1. Higher copy (pSB3K3) 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. Definitions and Measures of Performance for Standard Biological Parts Engineering Biological Systems Requirement 1: Signal Carrier l cI-857 O Lac RBS T CI LacI LacI  CI inverter CI LacI PoPS Inv.1 PoPS IN PoPS OUT Polymerase Per Second=PoPS Ribosome Per Second=RiPS RiPS Inv.1 RiPS IN RiPS OUT Protein Concentration l cI RBS T OlOl cI PoPS OUT PoPS IN RiPS OUT RiPS IN l cI T cI mRNA DNA RBS OlOl Jennifer C. Braff, Caitlin M. Conboy, and Drew Endy Acknowledgements 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 Next Steps Va lid ati on of St an da rd C on dit io ns 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.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 DensityFluorescence 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. 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. An Illustration of Part Composition & Functional Composition: Requirements of Composable Parts: 1) Matched signal carriers, levels, and timing. 2) Characterized Parts 3) Predictable device/system function IN OUT IN OUT l cI RBS T OlOl cI l cI RBS T OlOl cI TetR RBS T OlOl TetR T OlOl RBS In contrast to protein concentration, polymerase and ribosome transit rates are fungible, part-independent signal carriers. Requirement 2: Characterized Parts GFP Expression Devices Estimating PoPS and RiPS t l = RiPS per mRNA copy dP/dt = 0, t l = (  P+d P P)/R t r = PoPS per DNA copy dR/dt = 0, t r = (  R+d R R)/D 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 Requirement 3: Predictable Device/ System Function 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 Protein Generator Model t l = RiPS per mRNA copy t r = PoPS per DNA copy dP/dt = t l R-  P-d P P dR/dt = t r D-  R-d R R dD/dt = rD-  D dP/dt = 0, t l = (  P+d P P)/R dR/dt = 0, t r = (  R+d R R)/D dD/dt = 0, rD =  D Steady State: Rate Equations: 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 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 d P <<  for GFP in this system pSB3K3: p15A origin Med-copy plasmid pSB4A3: pSC101 origin low-copy plasmid BBa_I7100: BBa_I7101: P tet.strong RBS.GFP.terminator P tet.med RBS.GFP.terminator Variable RiPS Constructs: Variable PoPS Constructs: Variable Copy Number: 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 Characterized Under Standard Conditions effluent bubbler media Validation of Steady State 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 DensityFluorescence DNA Per Cell Quantification Method: Image quantification of SybrGold- stained, linearized plasmid DNA Steady State Plasmid Copy Number (Error bars indicate SD; N=18) Protein Per Cell Quantification Method: Quantitative Western Blot GFP standards pSB3K3- I7101 pSB4A3- I7101 Steady State Protein Levels (Error bars indicate SD) mRNA Half-life Measurement mRNA Per Cell Quantification Method: Quantitative Northern Blot And Real-time RT-PCR. Steady State mRNA Levels (Error bars indicate SD) Conclusions R0040.B0030.E0040.B0015 R0040.B0032.E0040.B0015 BBa_I7107: BBa_I7109: P LlacO1.med RBS.GFP.terminator P 22cII.med RBS.GFP.terminator R0011.B0032.E0040.B0015 R0053.B0032.E0040.B0015 (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. Method: Transcription arrest with Rifampicin. Real-time RT-PCR. mRNA Half-life (R^2 value) Non-Composable Parts: I7108 (R0053.B0030.E0040.B0015) Medium strength promoter combined with strong RBS in protein (GFP) generator yields background level of fluorescence. RBS 5’ UTR … 3’ mixed site 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 # MFOLD mRNA secondary structure prediction for first 45 bases of I7108 mRNA: dG = -11.1 kcal/mol degradation (d P ) degradation (d R ) replication (r) Protein DNA dilution (  ) transcription (t r ) translation (t l ) mRNA Exogenous control DNA Standard CurvepSB4A3-I7101 pSB3K3-I7101 pSB4A3- I7101 pSB3K3- I7101 pSB4A3- I7101 pSB3K3- I7101 pSB4A3- I7101 Standard Curves Exogenous pheB Control Low copy (pSB4A3)

2. Conclusions: (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 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. (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 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.