0.0E+002.0E+054.0E+056.0E+058.0E+051.0E+061.2E+061.4E+061.6E+06

I7100 I7101 I7107 I7109

GFP/cell

HighLowAverage

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-857OLacRBS T

CILacI

LacI CI inverter

CILacI

PoPSInv.1

PoPSIN PoPSOUT

Polymerase Per Second=PoPS

Ribosome Per Second=RiPS

RiPSInv.1

RiPSIN RiPSOUT

Protein Concentration

l cIRBS T Ol

cI

PoPSOUTPoPSIN

RiPSOUTRiPSIN

l cI T

cI

mRNA

DNA RBSOl

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

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.

• 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 Parts3) Predictable device/system function

ININ

OU

T

OU

T

IN

OU

T

l cIRBST

Ol

cI

l cIRBST

Ol

cI

TetRRBST

Ol

TetR

TetRT

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 DevicesQuickTime™ and a

TIFF (Uncompressed) decompressorare needed to see this picture.

Estimating PoPS and RiPS

tl = RiPS per mRNA copy

dP/dt = 0, tl = (P+dPP)/Rtr = 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 aTIFF (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 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

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 DNASteady 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.terminatorR0011.B0032.E0040.B0015 R0053.B0032.E0040.B0015

QuickTime™ and aTIFF (Uncompressed) decompressorare needed to see this picture.QuickTime™ and aTIFF (Uncompressed) decompressorare needed to see this picture.QuickTime™ and aTIFF (Uncompressed) decompressorare needed to see this picture.QuickTime™ and aTIFF (Uncompressed) decompressorare needed to see this picture.

(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 withRifampicin. 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 #

mR

NA

Ou

tpu

t

Low PoPSdc

Medium PoPSdc

High PoPSdc

Pro

tein

Ou

tpu

t

mRNA copy #

Low RiPSmc

Medium RiPSmc

High RiPSmcPoPS 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)

highlowave

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)

highlowave

Exo

gen

ous

con

trol

DN

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)

highlowave

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

Exo

geno

us p

heB

Con

trol

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

HighLowAverage

Low copy (pSB4A3)

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 componentsto be assembled in various combinations with resulting system behaviorthat is predictable. Ideal composable components are(1) functionally independent and (2) stateless.