taming biology as an engineering discipline tom knight [email protected] darpa flexible...
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Taming Biology as an Engineering Discipline
Tom Knight
DARPA Flexible Microbial Manufacturing MeetingFebruary 23, 2011
Simplicity is the greatest sophistication.-- Leonardo da Vinci
Science and Engineering
Engineered organisms
Knowledge & understandingExcellent models
Science &Systems Biologyof natural organisms
Engineering &Synthetic Biology
PartsRepository
De novo DNAsynthesis
Revisedknowledgeand newtechniques
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
catabolism anabolism
Design information
Design, Build, Test Loop
Design, Build, Test Loop
Build
• Gene synthesis on exponential improvement
• Standard parts are understood and availableBiobrick standard partsregistry.org IGEM competition igem.org
• Assembly of standard parts is routineBiobrick 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
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 measureRefactor the genome to standardize parts, re-
arrange for modular replacementDevelop 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
Mfl500 Mfl669Mfl009, Mfl033,Mfl318, Mfl312
Mfl666, Mfl667, Mfl668
Mfl023, Mfl024,Mfl025, Mfl026
ribose ABC transporter
glucosesucrose trehalose xylose
unknownfructose
sn-glycerol-3-phosphate ABC transporter
Mfl254, Mfl180, Mfl514, Mfl174, Mfl644, Mfl200, Mfl504, Mfl578, Mfl577, Mfl502, Mfl120, Mfl468, Mfl175, Mfl259Mfl039, Mfl040, Mfl041, Mfl042, Mfl043, Mfl044, Mfl596, Mfl281
Glycolysis
Mfl497 Mfl515, Mfl526 Mfl499 Mfl317?, Mfl313? ?
Mfl181
beta-glucoside
Mfl009, Mfl011, Mfl012, Mfl425, Mfl615, Mfl034, Mfl617, Mfl430, Mfl313?
PTS II SystemMfl519, 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/uracilpermease
DNA RNAMfl027, 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/putrescineABC transporter
unknown amino acidABC transporterglutamine
ABC transporter
oligopeptide ABC transporter
arginine/ornithineantiporter lysine
APC transporteralanine/Na+ symporter
glutamate/Na+symporter
Mfl016, Mfl664
putrescine/ornithineAPC 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 RNA transfer 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 SynthesisMfl230, 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
hypotheticallipoproteins
phospholipid membrane
Mfl063, Mfl065, Mfl038,Mfl388
Mfl186 formate/nitratetransporter
Mfl060, Mfl167, Mfl383, Mfl250
Formyl-THF Synthesis
THF?
x57hypothetical transmembrane proteins
met-tRNA formylationMfl409, Mfl569
Mfl152, Mfl153, Mfl154
Mfl233, Mfl234, Mfl235
Mfl571, Mfl572
Mfl356, Mfl496, Mfl217
Mfl064, Mfl178Nfl289, Mfl037, Mfl653, Mfl193
Mfl109, Mfl110, Mfl111, Mfl112, Mfl113, Mfl114,
Mfl115, Mfl116
ATP Synthase Complex
ATP ADP
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, FADMfl283, Mfl334
Mfl193
Mfl057, Mfl068, Mfl142,Mfl090,
Mfl275
Mfl347, Mfl558
G. Fournier02/23/04
x13+
unknown substrate transporters
PRPP
niacin?