Definitions and Measures of Performance for Standard Biological Parts Jennifer C. Braff, Caitlin M. Conboy, and Drew Endy Requirement 2: Characterized Parts R0040.B0032.E0040.B0015 Ptet.med RBS.GFP.terminator BBa_I7109: R0053.B0032.E0040.B0015 P22cII.med RBS.GFP.terminator pSB3K3: p15A origin Med-copy plasmid 200 0 pSB4A3: pSC101 origin low-copy plasmid Method: Transcription arrest with Rifampicin. Real-time RT-PCR. • Growth Conditions: Steady state continuous culture in a sixchamber chemostat (20 mL/chamber) Dilution rate = 0.75 hr-1, doubling time ~56 minutes. Temperature: 37º C • Strain: E. coli MC4100 • Media: M9 minimal media supplemented with 0.4% glycerol, 0.1% casamino acids, 1% thiamine hydrochloride 100.0% y = 1.071e-0.7043x 2 R = 0.9638 10.0% 1.0% 0.1% -4 0 4 8 12 Time (min post rifampicin addition) 5 4 2 RBS l cI 1 Ol RBS TetR IN IN Validation of Steady State Ol Optical Density 3.5 1200 3 1000 In contrast to protein concentration, polymerase and ribosome transit rates are fungible, part-independent signal carriers. OD 600 2.5 pSB3A3-1(b) pSB4A3-1(c) pSB3K3-1(b) pSB3K3-1(c) 2 1.5 1 0.5 Protein Concentration 0 CI LacI l cI-857 20 30 • PoPS per DNA copy insensitive to DNA copy #, RBS strength, and DNA sequence • PoPS per DNA copy varies predictably with promoter strength • Steady state mRNA and protein levels scale predictably with PoPS per DNA copy, within a functional range 800 600 400 200 12 40 Time (hours) LacI OLacRBS 10 17 22 27 Time (hours) CI LacI CI inverter T PoPS scale with DNA copy # T RBS l cI PoPSIN Ol PoPS Inv.1 PoPSOUT Standard Curve Ribosome Per Second=RiPS RiPSOUT mRNA RiPSIN cI DNA pSB4A3I7101 l cI T RiPS Inv.1 RiPSOUT 80 pSB4A3-I7101 cI RiPSIN Steady State Plasmid Copy Number (Error bars indicate SD; N=18) pSB3K3-I7101 I7 10 9 1.2 1.0 high low ave 0.8 0.6 0.4 high low ave 0.2 0.0 PoPS and RiPS estimates are consistent with qualitative predictions for devices on a low copy plasmid. When expressed from a higher copy plasmid, device behavior is not as predicted. Note: PoPS estimates assume DNA copy number unchanged between constructs. RiPS estimates assume dP << for GFP in this system Conclusions • RiPS per mRNA copy insensitive to DNA and mRNA copy #, and mRNA sequence • RiPS per mRNA copy varies predictably with RBS strength • Steady state protein levels scale predictably with RiPS per mRNA copy, within a functional range RiPS scale with mRNA copy # Medium PoPSdc High RiPSmc Medium RiPSmc Low PoPSdc Low RiPSmc DNA copy # 60 50 high low ave 40 30 20 MFOLD mRNA secondary structure prediction for first 45 bases of I7108 mRNA: dG = -11.1 kcal/mol mixed site 10 RBS 0 pSB3K3I7101 5’ UTR (1) This work describes a set of protein generator devices constructed from standard biological parts, characterized in terms of mean steady-state DNA, RNA, and protein copies per cell. (2) By characterizing devices with variable promoter and ribosome binding site strength, we have defined a range of PoPS and RiPS that engineered biological devices of this type might send and receive. (3) We have begun to qualitatively evaluate part composability across a set of standard BioBrick vectors, promoters, and ribosome binding sites and asses the extent to which characteristics of these devices are consistent with our understanding of their component parts. (4) Where parts in combination yield devices with surprising characteristics (i.e. evidence of part “non-composability”), we use these observations to develop design principles for the specification of future parts with improved composability. Next Steps mRNA copy # Medium strength promoter combined with strong RBS in protein (GFP) generator yields background level of fluorescence. pSB4A3I7101 OlRBS 1.0E+05 Non-Composable Parts: I7108 (R0053.B0030.E0040.B0015) 70 DNA (copies/cell) PoPSOUT PoPSIN Method: Image quantification of SybrGold- stained, linearized plasmid DNA 1.5E+05 High PoPSdc DNA Per Cell Quantification Polymerase Per Second=PoPS High Low Average 2.0E+05 Requirement 3: Predictable Device/ System Function 0 0 2.5E+05 1.4 0.0E+00 Fluorescence GFP (gmc) Requirement 1: Signal Carrier Low copy (pSB4A3) 0.0 dP/dt = 0, tl = (P+dPP)/R tl = RiPS per mRNA copy 5.0E+04 Cultures containing GFP expression devices I7100 and I7101, grown in chemostat under standard operating conditions exhibit stable