Transcript Definitions and Measures of Performance for Standard

Definitions and Measures of Performance for Standard Biological Parts
Jennifer C. Braff, Caitlin M. Conboy, and Drew Endy
Requirement 2: Characterized Parts
transcription (tr)
mRNA Half-life Measurement
I7
10
0
3.5E+05
An Illustration of Part Composition & Functional Composition:
T
TetR
RBS
Ol
RBS
l cI
T
Ol RBS
TetR
GFP/cell
T
OUT
IN
IN
Validation of Steady State
Ol
Optical Density
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
GFP (gmc)
Requirement 1: Signal Carrier
1200
0.5
Protein Concentration
0
CI
LacI
LacI
OLacRBS
l cI-857
10
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)
CI
17
22
27
Time (hours)
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
DNA (copies/cell)
PoPSOUT
PoPSIN
Method: Image quantification
of SybrGold- stained,
linearized plasmid DNA
OlRBS
RiPS scale with mRNA copy #
Medium PoPSdc
High RiPSmc
Medium RiPSmc
0.4
0.2
9
10
A3
B4
pS
A3
I7
10
-
I7
10
9
7
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
Low PoPSdc
Low RiPSmc
DNA copy #
mRNA copy #
Medium strength promoter combined with strong RBS in protein (GFP)
generator yields background level of fluorescence.
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
Non-Composable Parts: I7108 (R0053.B0030.E0040.B0015)
70
pSB4A3I7101
0.6
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
High PoPSdc
DNA Per Cell Quantification
Polymerase Per Second=PoPS
1.0E+05
Requirement 3: Predictable Device/
System Function
0
0
1.5E+05
high
low
ave
0.0E+00
Fluorescence
3.5
High
Low
Average
2.0E+05
0.8
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
2.5E+05
I7
10
0
I7
10
1
Ol
GFP fluorescence (GMC)
T
Low copy (pSB4A3)
3.0E+05
TetR
cI
OUT
l cI
OUT
RBS
TetR
0.0
1.0
B4
GFP standards
0.1
3-
pSB3K3- pSB4A3I7101
I7101
High
Low
Average
high
low
ave
0.1
pS
B3
K
1) Matched signal carriers, levels, and timing.
2) Characterized Parts
3) Predictable device/system function
Higher copy (pSB3K3)
I7
10
9
Method: Quantitative Western Blot
GFP/cell
Requirements of Composable Parts:
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
I7
10
7
effluent
0.2
1.2
I7
Steady State Protein Levels
(Error bars indicate SD)
0.2
1.4
pS
bubbler
0.3
7
Protein Per Cell Quantification
dP/dt = 0, tl = (P+dPP)/R
tl = RiPS per mRNA copy
3-
Time (min post rifampicin addition)
dR/dt = 0, tr = (R+dRR)/D
tr = PoPS per DNA copy
3-
12
Estimating PoPS and RiPS
00 100 101 101 107 107 109 109
1
I7 -I7 -I7 -I7 -I7 -I7 -I7 -I7
A3 3K3 4A3 3K3 4A3 3K3 4A3 3K3
4
B SB SB SB SB SB SB SB
pS
p
p
p
p
p
p
p
RiPSmc (Protein/RNA*s)
8
0
B3
K
4
0.98
(0.96)
1
dP/dt = 0, tl = (P+dPP)/R
dR/dt = 0, tr = (R+dRR)/D
dD/dt = 0, rD = D
tl = RiPS per mRNA copy
tr = PoPS per DNA copy
10
9
0
2
2.18
(0.73) 1.55
(0.83)
I7
-4
2.19 2.24
(0.79) (0.74)
3
3-
0.1%
3.08
(0.59)
10
7
1.0%
3.13
(0.72)
I7
10.0%
4
pS
B4
A
R = 0.9638
5
10
9
2
I7
y = 1.071e-0.7043x
5.36
(0.30)
6
3-
100.0%
Steady State:
Rate Equations:
dP/dt = tlR-P-dPP
dR/dt = trD-R-dRR
dD/dt = rD-D
pS
B4
A
• 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
mRNA Half-life
(R^2 value)
dilution ()
DNA
pS
Method: Transcription arrest with
Rifampicin. Real-time RT-PCR.
replication (r)
3-
pSB4A3: pSC101 origin
low-copy plasmid
Characterized Under Standard
Conditions
dilution ()
mRNA
Q uic kTi
e™ and a ompr es sor
TI FF
ncom
prtmess
ar e ( U
needed
o seeed) t dec
his pic t ur e.
media
cI
degradation (dR)
10
7
pSB3K3: p15A origin
Med-copy plasmid
translation (tl)
I7
Variable Copy Number:
Q uic kTi
e™ and a ompr
Q uic kTi
e™ and a omprTI
Q uic kTi
e™ and a ompr es sor
TI FF
ncom
prtmess
es( U
sor
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prtmess
esFF
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prtmess
ar e ( U
needed
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ar
uree.
needed
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arsor
e (U
needed
o seeed) t dec
his pic t ur e.
dilution ()
Protein
10
R0053.B0032.E0040.B0015
P22cII.med RBS.GFP.terminator
0
pS
B3
K
R0011.B0032.E0040.B0015
PLlacO1.med RBS.GFP.terminator
degradation (dP)
B3
K
BBa_I7109:
200
PoPSdc (RNA/DNA*s)
BBa_I7107:
400
mRNA Half-life (min)
Variable PoPS Constructs:
HIGH
LOW
AVE
600
I7
Quic kT ime™ and a
T IFF (Uncompres sed) decompres sor
are needed to s ee this picture.
800
pS
R0040.B0032.E0040.B0015
Ptet.med RBS.GFP.terminator
1000
mRNA (copies/cell)
pS
B3
K3
-I
71
pS
07
B3
K3
-I
71
pS
09
B4
A3
-I
71
pS
07
B4
A3
-I
71
09
Standard
Curves
BBa_I7101:
R0040.B0030.E0040.B0015
Ptet.strong RBS.GFP.terminator
pSB4A3- pSB3K3-pSB4A3- pSB3K3I7101
I7101
I7101
I7101
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
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 kT ime™ and a
T IFF (Uncompres sed) decompres sor
are needed to s ee this picture.
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.