iGEM World Jamboree Talk v14.5 DI

7/28/2019 iGEM World Jamboree Talk v14.5 DI

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RNA Strand Displacement forInformation Processing in

Mammalian Cells

1

World Jamboree

11.04.2012


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Motivation Mechanism Processing InterfacingProduction Key Reaction Conclusion Human Practices

The Importance of Smaller Parts

DNA required to encode logic circuits

Numberofgates

Circuits with

typical sizedparts

Circuits with

10X smallerparts

2

SynBio Challenge: Make more sophisticated

circuits to control cell behavior

1

23

Current Transcriptional Parts:

~10X Smaller Parts:

11 12 13 14 15 16 17 18 19 20

1 2 3 4 5 6 7 8 9 10

21 22 23 24 25 26 27 28 29 30

Motivation

Greater sophistication per unit DNA Better delivery with payload limits Less energy cost


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0

5

10

15

20

25

30

35

40

2000 2002 2004 2006 2008 2010 2012

Year

Progression of Circuit Sophistication Over Time

3

Analysis of strand displacement publications from Pierce,

Winfree, and Yurke groups

NumberofPromoters/dsGates

Adapted from Purnick et al.,Nature MCB 2009

Moon et al.,Nature 2012

Qian et al.,Science 2011

Transcription-translational circuits

In vitro strand displacement circuits

Motivation

AIM: Demonstrate strand displacement

computation in cells

In vitro = In solution

In vivo = In cells


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Signals and Computation

0 0

1 1

Low activatorInput signal = 0

Low proteinOutput signal = 0

High activator

Input signal = 1

High protein

Output signal = 1

Mechanism

BUFFER

BUFFER NOT OR AND

In traditional transcriptional circuits:


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Signals and Computation: Strand Displacement

1 1

Low signal

High signal

Input signal = 0 Output signal = 0

Input signal = 1 Output signal = 1

Mechanism

BUFFER

0 0

High signal (single-stranded DNA): 1Low signal (double-stranded DNA): 0

(We use fluorescence as a proxy to indicate signal level)


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Mechanism of Toehold-Mediated

Strand Displacement

6

Input

Signal

S1

S2

S3

S2*

S2

Gate

Output

Signal

BUFFER

Mechanism

15 nt


7/54Motivation Mechanism Processing InterfacingProduction Key Reaction Conclusion Human Practices

Mechanism of Toehold-Mediated Strand

Displacement

7

Input

Signal

S1

S2

S3

S2*

S2

Gate

Output

Signal

S2TInput

Signal

S1

S2*

S2

S3

Gate

Output

Signal

T* T*

T

Output

Signal

S2

T

S3

Gate

S2*T* T*

S2TInput

Signal

S1

Mechanism

BUFFER

Qian et al. 2011

15 nt

5 nt



Mechanism of Signal Cascading

8

Qian et al. 2011

Gate

S2*T* T*

S2TInput

Signal

S1

S2

T S3Input

Signal

BUFFER BUFFER

Mechanism



Mechanism of Signal Cascading

9

te

* T*

S2

T S3Input

Signal

S3*

S3

S4

Gate

Output

Signal

T* T*

T

S3*

Gate

T* T*

S2

T S3Input

Signal

S4

Output

Signal

T

S3

Mechanism

BUFFER BUFFER

Qian et al. 2011


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10

Overview ofIn Vivo Strand Displacement

Systems

Processing

Information

Sensing

Inputs

Actuating

Responses

Producing

Circuits



Processing Information

AND OR

True if both inputs true True if at least one input is true

Qian et al. 2011

?NOT

Inverts a signal

Nothing compatible

11

Processing



Our NOT Gate Using Strand Displacement

Dynamic Gate

(A)

Output (B) Output (B)

B is free to act downstream!

C is displaced.

Dynamic Gate(A)

Input Strand

Buffer (C)

Output (B)

Fuel / Catalyst (D)

12

Processing

Operation: Low Input High Output

NOT

0 1



Our NOT Gate Using Strand Displacement

13

B is trapped, cannot act downstream!

