rapid prototyping for biological design peter carr cba bits biology 5/1/14 this work is sponsored...

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


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)

Flu

ores

cenc

e (a

.u.)

Did it Work?

Bits-Bio-BitsConverter


Microfluidics

Ima

ge

So

urc

e: S

teph

en

Qu

ake

“Lab-on-a-Chip” to miniaturize thousands of experiments in parallel

Todd Thorsen

David Kong


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

-30

a

miR

-14

1

miR

-14

2(3

p)

miR

-14

6a

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


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


ExpressGenes


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


ExpressGenes


High-Throughput Architecture for Rapidly Prototyping New Enzymes

MeasureExpressGenes

0 50 1001502002503003504000

20

40

60

80

100IVT + DNA

IVT only

time (min)

Pro

tein

Pro

du

ctio

n

0 100 200 300 4000

100

200

300

400

500IVT + DNA

IVT only

time (min)

mR

NA

pro

du

ctio

n

Quantitate both mRNA and protein production in real time


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)

Flu

ore

scen

ce @

520

nm

(R

FU

)

0 20 40 600

1000

2000

3000Microfluidic

WT

time (min)

Flu

ore

scen

ce (

RF

U)


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



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



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



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…


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)

Pro

tein

Pro

du

ctio

n

ChemicalSynthesis

AssembleGenes


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)

Pro

tein

Pro

du

ctio

n

0 50 100 150 200 2500

200

400

600

800

1000

1200

1400

Time (min)

Pro

tein

Pro

du

ctio

n

0 50 100 150 200 2500

200

400

600

800

1000

1200

1400

Time (min)

Pro

tein

Pro

du

ctio

n

Modest code change (1)

Extreme code design (62, 20%)

3 edits restore


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


ExpressGenes

Sequence patient’s HIV diversity

Synthesize HIV proteins and test against antivirals

Administer personalized HIV

therapy


ChemicalSynthesis

AssembleGenes


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)

Pro

tein

Pro

du

ctio

n

0 50 100 150 200 2500

20

40

60

80

100

120

140

160

time (min)

Pro

tein

Pro

du

ctio

n

no drug

50 μM

Shown: Atazanavir response

0 50 100 150 200 2500

20

40

60

80

100

120

140

160

time (min)

Pro

tein

Pro

du

ctio

n

0 50 100 150 200 2500

20

40

60

80

100

120

140

160

time (min)

Pro

tein

Pro

du

ctio

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)

Pro

tein

Pro

du

ctio

n

0 50 100 150 200 2500

50

100

150

200

250

300

350

400

450

time (min)

Pro

tein

Pro

du

ctio

n

1 μM50 μM

no drug


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)

0 100 200 300 400

0

100

200

300

400

500IVT + DNA

IVT only

time (min)m

RN

A p

rodu

ctio

n


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

rapid prototyping for biological design peter carr cba bits biology 5/1/14 this work is sponsored...

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