Download Peter Carr

Rapid Prototyping for Biological
Design
Peter Carr
CBA Bits  Biology 5/1/14
This work is sponsored by the Assistant Secretary of Defense for Research & Engineering under Air Force
Contract #FA8721-05-C-0002. Opinions, interpretations, recommendations and conclusions are those of the
authors and are not necessarily endorsed by the United States Government.
DNA as Digital Communication
transmitter
receiver
G A T T A C A
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Bio
to Bits
Bits
to Bio
SEQUENCING
SYNTHESIS
The Biomolecular Prototyping Unit (BPU)
User Specs
Put DNA
to Work
Fluorescence
Bits-Bio
Build DNABits-Bio-Bits
Converter
(a.u.)
Did it Work?
Time (hrs)
Chemical
Synthesis
DNA Error
Correction
Assemble
Genes
Express
Genes
Integration for rapid design-build-test
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Measure
Image Source: Stephen Quake
Microfluidics
Todd Thorsen
David Kong
“Lab-on-a-Chip” to miniaturize thousands of experiments
in parallel
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Re-programming Diseased Cells
Self-ID and Self-Destruct
DNA for classifier circuit
cancer
cell
normal
cell
no
match
match
cell death
miR-21
miR-17-30a
miR-141
miR-142(3p)
miR-146a
no effect
hi
hi
lo
lo
lo
Weiss, Benenson et al. (2011) Science
Genetic circuit
measures a cell’s
molecular fingerprint
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1 1 0 0 0
match
Microfluidic Gene Assembler (MGA)
1 1 0 0 0
Prototype Microfluidic
Reactionware
Fingerprint to match
Need one of these DNA
designs to make each
measurement (joining
>15 different DNA parts)
Inexpensive Portable
Microfluidic Controller
Chemical
Synthesis
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DNA Error
Correction
Assemble
Genes
Express
Genes
Measure
With Weiss, Babb, Gam
Advanced Decontaminants
Biomolecular Prototyping
Unit (BPU) to enable the
design of CWA-destroying
enzymes
Chemical
Synthesis
DNA Error
Correction
Assemble
Genes
Express
Genes
Measure
Bio-engineered Decon
Desired Features:
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•
•
•
Specificity to target chemicals of interest
•
Robustness to complex environments
Catalysis for reduced decontaminant volumes
Motility to access threat within porous
materials
High-Throughput Architecture for Rapidly
Prototyping New Enzymes
Express
Genes
IVT + DNA
IVT + DNA
400
Protein Production
mRNA production
500
Measure
IVT only
300
200
100
100
IVT only
80
60
40
20
0
0
0
200
time (min)
400
0
200
time (min)
Quantitate both mRNA and
protein production in real time
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400
Microfluidic Measurement of Enzyme Activity
Thiol detection
agent
Bond broken
O
O
P
O
2-ethylthioethane thiol
OH
demeton-S
Microfluidic
WT
1.75hr
DS6
0hr
Fluorescence (RFU)
0hr
3000
Fluorescent
product
+
WT
DS6
2000
1000
1.75hr
0
Fluorescence @ 520nm (RFU)
OPH
Benchtop
2900
WT
DS6
2400
1900
1400
900
0
20
40
time (min)
60
0
20
time (min)
40
Can quantify (even weak) enzyme activity in microfluidic
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Why Engineer the Genetic code?
Nature’s Code
TTT
TCT
TAT
TGT
TTT
TAT
Phe TCT
Tyr TGT
Cys
TTT TTC Phe TCT TCC
TAT TAC Tyr TGT TGC Cys
Ser TAC Tyr TGT TGC Cys
TTT TTC Phe TCT TCC
TAT
TTC Phe
TCC TCA Ser TAC TAA
TGC TGA
TTA
stop
stop
Tyr
Cys
stop
stop
Leu TCA Ser TAC TAA
TTC TTA
TCC
TGC TGA
TTA TTG Leu TCA TCG
TAA TAG
stop stop
TGA TGG
stop Trp
Ser
TTA TTG Leu TCA TCG
TAA TAG
stop stop
TGA TGG
stop Trp
TTG Leu
TCG CCT
TAG CAT
stop
TGG CGT
Trp
CTT
His CGT
TTG CTT
TCG CCT
TAG CAT
stop
TGG
Trp
CTT CTC
CCT CCC
CAT CAC His CGT CGC
Leu CCC
Pro CAC His CGT CGC
Arg
CTT CTC
CCT
CAT
CTC CTA Leu CCC CCA Pro CAC CAA
CGC CGA Arg
His
Gln CGA Arg
CTC CTA Leu CCC CCA Pro CAC CAA