C is stable.

Dynamic Gate(A)

Input Strand

Buffer (C)

Output (B)

Fuel / Catalyst (D)

Dynamic Gate

(A)

Output (B)

Input Strand

Operation: High Input Low Output

Processing

NOT

1 0



Our NOT Gate In Vitro

TestSimulateDesign

Cooperative Hybridization at Low

ConcentrationsInitial Simulation

Input

O

utput

Input

Output

Initial Transfer FunctionNOT Gate Transfer Function

Simulations using Visual DSD; experiment by Jon, Giulio

Processing

Visual DSD

Using ODEs

9 reactions per NOT gate

Plate reader studies

Measuring fluorescence

100 uL reactions

14


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15

Producing Circuits

Processing

Information

Sensing

Inputs

Actuating

Responses

Producing

Circuits



In Vivo Implementation Choices

Utilize RNA Mammalian Cells

Tl* Sl*

T S

In vitro In vivo

24 hours

1 cell cycle

Slow dilution rate

Natural RNAi pathway

RNA more stable

Can be produced continuously

Provide for dynamic circuit operation

Production

16


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18

Interfacing With the Cell

Processing

Information

Sensing

Inputs

Actuating

Responses

Producing

Circuits



Sensing mRNA Inputs

Short RNA

Output

Signal

+

19

Design rules orthogonality

three-letter code

accessibility

Downstream

Input

Signal

Sensor

Input mRNA

+

Fuel

Output

Gate

Gate

Interfacing



Sensing mRNA for Cellular Interfacing

20

NUPACK Rendering of eBFP2 mRNA

Interfacing


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22

Using Strand Displacement: Feasible

Processing

Information

Sensing

Inputs

Actuating

Responses

Producing

Circuits


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The Key Reaction Behind It All

24

Input Strand

Gate

Input StrandOutput Strand

Output Strand

High green / High red

Key Reaction

REPORTER

1 1


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The Key Reaction: In Vitro with RNA

Input Strand

S6TT

T* S6*

S6

Fluorescent Complex

+ +

S6

T* S6*

S6

WasteReporterIncorrect Input

Strand

S1

Data collected by Eerik/Chelsea/Felix 25

Key Reaction

REPORTER

1 1


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Video Here

HEK293

cell

Vesicle

Tagged

RNA

In Vivo RNA Delivery

T = 0h T = 2h T = 3h T = 4h

Experiment by Katie


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Motivation Mechanism Processing InterfacingProduction Key Reaction Conclusion Human Practices27

The Key Reaction with RNA in Cells: Iteration 1

Input Strand

ST + +

T* S*

S

Reporter

T* S*

T S

Fluorescent Complex

S

Waste

Transfection by Katie, FACS by NathanKey Reaction

REPORTER

1 1


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Design Test

Key Reaction


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An Example Future Cancer Detect and Destroy Circuit


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An Example Future Cancer Detect-and-Destroy Circuit

Enabled by Our Results

S8T

S7w

7,8

G9:9,10

T*S9*T*

S9 T

S10

w9,fS9 T

Sf

S9*T*s8*

S9

Th8,9:9

G8:8,9

T*S8*T*

S8 T

S9

Th5:8,7

m5*T5*

m5 S8* T* s7*

S8

Th6:8,7

m6*T6*

m6 S8* T* s7*

S8

G1:1,8

T*m1*T1*

m1 T

S8

G2:2,8

T*m2*T2*

m2 T

S8

G3:3,8

T*m3*T3*

m3 T

S8

Th4:8,7

m4*T4*

m4 S8* T* s7*

S8

Processing Actuation

Killer

protein

production

ANXA2CD-55

SEL1LBRCA2

INK4A

CEACAM-6

Conclusion

ANXA2

CD-55

CEACAM-6

SEL1L

BRCA2

INK4A

Sensing

Pancreatic Cancer (PDAC)