cell density and GFP fluorescence. This allows us to assume a constant dilution rate () and protein level (dP/dt = 0) when modeling this system. IN 0.1 3.0E+05 GFP/cell T I7 10 7 3.5E+05 0.1 High Low Average dP/dt = 0, tl = (P+dPP)/R dR/dt = 0, tr = (R+dRR)/D dD/dt = 0, rD = D dR/dt = 0, tr = (R+dRR)/D tr = PoPS per DNA copy 0.2 Higher copy (pSB3K3) I7 10 0 GFP standards GFP/cell pSB3K3- pSB4A3I7101 I7101 1.6E+06 1.4E+06 1.2E+06 1.0E+06 8.0E+05 6.0E+05 4.0E+05 2.0E+05 0.0E+00 Steady State: Estimating PoPS and RiPS 0.2 Steady State Protein Levels (Error bars indicate SD) dilution () DNA tl = RiPS per mRNA copy tr = PoPS per DNA copy 0 00 100 101 101 107 107 109 109 1 I7 -I7 -I7 -I7 -I7 -I7 -I7 -I7 3 3 3 3 3 3 3 3 A K A K A K A K B4 SB3 SB4 SB3 SB4 SB3 SB4 SB3 S p p p p p p p p bubbler TetR T 2.18 (0.73) 1.55 (0.83) 0.98 (0.96) I7 10 0 I7 10 1 Ol OUT OUT T TetR RBS 3.08 (0.59) 2.19 2.24 (0.79) (0.74) 3 GFP fluorescence (GMC) Ol 3.13 (0.72) Protein Per Cell Quantification Method: Quantitative Western Blot OUT T 5.36 (0.30) 6 An Illustration of Part Composition & Functional Composition: l cI dilution () mRNA Rate Equations: dP/dt = tlR-P-dPP dR/dt = trD-R-dRR dD/dt = rD-D 0.3 1) Matched signal carriers, levels, and timing. 2) Characterized Parts 3) Predictable device/system function RBS replication (r) mRNA Half-life (R^2 value) Requirements of Composable Parts: cI degradation (dR) transcription (tr) effluent TetR translation (tl) mRNA Half-life Measurement Characterized Under Standard Conditions dilution () Protein Q ui ck Ti m e ™ an d a TF I F ( Un co m p re ss ed ) d ec o m pr e ss or a re ne ed ed t o s ee th i s pi c t ur e. media cI degradation (dP) pS RiPSmc (Protein/RNA*s) B3 K3 pS I7 10 B3 7 K3 pS I7 10 B4 9 A3 pS I7 10 B4 7 A3 I7 10 9 Variable Copy Number: Q ui ck Ti m e ™ an d a Q ui ck Ti m e ™ an d a Q ui ck Ti m e ™ an d a TF I F ( Un co m p re ss ed ) d ec T IFo Fm(prUne co ss m orp re ss ed ) d ec o TmIFprFe (ss Un orco m p re ss ed ) d ec o m pr e ss or a re ne ed ed t o s ee th i s pia cre t ur nee.ed ed t o s ee th i s pi c taurree. ne ed ed t o s ee th i s pi c t ur e. 400 pS PoPSdc (RNA/DNA*s) B3 K3 pS I7 10 B3 7 K3 pS I7 10 B4 9 A3 pS I7 10 B4 7 A3 I7 10 9 R0011.B0032.E0040.B0015 PLlacO1.med RBS.GFP.terminator HIGH LOW AVE 600 B3 K3 -I 71 pS 07 B3 K3 -I 71 pS 09 B4 A3 -I 71 pS 07 B4 A3 -I 71 09 BBa_I7107: 800 mRNA Half-life (min) Variable PoPS Constructs: 1000 ODE model of gene expression suggests that RiPS and PoPS can be determined for a simple protein generator from measurements of 1) per cell DNA, mRNA, and protein levels 2) mRNA and protein degradation rates 3) steady state growth rate pS Quic kTime™ and a TIFF (Unc ompres sed) dec ompres sor are needed to see this pic ture. mRNA (copies/cell) Standard Curves BBa_I7101: R0040.B0030.E0040.B0015 Ptet.strong RBS.GFP.terminator pSB4A3- pSB3K3-pSB4A3- pSB3K3I7101 I7101 I7101 I7101 Steady State mRNA Levels (Error bars indicate SD) I7 10 7 I7 10 9 Pieces of DNA encoding biological function can be defined as parts and readily combined into larger systems. To be most useful, parts must be composable, i.e. it must be possible for (1) one part to be combined with any other part such that (2) the resulting composite system behaves as expected. Quic kTime™ and a TIFF (Unc ompres sed) dec ompres sor are needed to see this pic ture. Variable RiPS Constructs: BBa_I7100: Method: Quantitative Northern Blot And Real-time RT-PCR. I7 10 1 Engineering Biological Systems GFP Expression Devices Exogenous pheB Control We are working to enable the engineering of integrated biological systems. Specifically, we would like to be able to build systems using standard parts that, when combined, have reliable and predictable behavior. Here, we define standard characteristics for describing the absolute physical performance of genetic parts that control gene expression. The first characteristic, PoPS, defines the level of transcription as the number of RNA polymerase molecules that pass a point on DNA each second, on a per DNA copy basis (PoPS = Polymerase Per Second; PoPSdc = PoPS per DNA copy). The second characteristic, RiPS, defines the level of translation as the number of ribosome molecules that pass a point on mRNA each second, on a per mRNA copy basis (RiPS = Ribosomes Per Second; RiPSmc = RiPS per