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Rapid Prototyping for Biological Design. Peter Carr CBA Bits Biology 5/1/14. - PowerPoint PPT Presentation
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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.
CBA Bits Bio-2Carr 5/1/14
DNA as Digital Communication
receivertransmitter
Bioto Bits
Bitsto Bio
G A T T A C A
SEQUENCING SYNTHESIS
CBA Bits Bio-3Carr 5/1/14
The Biomolecular Prototyping Unit (BPU)
ChemicalSynthesis
AssembleGenes
DNA ErrorCorrection Measure
ExpressGenes
Integration for rapid design-build-test
Build DNA Put DNA to Work
Bits-BioConverter
User Specs
Time (hrs)
Fluo
resc
ence
(a.u
.)
Did it Work?
Bits-Bio-BitsConverter
CBA Bits Bio-4Carr 5/1/14
Microfluidics
Imag
e S
ourc
e: S
teph
en Q
uake
“Lab-on-a-Chip” to miniaturize thousands of experiments in parallel
Todd Thorsen
David Kong
CBA Bits Bio-5Carr 5/1/14
Re-programming Diseased CellsSelf-ID and Self-Destruct
DNA for classifier circuit
match
cell death
no match
no effect
cancer cellnormal
cell
miR
-21
miR
-17-
30a
miR
-141
miR
-142
(3p)
miR
-146
a
hi hi lo lo lo
1 1 0 0 0
Weiss, Benenson et al. (2011) Science
Genetic circuit measures a cell’s
molecular fingerprint match
CBA Bits Bio-6Carr 5/1/14
Microfluidic Gene Assembler (MGA)
Prototype Microfluidic Reactionware
Inexpensive Portable Microfluidic Controller
1 1 0 0 0 Fingerprint to match
Need one of these DNA designs to make each measurement (joining
>15 different DNA parts)
With Weiss, Babb, Gam
ChemicalSynthesis
AssembleGenes
DNA ErrorCorrection Measure
ExpressGenes
CBA Bits Bio-7Carr 5/1/14
Advanced Decontaminants
Desired Features:• Specificity to target chemicals of interest• Catalysis for reduced decontaminant volumes• Motility to access threat within porous materials• Robustness to complex environments
Bio-engineered Decon
Biomolecular Prototyping Unit (BPU) to enable the
design of CWA-destroying enzymes
ChemicalSynthesis
AssembleGenes
DNA ErrorCorrection Measure
ExpressGenes
CBA Bits Bio-8Carr 5/1/14
High-Throughput Architecture for Rapidly Prototyping New Enzymes
MeasureExpressGenes
0 50 1001502002503003504000
20
40
60
80
100IVT + DNA
IVT only
time (min)
Prot
ein
Prod
uctio
n
0 100 200 300 4000
100
200
300
400
500IVT + DNA
IVT only
time (min)
mRN
A pr
oduc
tion
Quantitate both mRNA and protein production in real time
CBA Bits Bio-9Carr 5/1/14
Microfluidic Measurement of Enzyme Activity
Can quantify (even weak) enzyme activity in microfluidic
WT0hr
1.75hr 1.75hr
DS60hr
P
OH
O
O
O
demeton-S
OPH +2-ethylthioethane thiol
Bond broken
Fluorescent product
Thiol detection agent
0 5 10 15 20 25 30 35 40 45 50900
1400
1900
2400
2900 BenchtopWT
DS6
time (min)
Fluo
resc
ence
@ 5
20nm
(RFU
)
0 20 40 600
1000
2000
3000MicrofluidicWT
time (min)
Fluo
resc
ence
(RFU
)
CBA Bits Bio-10Carr 5/1/14
TTTPhe
TCT
Ser
TATTyr
TGTCys
TTC TCC TAC TGC
TTALeu
TCA TAA stop TGA stop
TTG TCG TAG stop TGG Trp
CTT
Leu
CCT
Pro
CATHis
CGT
ArgCTC CCC CAC CGC
CTA CCA CAAGln
CGA
CTG CCG CAG CGG
ATT
Ile
ACT
Thr
AATAsn
AGTSer
ATC ACC AAC AGC
ATA ACA AAALys
AGAArg
ATG Met ACG AAG AGG
GTT
Val
GCT
Ala
GATAsp
GGT
GlyGTC GCC GAC GGC
GTA GCA GAAGlu
GGA
GTG GCG GAG GGG
Why Engineer the Genetic code?
