315, Mfl325,Mfl482 x13+ PRPP cardiolipin/ phospholipids membrane synthesis Purine/Pyrimidine Salvage phospholipid membrane Identified Metabolic Pathways in Mesoplasma florum Mfl074, Mfl075, Mfl276, Mfl665, Mfl463, Mfl144, Mfl342, Mfl343, Mfl170, Mfl195, Mfl372 Mfl419, Mfl676, Mfl635, Mfl119, Mfl107, Mfl679, Mfl306, Mfl648, Mfl143, Mfl466, Mfl198, Mfl556, Mfl385 Mfl076, Mfl121, Mfl639, Mfl528, Mfl530, Mfl529, Mfl547, Mfl375 niacin? Mfl063, Mfl065, Mfl038, Mfl388 xanthine/uracil permease Pyridine Nucleotide Cycling variable surface lipoproteins Mfl413, Mfl658 Mfl444, Mfl446, Mfl451 Mfl340, Mfl373, Mfl521, Mfl588 Mfl583, Mfl288, Mfl002, Mfl678, Mfl675, Mfl582, Mfl055, Mfl328 Mfl150, Mfl598, Mfl597, Mfl270, Mfl649 hypothetical lipoproteins DNA Polymerase RNA Polymerase x22 competence/ DNA transport Mfl047, Mfl048, Mfl475 Electron Carrier Pathways DNA RNA K+, Na+ transporter Mfl027, Mfl369 Flavin Synthesis Mfl064, Mfl178 Nfl289, Mfl037, Mfl653, Mfl193 NAD+ Mfl165, Mfl166 ribosomal RNA transfer RNA degradation FMN, FAD Mfl193 Mfl563, Mfl548, Mfl088, Mfl258, Mfl329, Mfl374, Mfl541, Mfl005, Mfl647, Mfl231, Mfl209 Mfl029, Mfl412, Mfl540, Mfl014, Mfl196,Mfl156, Mfl282, Mfl387, Mfl682, Mfl673, Mfl077, rnpRNA Mfl283, Mfl334 malate transporter? hypothetical transmembrane proteins NADP Mfl378 x57 Ribosome metal ion transporter Signal Recognition Particle (SRP) riboflavin? Mfl356, Mfl496, Mfl217 messenger RNA tRNA aminoacylation NADPH NADH protein secretion (ftsY) srpRNA, Mfl479 23sRNA, 16sRNA, 5sRNA, Mfl122, Mfl149, Mfl624, Mfl148, Mfl136, Mfl284, Mfl542, Mfl132, Mfl082, Mfl127, Mfl561, Mfl368.1, Mfl362.1, Mfl129, Mfl586, Mfl140, Mfl080, Mfl623, Mfl137, Mfl492, Mfl406 Mfl608, Mfl602, Mfl609, Mfl493, Mfl133, Mfl141, Mfl130, Mfl151, Mfl139, Mfl539, Mfl126, Mfl190, Mfl441, Mfl128, Mfl125, Mfl134, Mfl439, Mfl227, Mfl131, Mfl123, Mfl638, Mfl396, Mfl089, Mfl380, Mfl682.1, Mfl189, Mfl147, Mfl124, Mfl135, Mfl138, Mfl601, Mfl083, Mfl294, Mfl440? cobalt ABC transporter Mfl237 Mfl152, Mfl153, Mfl154 proteins Formyl-THF Synthesis Export Mfl613, Mfl554, Mfl480, Mfl087, Mfl651, Mfl268, Mfl366, Mfl389, Mfl490, Mfl030, Mfl036, Mfl399, Mfl398, Mfl589, Mfl017, Mfl476, Mfl177, Mfl192, Mfl587, Mfl355 Mfl086, Mfl162, Mfl163, Mfl161 phosphonate ABC transporter met-tRNA formylation Mfl571, Mfl572 Mfl060, Mfl167, Mfl383, Mfl250 protein translocation complex (Sec) Mfl057, Mfl068, Mfl142,Mfl090, Mfl275 Mfl409, Mfl569 phosphate ABC transporter Mfl233, Mfl234, Mfl235 degradation THF? Mfl186 formate/nitrate transporter amino acids intraconversion? Mfl094, Mfl095, Mfl096, Mfl097, Mfl098 Mfl418, Mfl404, Mfl241, Mfl287, Mfl659, Mfl263, Mfl402, Mfl484, Mfl494, Mfl210, tmRNA Mfl509, Mfl510, Mfl511 spermidine/putrescine ABC transporter oligopeptide ABC transporter Mfl016, Mfl664 putrescine/ornithine APC transporter Mfl015 Mfl182, Mfl183, Mfl184 Mfl019 Mfl605 arginine/ornithine antiporter Mfl557 Mfl652 unknown amino acid ABC transporter glutamine ABC transporter lysine APC transporter alanine/Na+ symporter glutamate/Na+ symporter Amino Acid Transport