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40MammoBlocks

New MammoBlocks (RFC 65) / BioBricks

Best 22 Parts Submitted To Registry

10 Regulatory

Composite Parts

BBa_K779400 BBa_K779405

BBa_K779401 BBa_K779406

BBa_K779402 BBa_K779407

BBa_K779403 BBa_K779408

BBa_K779404 BBa_K779409

37Biobricks

37 Logic Parts for Strand

Displacement

BBa_K779500 BBa_K779501 BBa_K779502

BBa_K779503 BBa_K779504 BBa_K779100

BBa_K779101 BBa_K779102 BBa_K779103

BBa_K779104 BBa_K779105 BBa_K779106

BBa_K779107 BBa_K779108 BBa_K779109

BBa_K779110 BBa_K779111 BBa_K779112

BBa_K779113 BBa_K779114 BBa_K779115

BBa_K779116 BBa_K779117 BBa_K779118

BBa_K779119 BBa_K779120 BBa_K779121

BBa_K779122 BBa_K779123 BBa_K779124

BBa_K779125 BBa_K779126 BBa_K779127

BBa_K779128 BBa_K779129 BBa_K779130BBa_K779131

3 PromotersBBa_K779200

BBa_K779201

BBa_K779202

4 Hammerhead Ribozyme

Coding Sequences

BBa_K779315

BBa_K779316

BBa_K779317

Bba_K779318

13 Reporters

BBa_K779300 BBa_K779307

BBa_K779301 BBa_K779309

BBa_K779302 BBa_K779310

BBa_K779303 BBa_K779311

BBa_K779304 BBa_K779312

BBa_K779306 BBa-K779313BBa_K779314

BBa_K779305BBa_K779308

2 Transcriptional

Regulators

10 Generators

BBa_K779600 BBa_K779601

BBa_K779602 BBa_K779603

BBa_K779604 BBa_K779605

BBa_K779606 BBa_K779607BBa_K779608 BBa_K779609


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Outreach: Middle School, High School, College

Splash: Educating local high school students

MIT Educational Studies Program

Coming soon! November 17, 2012

Wellesley: Building multi-university communities

Use case for Wellesley HCI software

Bridging gap between tool designer and end-user

Summer HSSP: Educating local middle school students Biology Lecture Series: Synthetic Biology

Focus on applications, practices, and opportunities in synthetic biology

33

MIT: January Term Synthetic Biology Class

Engineer Your Own Bacteria 2 week lecture, wet lab

Human Practices


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Acknowledgments

MIT iGEM 2012 TeamFelix Sun, Giulio Alighieri, Eta Atolia, Katie Bodner, Jonathan Elzur, Keren Greenbaum, Divya Israni, Lealia

Xiong, Chelsea Voss, Kristjan Eerik Kaseniit, Nathan Kipniss, Jenna Klein, Robert Learsch, Wilson Louie,Alaa Siam, Linh Vuong

Ron Weiss (faculty)

Jonathan Babb

Deepak Mishra

Coordinators:

Lab Shift Monitors:

Additional thanks to:

Jameel Zayed

Kenneth H. Hu

Leanna S. Morishini

Mariya Barch

Mark Andrew Keibler

Nathan S. Lachenmyer

Sebastien Lemire

Lulu Qian

Peter Andrew Carr

Nevin M. Summers

Timothy Lu

Domitilla Del

Vecchio

Alice M. Rushforth

Roger KammBU-Wellesley iGEM Team

Thanks to our sponsors for their generous support!

Christopher Voigt

Feng Zhang

Jacquin Niles

Kristala L. Jones

Prather

Rahul Sarpeshkar

Narendra Maheshri

Natalie Kuldell

NOT Gate Transfer Function


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PROCESSING

CIRCUIT PRODUCTION

CELL INTERFACING

KEY REACTION

Thank you!

Questions?


