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

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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

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High-Throughput Architecture for Rapidly Prototyping New Enzymes

MeasureExpressGenes

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100IVT + DNA

IVT only

time (min)

Prot

ein

Prod

uctio

n

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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

)

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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?

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1400

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Prot

ein

Prod

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n

ChemicalSynthesis

AssembleGenes

DNA ErrorCorrection Measure

ExpressGenes

Test downselected codes in vitroInstall new

codes in vivo

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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

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100

150

200

250

300

350

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450

time (min)

Prot

ein

Prod

uctio

n

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160

time (min)

Prot

ein

Prod

uctio

n

no drug

50 μM

Shown: Atazanavir response

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ein

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ein

Prod

uctio

n

HIV Protease Reference Multidrug Resistant Mutant

1 μM

0 50 100 150 200 2500

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250

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time (min)

Prot

ein

Prod

uctio

n

0 50 100 150 200 2500

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100

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300

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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