Metrology & Predictive Design For Synthetic Biology Jacob Beal November, 2013.
-
Upload
caroline-casey -
Category
Documents
-
view
215 -
download
0
Transcript of Metrology & Predictive Design For Synthetic Biology Jacob Beal November, 2013.
Bringing Wet & Dry Together…
Computer sciencetools and models
IPTG not green
LacI
AIPTG
BGFPoutputs outputs outputsarg0arg0
LacI A
IPTG
B GFP
O(t)P(I,t) +
Delay
I(t)
Dilution& decay
Deeper Understanding
Reliable Engineering
Outline
• Vision and Motivation• Proto BioCompiler• Calibrating Flow Cytometry• Building EQuIP Models• Prediction & Validation
Why is this important?
• Breaking the complexity barrier:
• Multiplication of research impact• Reduction of barriers to entry
*Sampling of systems in publications with experimental circuits
1975 1980 1985 1990 1995 2000 2005 2010100
1,000
10,000
100,000
1,000,000
207
2,100 2,700
7,500 14,600
32,000
583,0001,080,000
Year
Leng
th in
bas
e pa
irs
DNA synthesis Circuit size ?
5
[Purnick & Weiss, ‘09]
Why a tool-chain?
Organism Level Description
Cells
This gap is too big to cross with a single method!
6
TASBE tool-chain
Organism Level Description
Abstract Genetic Regulatory Network
DNA Parts Sequence
Assembly Instructions
Cells
High level simulator
Coarse chemical simulator
Testing
High Level DescriptionIf detect explosives: emit signalIf signal > threshold: glow red
Detailed chemical simulator
Modular architecture also open for flexible choice of organisms, protocols, methods, …
7
RonWeiss
Douglas Densmore
Collaborators:
[Beal et al, ACS Syn. Bio. 2012]
A Tool-Chain Example
If detect explosives: emit signalIf signal > threshold: glow red
(def simple-sensor-actuator () (let ((x (test-sensor))) (debug x) (debug-2 (not x))))
Mammalian Target E. coli Target
8
A high-level program of a system that reacts depending on sensor output
[Beal et al, ACS Syn. Bio. 2012]
A Tool-Chain Example
If detect explosives: emit signalIf signal > threshold: glow red
Mammalian Target E. coli Target
9
Program instantiated for two target platforms
[Beal et al, ACS Syn. Bio. 2012]
A Tool-Chain Example
If detect explosives: emit signalIf signal > threshold: glow red
Mammalian Target E. coli Target
10
Abstract genetic regulatory networks
[Beal et al, ACS Syn. Bio. 2012]
A Tool-Chain Example
If detect explosives: emit signalIf signal > threshold: glow red
Mammalian Target E. coli Target
11
Automated part selection using database of known part behaviors
[Beal et al, ACS Syn. Bio. 2012]
A Tool-Chain Example
If detect explosives: emit signalIf signal > threshold: glow red
Mammalian Target E. coli Target
12
Automated assembly step selection for two different platform-specific assembly protocols
[Beal et al, ACS Syn. Bio. 2012]
A Tool-Chain Example
If detect explosives: emit signalIf signal > threshold: glow red
Mammalian Target E. coli Target
Uninduced Uninduced
Induced Induced
13
Resulting cells demonstrating expected behavior
[Beal et al, ACS Syn. Bio. 2012]
Outline
• Vision and Motivation• Proto BioCompiler• Calibrating Flow Cytometry• Building EQuIP Models• Prediction & Validation
Focus: BioCompiler
Organism Level Description
Abstract Genetic Regulatory Network
DNA Parts Sequence
Assembly Instructions
Cells
High level simulator
Coarse chemical simulator
Testing
High Level DescriptionIf detect explosives: emit signalIf signal > threshold: glow red
Detailed chemical simulator
Compilation &Optimization
15
Other tools aiming athigh-level design:Cello, Eugene, GEC,GenoCAD, etc.
[Beal, Lu, Weiss, 2011]
Operators translated to motifs:
IPTG not green
LacI
AIPTG
B
GFPoutputs outputs outputsarg0arg0
LacI A
IPTG
B GFP
Motif-Based Compilation
17
(def sr-latch (s r) (letfed+ ((o boolean (not (or r o-bar))) (o-bar boolean (not (or s o)))) o))
(green (sr-latch (aTc) (IPTG)))
Design Optimization
LacI B
IPTG
I
G
IF
GFP
DE1 E2
A
aTcJ
HC
JTetR
Unoptimized: 15 functional units, 13 transcription factors 18
GFP
Design Optimization
LacIF
IPTG
TetR
H
aTc
F
Final Optimized:5 functional units4 transcription factors
(def sr-latch (s r) (letfed+ ((o boolean (not (or r o-bar))) (o-bar boolean (not (or s o)))) o))
(green (sr-latch (aTc) (IPTG)))
Unoptimized: 15 functional units, 13 transcription factors
H
19
Complex Example: 4-bit Counter
(4-bit-counter)
compiled for mammalian
Optimized compiler already outperforms human designers
Barriers & Emerging Solutions:
• Barrier: Availability of High-Gain Devices– Emerging Solution: combinatorial device libraries
based on TALs, ZFs, miRNAs
• Barrier: Characterization of Devices– Emerging solution: TASBE characterization method
• Barrier: Predictability of Biological Circuits– Emerging solution: EQuIP prediction method
Outline
• Vision and Motivation• Proto BioCompiler• Calibrating Flow Cytometry TASBE
Method• Building EQuIP Models• Prediction & Validation
[Beal et al., Technical Report: MIT-CSAIL-TR-2012-008, 2012]
First, some metrology…
Unit mismatch!