• Existing code assignments are saturated
• Add in new chemical functions not seen in nature
• Better control over engineered organisms
• Block viral infection
TTTPhe
TCT
Ser
TATTyr
TGTCys
TTC TCC TAC TGC
TTALeu
TCA TAA stop TGA stop
TTG TCG TAG stop TGG Trp
CTT
Leu
CCT
Pro
CATHis
CGT
ArgCTC CCC CAC CGC
CTA CCA CAAGln
CGA
CTG CCG CAG CGG
ATT
Ile
ACT
Thr
AATAsn
AGTSer
ATC ACC AAC AGC
ATA ACA AAALys
AGAArg
ATG Met ACG AAG AGG
GTT
Val
GCT
Ala
GATAsp
GGT
GlyGTC GCC GAC GGC
GTA GCA GAAGlu
GGA
GTG GCG GAG GGG
Nature’s Code
3-letter words with a choice of 4 letters =a dictionary of 64 words
TTTPhe
TCT
Ser
TATTyr
TGTCys
TTC TCC TAC TGC
TTALeu
TCA TAA stop TGA stop
TTG TCG TAG stop TGG Trp
CTT
Leu
CCT
Pro
CATHis
CGT
ArgCTC CCC CAC CGC
CTA CCA CAAGln
CGA
CTG CCG CAG CGG
ATT
Ile
ACT
Thr
AATAsn
AGTSer
ATC ACC AAC AGC
ATA ACA AAALys
AGAArg
ATG Met ACG AAG AGG
GTT
Val
GCT
Ala
GATAsp
GGT
GlyGTC GCC GAC GGC
GTA GCA GAAGlu
GGA
GTG GCG GAG GGG
TTTPhe
TCT
Ser
TATTyr
TGTCys
TTC TCC TAC TGC
TTALeu
TCA TAA stop TGA stop
TTG TCG TAG stop TGG Trp
CTT
Leu
CCT
Pro
CATHis
CGT
ArgCTC CCC CAC CGC
CTA CCA CAAGln
CGA
CTG CCG CAG CGG
ATT
Ile
ACT
Thr
AATAsn
AGTSer
ATC ACC AAC AGC
ATA ACA AAALys
AGAArg
ATG Met ACG AAG AGG
GTT
Val
GCT
Ala
GATAsp
GGT
GlyGTC GCC GAC GGC
GTA GCA GAAGlu
GGA
GTG GCG GAG GGG
with Jacobson, Church, Isaacs, Wang, LaJoie, Sterling…
CBA Bits Bio-11Carr 5/1/14
in silico code simulation and design
Prototyping Alternate Genetic Codes
Can we predict the effect of a new genetic code on a cell?
0 50 100 150 200 2500
200
400
600
800
1000
1200
1400
Time (min)
Prot
ein
Prod
uctio
n
ChemicalSynthesis
AssembleGenes
DNA ErrorCorrection Measure
ExpressGenes
Test downselected codes in vitroInstall new
codes in vivo
0 50 100 150 200 2500
200
400
600
800
1000
1200
1400
Time (min)
Prot
ein
Prod
uctio
n
0 50 100 150 200 2500
200
400
600
800
1000
1200
1400
Time (min)
Prot
ein
Prod
uctio
n
0 50 100 150 200 2500
200
400
600
800
1000
1200
1400
Time (min)
Prot
ein
Prod
uctio
n
Modest code change (1)
Extreme code design (62, 20%)
3 edits restore
CBA Bits Bio-12Carr 5/1/14
Personalized Antiviral Therapies
HIV-infectedpatient
(unique)
HIV virus sequence diversity
Doctor chooses among many drugs
and regimens
Patient IS the experiment:
Mileage may vary
ChemicalSynthesis
AssembleGenes
DNA ErrorCorrection Measure
ExpressGenes
Sequence patient’s HIV diversity
Synthesize HIV proteins and test against antivirals
Administer personalized HIV
therapy
CBA Bits Bio-13Carr 5/1/14
ChemicalSynthesis
AssembleGenes
DNA ErrorCorrection Measure
ExpressGenes
Personalized Antiviral Therapies
Sequence patient’s HIV diversity
Synthesize HIV PROTEASE variants, test against antivirals
Administer personalized HIV
therapy
0 50 100 150 200 2500
50
100
150
200
250
300
350
400
450
time (min)
Prot
ein
Prod
uctio
n
0 50 100 150 200 2500
20
40
60
80
100
120
140
160
time (min)
Prot
ein
Prod
uctio
n
no drug
50 μM
Shown: Atazanavir response
0 50 100 150 200 2500
20
40
60
80
100
120
140
160
time (min)
Prot
ein
Prod
uctio
n
0 50 100 150 200 2500
20
40
60
80
100
120
140
160
time (min)
Prot
ein
Prod
uctio
n
HIV Protease Reference Multidrug Resistant Mutant
1 μM
0 50 100 150 200 2500
50
100
150
200
250
300
350
400
450
time (min)
Prot
ein
Prod
uctio
n
0 50 100 150 200 2500
50
100
150
200
250
300
350
400
450
time (min)
Prot
ein
Prod
uctio
n1 μM
50 μM
no drug
CBA Bits Bio-14Carr 5/1/14
Integration
User Specs
OligonucleotideSynthesis
AssembleGenes
DNA ErrorCorrection MeasureExpress
Genes
Parallel microfluidicgene synthesis
(Kong, Carr, Jacobson)
MutS error correction(Carr, Jacobson)
PEC Synthesis(Chow, Jacobson)
Microfluidic Gene Assembler(Kong, Thorsen, Carr)
Microfluidic gene to protein to assay(Kong, Carr, Jacboson)
Microfluidic Assays(Kong, Thorsen, Carr)
On-Chip Central Dogma(Kong, Thorsen, Carr)
0 100 200 300 4000
100
200
300
400
500IVT + DNAIVT only
time (min)m
RNA
prod
uctio
n
CBA Bits Bio-15Carr 5/1/14
Acknowledgements
MIT Lincoln LaboratoryDavid Kong
Todd ThorsenScott Wick
Kim Hamad-SchifferliBea Yu
Whitney YoungVlad LibermanMichael SworinTed FedynyshynEric Schwoebel
Sandra DeneaultDarrell Ricke
Anna Shcherbina
CollaboratorsJoe 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)
FundingASD (R&E)
DARPANSFNIH