Reliable parts to make biology easy to engineer

Vivek K Mutalik, PhD

Scientist, Physical Biosciences Division

Lawrence Berkeley National Lab

BIO Pacific Rim Summit, San Diego

2013

Synthetic Biology:

Food

Energy

Environment

Agriculture

Health

Chemicals

Biological complexity

http://krieger.jhu.edu/magazine/sp11/f2.html

Engineering living systems for useful purposes……

Build devices, systems, pathways, iGEM

Voigt et al 2008; Collins, Nat Genetics 2010 Watanabe et al., Nat Chem Bio 2006, Hasty lab 2011

Application

Empirical ad hoc approach

Ite

rate

Numerous attempts

to get what is desired

Coupled Design/Fabrication

Current SOA

Most Biotech projects are Herculean

1. Parts are not well defined & characterized 2. Parts are incompatible, not composable 3. Parts, circuits are impacted by context 4. Circuitry is unpredictable 5. Biology is complex

Standardized approach to engineering biology

Tools for Design, Modeling and

Simulation Libraries of standard, well characterized biological parts

Fast, cheap DNA synthesis/assembly

Methods and standards for measurement that enable

rapid system characterization and refinement

Parts/Devices

Feedback/Debug

Application

Characterize/Assay

Specification

Design/Modeling

Fabrication

Technology Needs: Bio-Foundry/BioFAB

Genome Scale Engineering Variety of standard chasses

7

A public-benefit facility producing the parts, tools & standards powering future of biotechnology.

BIOFAB

Aim: To make engineering of biology easier Pilot Project on well studied organism: Gene expression in E coli

Drew Endy Adam Arkin Jay Keasling

Challenges: Standardizing parts and junctions

BioFAB C. dog. Projects are based on a genetic layout architecture known as the Expression Operating Unit (EOU)

EOUs serve as independent expression elements that

begin to enable forward engineering at the genome

scale

We must develop and validate junction architectures

We must define and produce primary functional

elements

BioFAB’s Promoter Library

00.10.20.30.40.50.60.70.80.9

1

MP

L_1

9

MP

L_7

4

MP

L_8

8

RP

L_1

78

RP

L_1

53

MP

L_1

02

RP

L_1

34

MP

L_9

5

RP

L_9

0

RP

L_4

6

MP

L_5

6

RP

L_6

2

% lo

g10(a

ctivity)

with

re

sp

ect to

ma

x

Rank ordered 325 promoters

pT7A1

pTrc

pT5N25

pU56D46

pNW535

Module 3

-10 & Disc

Module 2

Spacer

Module 1

-35 & UP

+1 -10 -35 UP

0

10

20

30

40

50

60

70

80

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9 1

# o

f ca

nd

idat

es

Deciles of max. activity

-35N -10N +1

pTrc

Modular Promoter Library Randomized Promoter Library

Mutalik et al., Nature methods, 2013

Goal: Full panel across 10 “strengths” with depth at each strength

BioFAB’s Terminator Library

Cambray et al., Nucleic Acid Research 2013

% Termination efficiency (+/- global SEM)

Linear regression model built on Terminators shows a good

correlation with experimental data

Can we engineer parts and reliable junctions?

Nucleus

Synthetic light collecting structure

% term

ination e

ffic

iency

Rank ordered 75 Terminators

Promoters Terminators

Rank ordered 325 promoters

How do we engineer a

‘reliable’ RBS Library ?

Nucleus

Synthetic light collecting structure

Reliable

UTR-CDS junction

Genes-of-Interest

G1 G2 G3 G4 G5 G6

Mea

n N

orm

aliz

ed E

xpre

ssio

n

RBS1

RBS2

RBS3

RBS4

How do we engineer a ‘reliable’ RBS Library ?

BioFAB’s Bicistronic design library

Nucleus

Synthetic light collecting structure

Reliable

UTR-CDS junction

Bicistronic design library

Translational coupling: Yanofsky lab, 1980 (trp operon) Rosenberg lab 1982 (gal operon) Bicistronic designs: Schoner etal, 1984 Makoff and Smallwood, 1990

Bicistronic design performs reliably across genetic contexts 1

4 G

OI*2

2 B

CD

= 3

08

14

GO

I*22

MC

D =

30

8

Current state of the art BioFAB approach

Mutalik et al., Nature methods, 2013

Standardized Promoters + BCDs driving 2 genes

14 Promoters*22 BCD = 308 14 Promoters*22 BCD = 308

Mutalik et al., Nature methods, 2013

Improvements in part-part junction and reliability

y = 1.0019x + 1.5094 R² = 0.8917

2

3

4

5

6

7

8

9

10

11

2 4 6 8 10

GFP

Flu

ore

sce

nce

(A

U, l

og2

)

RFP Fluorescence (AU, log2)

y = 0.7036x + 1.9614 R² = 0.3858

2

3

4

5

6

7

8

9

10

11

2 4 6 8 10

GFP

Flu

ore

sce

nce

(A

U, L

og2

)

RFP Fluorescence (AU, Log2)

Irregular P-U-CDS Junctions Standardized P-BCD-CDS Junctions

Typical Practice BioFAB Standard Parts

Mutalik et al., Nature methods, 2013

Construct Design and Sequence

All data freely accessible @ www.biofab.org/data

(1) We have systematically tested key “parts” when reused in combination in E coli

(2) Demonstrate that standardization and defined junctions aid in reusable reliable parts, and enable precision gene expression

(3) This work provides an avenue for developing tools to access and visualize data and refining biophysical models

(4) Lessons for refactoring: eg., T7 Phage genome, Nitrogen fixation cluster

(5) Needs further work: Operon engg., Solubility issues etc

(6) LBNL’s Bio-Foundry/BioFAB

Significance and summary

JBEI Jay Keasling Nathan Hillson

SynBERC Leonard Katz

BioFAB Adam Arkin and Drew Endy Guillaume Cambray Joao Guimaraes Colin Lam Quynh-Anh Mai Andrew Tran Morgan Paul Marc Juul Jenhan Tao Lance Martin Cesar Rodriguez and Gaymon Bennett

Acknowledgements

NSF

SynBERC LBNL, Cal

BioBrick Foundation Univ Stanford

Agilent DSM

Funding