0.0E+00 2.0E+05 4.0E+05 6.0E+05 8.0E+05 1.0E+06 1.2E+06 1.4E+06 1.6E+06
I7100 I7101 I7107 I7109
GFP/cell High Low Average 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 OLacRBS T CI LacI LacI CI inverter CI LacI PoPS Inv.1
PoPSIN PoPSOUT
Polymerase Per Second=PoPS
Ribosome Per Second=RiPS
RiPS Inv.1
RiPSIN RiPSOUT
Protein Concentration l cI RBS T Ol cI PoPSOUT PoPSIN RiPSOUT RiPSIN l cI T cI mRNA DNA OlRBS
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
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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 IN O U T O U T IN O U T l cI RBS T Ol cI l cI RBS T Ol cI TetR RBS T Ol TetR TetR T Ol TetR RBS
In contrast to protein concentration, polymerase and ribosome transit rates are fungible, part-independent signal carriers.
Requirement 2: Characterized Parts
GFP Expression Devices
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TIFF (Uncompressed) decompressor are needed to see this picture.
Estimating PoPS and RiPS
tl = RiPS per mRNA copy dP/dt = 0, tl = (P+dPP)/R
tr = PoPS per DNA copy dR/dt = 0, tr = (R+dRR)/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
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:
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 dP << for GFP in this system
pSB3K3: p15A origin
Med-copy plasmid
pSB4A3: pSC101 origin
low-copy plasmid
BBa_I7100: BBa_I7101:
Ptet.strong RBS.GFP.terminator Ptet.med RBS.GFP.terminator
QuickTime™ and a
TIFF (Uncompressed) decompressor are needed to see this picture.
Variable RiPS Constructs:
Variable PoPS Constructs:
Variable Copy Number:
• Growth Conditions: Steady state continuous culture in a 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 0 200 400 600 800 1000 1200 12 17 22 27 Time (hours) GFP (gmc)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.
0 0.5 1 1.5 2 2.5 3 3.5 0 10 20 30 40 Time (hours) OD 600 pSB3A3-1(b) pSB4A3-1(c) pSB3K3-1(b) pSB3K3-1(c)
Optical Density Fluorescence
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:
PLlacO1.med RBS.GFP.terminator P22cII.med RBS.GFP.terminator
R0011.B0032.E0040.B0015 R0053.B0032.E0040.B0015
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(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.
y = 1.071e-0.7043x R2 = 0.9638 0.1% 1.0% 10.0% 100.0% -4 0 4 8 12
Time (min post rifampicin addition) mRNA (relative copies/cell)
3.13 (0.72) 5.36 (0.30) 2.19 (0.79) 2.24 (0.74) 0.98 (0.96) 2.18 (0.73) 1.55 (0.83) 3.08 (0.59) 0 1 2 3 4 5 6
pSB4A3-I7100pSB3K3-I7100pSB4A3-I7101pSB3K3-I7101pSB4A3-I7107pSB3K3-I7107pSB4A3-I7109pSB3K3-I7109
mRNA Half-life (min)
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 # m R N A O u tp u t Low PoPSdc Medium PoPSdc High PoPSdc P ro te in O u tp u t 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
0.0E+00 2.0E+02 4.0E+02 6.0E+02 8.0E+02 1.0E+03 1.2E+03
I7108 I7109 neg construct GFP fluorescence (GMC) degradation (dP) degradation (dR) replication (r) Protein DNA dilution () dilution () dilution () transcription (tr) translation (tl) mRNA 0.0 0.1 0.1 0.2 0.2 0.3
pSB3K3- I7107pSB3K3- I7109pSB4A3- I7107pSB4A3- I7109
PoPSdc (RNA/DNA*s) high low ave 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4
pSB3K3- I7107pSB3K3- I7109pSB4A3- I7107pSB4A3- I7109
RiPSmc (Protein/RNA*s) high low ave E xo ge n ou s con tr ol D N A
Standard Curve pSB4A3-I7101
pSB3K3-I7101 pSB4A3-I7101 0 10 20 30 40 50 60 70 80 pSB4A3-I7101 pSB3K3-I7101 DNA (copies/cell) high low ave 0 200 400 600 800 1000
pSB3K3-I7107pSB3K3-I7109pSB4A3-I7107pSB4A3-I7109
mRNA (copies/cell) HIGH LOW AVE pSB3K3-I7101 pSB4A3-I7101 pSB3K3-I7101 pSB4A3-I7101 Standard Curves E xo ge no us p he B C on tr ol 0.0E+00 5.0E+04 1.0E+05 1.5E+05 2.0E+05 2.5E+05 3.0E+05 3.5E+05
I7100 I7101 I7107 I7109
GFP/cell
High Low Average
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