CGC
CTA CTG
CCA CCG
CAA CAG Gln CGA CGG
Leu
Pro
Arg
CTA CTG
CCA CCG
CAA CAG Gln CGA CGG
CTG ATT
CCG ACT
CAG AAT
CGG AGT
Gln
Asn AGT
Ser
CTG ATT
CCG ACT
CAG AAT
CGG
ATT ATC
AAT AAC Asn AGT AGC Ser
IleACT ACC
IleACT ACC
Thr AAC Asn AGT AGC Ser
ATT ATC
AAT
ATC ATAIle
ACC ACA Thr AAC Asn
AGC AGA
AAA
Ser
Lys AGA
Arg
ATC ATAIle
ACC ACA Thr AAC AAA
AGC
ATA ATG
ACA ACG
AAA AAG Lys AGA AGG Arg
Met
Thr
Met
ATA ATG
ACA ACG
AAA AAG Lys AGA AGG Arg
ATG GTT
Met
ACG GCT
AAG GAT
AGG GGT
Lys
Arg
Asp GGT
ATG GTT
Met
ACG GCT
AAG GAT
AGG
GTT GTC
GCT GCC
GAT GAC Asp GGT GGC
Val GCC
Ala GAC Asp GGT GGC
Gly
GTT GTC
GCT
GAT
GTC GTA Val GCC GCA Ala GAC GAA
GGC GGA Gly
Asp
Glu GGA Gly
GTC GTA Val GCC GCA Ala GAC GAA
GGC
GTA GTG
GCA GCG
GAA GAG Glu GGA GGG
Val
Ala
Gly
GTA GTG
GCA GCG
GAA GAG Glu GGA GGG
GTG
GCG
GAG Glu
GGG
GTG
GCG
GAG
GGG
• Existing code assignments
are saturated
• Add in new chemical
functions not seen in nature
• Better control over
engineered organisms
• Block viral infection
3-letter words with a choice of 4 letters =
a dictionary of 64 words
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with Jacobson, Church, Isaacs, Wang, LaJoie, Sterling…
Prototyping Alternate Genetic Codes
Can we predict the effect of a new genetic code on a cell?
Chemical
Synthesis
DNA Error
Correction
in silico code
simulation and design
Express
Genes
Assemble
Genes
Measure
Install new
codes in vivo
Test downselected codes in vitro
1400
Modest code change (1)
Protein Production
1200
1000
3 edits restore
800
600
400
200
Extreme code design (62, 20%)
0
0
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50
100
150
Time (min)
200
250
Personalized Antiviral Therapies
HIV-infected
patient
(unique)
HIV virus
sequence
diversity
Chemical
Synthesis
Sequence patient’s
HIV diversity
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DNA Error
Correction
Doctor chooses
among many drugs
and regimens
Assemble
Genes
Express
Genes
Synthesize HIV proteins and test
against antivirals
Patient IS the
experiment:
Mileage may vary
Measure
Administer
personalized HIV
therapy
Personalized Antiviral Therapies
Chemical
Synthesis
Sequence patient’s
HIV diversity
DNA Error
Correction
Express
Genes
Assemble
Genes
Administer
personalized HIV
therapy
Synthesize HIV PROTEASE variants,
test against antivirals
HIV Protease Reference
Multidrug Resistant Mutant
160
450
400
1 μM
no drug
120
Protein Production
140
Protein Production
Measure
100
80
50 μM
60
40
20
no drug
350
300
250
200
1 μM
150
50 μM
100
50
0
0
0
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50
100
150
time (min)
200
250
0
Shown: Atazanavir response
50
100
150
time (min)
200
250
Integration
Assemble
Genes
DNA Error
Correction
User Specs
PEC Synthesis
(Chow, Jacobson)
Parallel microfluidic
gene synthesis
(Kong, Carr, Jacobson)
Microfluidic Gene Assembler
(Kong, Thorsen, Carr)
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Express
Genes
Microfluidic gene to protein to assay
(Kong, Carr, Jacboson)
Measure
Microfluidic Assays
(Kong, Thorsen, Carr)
MutS error correction
(Carr, Jacobson)
On-Chip Central Dogma
(Kong, Thorsen, Carr)
500
mRNA production
Oligonucleotide
Synthesis
IVT +
DNA
400
300
200
100
0
0
200
time (min)
400
Acknowledgements
MIT Lincoln Laboratory
David Kong
Todd Thorsen
Scott Wick
Kim Hamad-Schifferli
Bea Yu
Whitney Young
Vlad Liberman
Michael Sworin
Ted Fedynyshyn
Eric Schwoebel
Sandra Deneault
Darrell Ricke
Anna Shcherbina
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Collaborators
Joe Jacobson (MIT)
Neil Gershenfeld (MIT)
Shuguang Zhang (MIT)
George Church (Harvard)
Ron Weiss (MIT)
Farren Isaacs (Yale)
Harris Wang (Columbia)
Marc LaJoie (Harvard)
Bram Sterling (Harvard)
Funding
ASD (R&E)
DARPA
NSF
NIH