Definitions and Measures of Performance for Standard Biological Parts Validation of Standard Conditions 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. Optical Density Fluorescence Jennifer C. Braff, Caitlin M. Conboy, and Drew Endy Protein Generator Model Abstract 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. Requirement 2: Characterized Parts GFP Expression Devices mRNA Per Cell Quantification Steady State mRNA Levels (Error bars indicate SD) 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 Method: Quantitative Northern Blot And Real-time RT-PCR. Variable RiPS Constructs: pSB3K3- I7101 pSB4A3- Standard Curves Exogenous pheB Control BBa_I7100: BBa_I7101: R0040.B0030.E0040.B0015 R0040.B0032.E0040.B0015 Ptet.strong RBS.GFP.terminator Ptet.med RBS.GFP.terminator degradation (dP) degradation (dR) replication (r) Protein DNA dilution () transcription (tr) translation (tl) mRNA Variable PoPS Constructs: BBa_I7107: BBa_I7109: PLlacO1.med RBS.GFP.terminator P22cII.med RBS.GFP.terminator R0011.B0032.E0040.B0015 R0053.B0032.E0040.B0015 mRNA Half-life Measurement Variable Copy Number: pSB3K3: p15A origin Med-copy plasmid pSB4A3: pSC101 origin low-copy plasmid Method: Transcription arrest with Rifampicin. Real-time RT-PCR. mRNA Half-life (R^2 value) tl = RiPS per mRNA copy tr = PoPS per DNA copy dP/dt = tlR-P-dPP dR/dt = trD-R-dRR dD/dt = rD-D dP/dt = 0, tl = (P+dPP)/R dR/dt = 0, tr = (R+dRR)/D dD/dt = 0, rD = D Steady State: Rate Equations: Characterized Under Standard Conditions Estimating PoPS and RiPS Growth Conditions: Steady state continuous culture in a six- chamber 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 Engineering Biological Systems tr = PoPS per DNA copy dR/dt = 0, tr = (R+dRR)/D tl = RiPS per mRNA copy dP/dt = 0, tl = (P+dPP)/R 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. Protein Per Cell Quantification Steady State Protein Levels (Error bars indicate SD) effluent bubbler media Higher copy (pSB3K3) Requirements of Composable Parts: Matched signal carriers, levels, and timing. Characterized Parts Predictable device/system function Method: Quantitative Western Blot GFP standards pSB3K3- I7101 pSB4A3- Low copy (pSB4A3) 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 An Illustration of Part Composition & Functional Composition: l cI RBS T Ol cI TetR Validation of Steady State Conclusions IN OUT 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. 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. 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. 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. 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. Requirement 3: Predictable Device/ System Function Optical Density Fluorescence Requirement 1: Signal Carrier 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 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 In contrast to protein concentration, polymerase and ribosome transit rates are fungible, part-independent signal carriers. Protein Concentration l cI-857 OLac RBS T CI LacI LacI  CI inverter CI LacI DNA copy # mRNA Output Low PoPSdc Medium PoPSdc High PoPSdc Protein Output mRNA copy # Low RiPSmc Medium RiPSmc High RiPSmc PoPS scale with DNA copy # RiPS scale with mRNA copy # DNA Per Cell Quantification Next Steps Polymerase Per Second=PoPS 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. Method: Image quantification of SybrGold- stained, linearized plasmid DNA Steady State Plasmid Copy Number (Error bars indicate SD; N=18) l cI RBS T Ol cI PoPSOUT PoPSIN PoPS Inv.1 PoPSIN PoPSOUT Non-Composable Parts: I7108 (R0053.B0030.E0040.B0015) Medium strength promoter combined with strong RBS in protein (GFP) generator yields background level of fluorescence. Exogenous control DNA Standard Curve pSB4A3-I7101 pSB3K3-I7101 pSB4A3- I7101 MFOLD mRNA secondary structure prediction for first 45 bases of I7108 mRNA: dG = -11.1 kcal/mol Acknowledgements Ribosome Per Second=RiPS RiPSOUT RiPSIN l cI T cI mRNA DNA RBS Ol RiPS Inv.1 RiPSIN RiPSOUT mixed site 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 RBS … 5’ UTR 3’

Conclusions: 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. 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. 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. 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. 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. 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. 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. 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 self-contained (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.