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Highly Scalable Parts Comparison

U6

187 bp

Hef1A

1174 bp

Cag

1718 bp

Transcriptional:

HH Gate:Output HH

216 bp

Strand Displacement:

Hef1A: TAL Effector

TAL-VP16 w/ 17bp DNA recognition

1174 + 2477 = 3651bp

Poly A / Terminator

527 bp

U6 terminator

6 bp

3651+527 = 4178bp 187+216+6 = 409bp


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38

Strand Displacement Reactions

Qian et al. 2011


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39

AND, OR Logic Using Strand Displacement

Qian et al. 2011


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40

Full NOT Gate Reaction Diagram

Case: Input present

low output signal

Irreversible

Trapped!

Case: No input present high output signalReversible

Downstream

Input (B)

B

No Input

Strand

Dynamic Gate (A)

Input Strand

Dynamic Gate (A)

Downstream Input (B) reacts:

Downstream

Input (B) Buffer (C)

Fuel / Catalyst (D)

Signal!


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Digital NOT Gate Behavior


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42

RFC65 Review: Recombination Cloning

pDEST

L1 L2

pENTR

Gene

LR

Kan

L4 R1

pENTR

Prom

pEXPR

Ori Kan Ori

Amp Marker

Amp Marker

R4 R2ccdBCm Term

B1 B2Gene TermPromB4

ll h d


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cleaves

43

Actuation: Controlling Hammerhead Activity

Hammerhead-Stem

(inactive)Input Strand Active Hammerhead

c.f.

+

cleaves

cleaves

Design by Divya & Eerik, Hammerhead adapted from Yen et al. 2004

d i f l i id i O


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Transduction of Nucleic Acids to Protein Outputs

Input Output Protein

None

mRNA is stable,

protein produced

mRNA unstable,no protein expression

GFP

GFP cleaves

A i T i H h d Cl


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45

Actuation: Testing Hammerhead Cleavage

Hef1A mKate Hef1A Hammerhead mKate Hef1A HammerheadmKate

Producing Components Using Transcription and


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Hammerhead

Hammerhead

Initial RNA Transcript

RNA Folds

T*

Output

TS2

S3

GateS2* T*

46

Producing Components Using Transcription and

Hammerheads

Hammerhead

Cleaves

Goal:

I Vit St d Di l t It ti 2


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47

In Vitro Strand Displacement: Iteration 2

Input Strand

SlongTlongTlong

Tlong* Slong*

Slong

Fluorescent Complex

+ +Slong/bulge

Tlong* Slong*

Slong/bulge

WasteReporter


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Th K R ti I Vi It ti 2 (B l )


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The Key Reaction: In Vivo Iteration 2 (Bulge)

Input Strand

SlongTlong + +

Tlong* Slong*

Slong

Reporter

Tlong* Slong*

Tlong Slong

Fluorescent Complex

Slong

Waste

Nucleofection by Giulio, FACS by Rob

Longer toehold

Longer hybridization domain

Incorporation of bulge region

Orthogonal sequences

REPORTER

1

O ti i i T f ti f 2 O M d RNA


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50

Optimizing Transfection of 2-O-Me dsRNA

I d ibl E i S t


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Inducible Expression Systems

Cellular-RNA-Compatible Actuation: Relieving miRNA


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p g

Repression with Decoys

Hef1A-LacO eYFP-4xFF4Hef1A mKate-Intronic

miR-FF4

U6-TetO Decoy FF4

TuD FF4

Slight Relief of miRNA Knock-Down of Reporter via Antisense Decoy RNA

1:0:1 Reporter:miRNA:Decoy



Antisense to miRNA

miRNA

Outreach: International


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Internationalization Project of Synthetic Biology

Launching a collaboration between MIT and Tel-AvivUniversity.

Bringing together high school Palestinians and Israelis

for three years to work on an iGEM technical and

entrepreneurial projects.

One-year pilot program to be launched this summer.

A college component and an incubator to follow.

Synthetic Biology Policy Research One student conducted synthetic biology policy research, with

Prof. Kenneth Oye of MITs Engineering Systems Division.

Work presented in SynBERC retreat and at a conference in the

Woodrow Wilson International Center for Scholars.

Outreach: International

Human Practices


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Documents

iGEM World Jamboree Talk v14.5 DI