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

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

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

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

Abstraction model Small core of standard parts Real world complexity Constructed complexity catabolismanabolism Design information

Design, Build, Test Loop

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

Ginkgo Fabrication Facility Tecan EVO Qiagen Biorobot 8000 Biomicrolab XL-20 Biomek FX Tecan EVO Biomek FX

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

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

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

Mfl214, Mfl187 Mfl516, Mfl527, Mfl187 Mfl500Mfl669 Mfl009, Mfl033, Mfl318, Mfl312 Mfl666, Mfl667, Mfl668 Mfl023, Mfl024, Mfl025, Mfl026 ribose ABC transporter glucose sucrosetrehalosexylose unknown fructose sn-glycerol-3-phosphate ABC transporter Mfl254, Mfl180, Mfl514, Mfl174, Mfl644, Mfl200, Mfl504, Mfl578, Mfl577, Mfl502, Mfl120, Mfl468, Mfl175, Mfl259 Mfl039, Mfl040, Mfl041, Mfl042, Mfl043, Mfl044, Mfl596, Mfl281 Glycolysis Mfl497Mfl515, Mfl526Mfl499Mfl317?, Mfl313?? Mfl181 beta-glucoside Mfl009, Mfl011, Mfl012, Mfl425, Mfl615, Mfl034, Mfl617, Mfl430, Mfl313? PTS II System Mfl519, Mfl565 chitin degradation Mfl223, Mfl640, Mfl642, Mfl105, Mfl349 Pentose-Phosphate Pathway glyceraldehyde-3-phosphate Mfl619, Mfl431, Mfl426 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 Purine/Pyrimidine Salvage glucose-6-phosphate ribose-5-phosphate Mfl413, Mfl658 xanthine/uracil permease DNARNA Mfl027, Mfl369 competence/ DNA transport DNA Polymerase degradation RNA Polymerase Mfl047, Mfl048, Mfl475 Mfl237 protein translocation complex (Sec) protein secretion (ftsY) srpRNA, Mfl479 Signal Recognition Particle (SRP) Ribosome Export Mfl182, Mfl183, Mfl184 Mfl509, Mfl510, Mfl511 Mfl652Mfl557 Mfl605Mfl019 Mfl094, Mfl095, Mfl096, Mfl097, Mfl098 Mfl015 spermidine/putrescine ABC transporter unknown amino acid ABC transporter glutamine ABC transporter oligopeptide ABC transporter arginine/ornithine antiporter lysine APC transporter alanine/Na+ symporter glutamate/Na+ symporter Mfl016, Mfl664 putrescine/ornithine APC transporter 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? proteins degradation Mfl418, Mfl404, Mfl241, Mfl287, Mfl659, Mfl263, Mfl402, Mfl484, Mfl494, Mfl210, tmRNA tRNA aminoacylation ribosomal RNAtransfer RNA messenger RNA Mfl029, Mfl412, Mfl540, Mfl014, Mfl196,Mfl156, Mfl282, Mfl387, Mfl682, Mfl673, Mfl077, rnpRNA Mfl563, Mfl548, Mfl088, Mfl258, Mfl329, Mfl374, Mfl541, Mfl005, Mfl647, Mfl231, Mfl209 Mfl613, Mfl554, Mfl480, Mfl087, Mfl651, Mfl268, Mfl366, Mfl389, Mfl490, Mfl030, Mfl036, Mfl399, Mfl398, Mfl589, Mfl017, Mfl476, Mfl177, Mfl192, Mfl587, Mfl355 Mfl086, Mfl162, Mfl163, Mfl161 amino acids Amino Acid Transport intraconversion? Mfl590, Mfl591 Lipid Synthesis Mfl230, Mfl382, Mfl286, Mfl663, Mfl465, Mfl626 fatty acid/lipid transporter Identified Metabolic Pathways in Mesoplasma florum Mfl384, Mfl593, Mfl046, Mfl052 L-lactate, acetate Mfl099, Mfl474,Mfl315, Mfl325,Mfl482 cardiolipin/ phospholipids membrane synthesis x22 Mfl444, Mfl446, Mfl451 variable surface lipoproteins hypothetical lipoproteins phospholipid membrane Mfl063, Mfl065, Mfl038, Mfl388 Mfl186 formate/nitrate transporter Mfl060, Mfl167, Mfl383, Mfl250 Formyl-THF Synthesis THF? x57 hypothetical transmembrane proteins met-tRNA formylation Mfl409, Mfl569 Mfl152, Mfl153, Mfl154 Mfl233, Mfl234, Mfl235 Mfl571, Mfl572 Mfl356, Mfl496, Mfl217 Mfl064, Mfl178 Nfl289, Mfl037, Mfl653, Mfl193 Mfl109, Mfl110, Mfl111, Mfl112, Mfl113, Mfl114, Mfl115, Mfl116 ATP Synthase Complex ATPADP phosphate ABC transporter phosphonate ABC transporter metal ion transporter Mfl583, Mfl288, Mfl002, Mfl678, Mfl675, Mfl582, Mfl055, Mfl328 Mfl150, Mfl598, Mfl597, Mfl270, Mfl649 acetyl-CoA cobalt ABC transporter Mfl165, Mfl166 K+, Na+ transporter Mfl378 malate transporter? Mfl340, Mfl373, Mfl521, Mfl588 Pyridine Nucleotide Cycling NAD+ Electron Carrier Pathways NADHNADPH NADP Flavin Synthesis riboflavin? FMN, FAD Mfl283, Mfl334 Mfl193 Mfl057, Mfl068, Mfl142,Mfl090, Mfl275 Mfl347, Mfl558 G. Fournier 02/23/04 x13+ unknown substrate transporters PRPP niacin?