mRNA copy). In theory, it should be possible to routinely combine devices that send and receive PoPS and RiPS signals to produce gene expression-based systems whose quantitative behavior is easy to predict. To begin to evaluate the utility of the PoPS and RIPS framework we are characterizing the performance of a simple gene expression device in E. coli growing at steady state under standard operating conditions; we are using a simple ordinary differential equation model to estimate the steady state PoPS and RiPS levels. Protein Generator Model mRNA Per Cell Quantification mRNA (relative copies/cell) Abstract • Employ quantitative single-cell techniques (e.g. polony, FCS) to validate DNA, mRNA, and protein per cell measurements and address cell to cell variability. • Integrate characterized parts into larger devices (ex. inverters) to evaluate predictability of device function. • Specify second generation standard biological parts according to design principles for improved composability. 1.2E+03 1.0E+03 Acknowledgements 8.0E+02 6.0E+02 4.0E+02 2.0E+02 0.0E+00 I7108 I7109 construct neg • • • • • Endy, Knight, and Sauer Labs MIT Synthetic Biology Working Group The MIT Registry of Standard Biological Parts External funding Sources: NSF, NIH, DARPA MIT Funding: CSBI, Biology, BE, CSAIL, EE & CS Conclusions: (1) This work allows us to describe a set of protein generator devices constructed from standard biological parts in terms of their steadystate DNA, RNA, and protein mean copies per cell. (2) By characterizing devices with strong and weak promoters and ribosome binding sites, we have defined a range of PoPS and RiPS that engineered biological devices of this type might send and receive. (3) We have begun to qualitatively evaluate part composability across a set of standard BioBrick vectors, promoters, and ribosome binding sites by evaluating the extent to which the characteristics of these devices are consistent with our understanding of their component parts. (4) Where parts in combination yield devices with surprising characteristics (i.e. evidence of part “non-composability”,) we use these observations to guide the development of design principles that will underlie the specification of future parts with improved composability. (1) This work allows us to describe a set of protein generator devices constructed from standard biological parts in terms of their steady-state DNA, RNA, and protein mean copies per cell. (2) By characterizing devices with strong and weak promoters and ribosome binding sites, we have defined a range of PoPS and RiPS that engineered biological devices of this type might send and receive. (3) We have begun to qualitatively evaluate part composability across a set of standard BioBrick vectors, promoters, and ribosome binding sites by evaluating the extent to which the characteristics of these devices are consistent with our understanding of their component parts. (4) Where parts in combination yield devices with surprising characteristics (i.e. evidence of part “non-composability”,) we use these observations to guide the development of design principles that will underlie the specification of future parts with improved composability. Composability is a system design principle that deals with the inter-relationships of components. A highly composable system provides recombinant components that can be selected and assembled in various combinations to satisfy specific user requirements. The essential attributes that make a component composable are: 1) It is selfcontained (i.e., it can be deployed independently - note that it may cooperate with other components at run-time, but dependent components are either replaceable.) 2) It is stateless (i.e., it treats each request as an independent transaction, unrelated to any previous request) ~~Wikipedia, 10-17-05. Composability is a system design principle which allows components to be assembled in various combinations with resulting system behavior that is predictable. Ideal composable components are (1) functionally independent and (2) stateless. Composability is a system design principle which allows components to be assembled in various combinations with resulting system behavior that is predictable. Ideal composable components are (1) functionally independent and (2) stateless.