Output[R2]
[R1][O
utpu
t][R2]
R2R1Arbitrary Red
Arbi
trar
y Bl
ue
Arbitrary RedAr
bitr
ary
Blue
How Flow Cytometry Works
Challenges:• Autofluorescence• Variation in measurements• Spectral overlap• Time Contamination
• Lots of data points!• Different protein fluorescence• Individual cells behave (very)
differently
Fluorescent Beads Absolute Units
Run beads every time: flow cytometers drift up to 20 percent!
Also can detect instrument problems, mistakes in settings
SpheroTech RCP-30-5A
Compensating for Spectral Overlap
Strong positive control used for each colorNote: only linear when autofluorescence subtracted
Translating Fluorescence to MEFL
• Only FITC channel (e.g. GFP) goes directly
• Others obtained from triple/dual constitutive controls• Must have exact same constitutive promoter! • Must have a FITC control protein!
Outline
• Vision and Motivation• Proto BioCompiler• Calibrating Flow Cytometry• Building EQuIP Models• Prediction & Validation
[Davidsohn et al., IWBDA, 2013]
TASBE Characterization Method
Transient cotransfection of 5 plasmids
Calibrated flow cytometry
Analysis by copy-count subpopulations
OutputDox R1
pCAG
Dox
T2ArtTA3 VP16Gal4 pTREEBFP2
pTRER1
pUAS-Rep1EYFPpCAGmkate
pCAG
Multi-plasmid cotransfection!?!
• Avoids all problems with adjacency, plasmid size, sequence validations
• Variation appears to be independent
Result: Input/Output Relations
R1 = TAL14 R1 = TAL21
32
Transfer curve for TAL 14 Transfer curve for TAL 21
Expression Dynamics
Fraction Active Mean Expression
Results division rate, mean expression time, production scaling factor
EQuIP model
Model = first-order discrete-time approximation
Output(t)
Regulated Production
Input(t)
Loss
Input
ΔOut
put …
λ
Time
Outline
• Vision and Motivation• Proto BioCompiler• Calibrating Flow Cytometry• Building EQuIP Models• Prediction & Validation
[Davidsohn et al., IWBDA, 2013]
EQuIP Prediction
pCAG
Dox
T2ArtTA3 VP16Gal4 pTREEBFP2
pTRER1
pUAS-Rep1 pUAS-Rep2EYFPR2pCAGmkate
pCAG
R2 OutputDox R1
+
Regulated Production
Input(t)
Loss
Input
ΔOut
put
…
λ
Time Output(t)
Regulated Production
Loss
Input
ΔOut
put
…
λ
Time
Incremental Discrete Simulation[TAL21] [TAL14] [OFP]
TAL14 OFP
TAL21state
TAL14production TAL14
state
OFPproduction OFP
statetime
hour 1
hour 2
hour 46
loss
production
production
loss
production +
-
+
+ +
+
+
-
…
- -
-
…
-
…
High Quality Cascade Predictions
TAL14 TAL21 TAL21 TAL14
Circles = EQuIP predictionsCrosses = Experimental Data
1.6x mean error on 1000x range!
Summary
Automation supports design and debugging of biological devices, sensors, actuators, circuits– BioCompiler automates regulatory network design– TASBE method calibrates flow cytometry data– Cotransfected test circuits give good models– EQuIP accurately predicts cascade behavior from
models of individual repressors
Looking forward:• Real measurements good engineering• Bigger, better circuits on more platforms
[multiple manuscripts in preparation]
Acknowledgements:
Aaron Adler
Joseph Loyall
Rick Schantz
Fusun Yaman
Ron Weiss
Jonathan Babb
Noah Davidsohn
Ting Lu
Douglas Densmore
Evan Appleton
Swapnil Bhatia
Traci Haddock
Chenkai Liu
Viktor Vasilev
40
Characterization Tools Online!
Register: individual accounts or group account?
Anonymous access also available (but not private)
• On first use, you will have to terms of service
• Your data is secure, and can’t be shared on site.
• FireFox recommended; Chrome has an image-display bug.
https://synbiotools.bbn.com/