DNA Matters

Constructing Genes,

Enzymes and

Pathways by the Plate

Leda Notchey

May 19, 2015

Gen9 Proprietary Information and Technology

Agenda

1. The Synthetic Biology Opportunity

2. The Gen9 Advantage

3. Working with Gen9:

o Custom Gene Synthesis

o Variant Libraries

2

Genes Pathways Genomes

Gen9 Proprietary Information and Technology

Agenda

1. The Synthetic Biology Opportunity

2. The Gen9 Advantage

3. Working with Gen9:

o Custom Gene Synthesis

o Variant Libraries

3

Genes Pathways Genomes

Gen9 Proprietary Information and Technology

The Synthetic Biology Opportunity

4

New Sensors

Resistant Plants

Low Cost Fuels

Cutting-edge

Materials

Better Drugs

The Need The Problem The Solution

Researchers have the

DNA sequence

information needed

and the computing

power to design all of

these.

There is a lack high

quality, low cost parts

needed to build

them.

High value,

biologically-based

engineered products

The Rational Design

of vast numbers of

genes thru Next Gen

Gene Synthesis

Using semiconductor

technology, Gen9

developed a HT

process to build DNA

at a very high quality

and low cost

Gen9 Proprietary Information and Technology

Today there is a 10,000X discrepancy between

what is sequenced vs. what is synthesized

5

Capacity in Base Pairs Per Year

3 Trillion

Annual Sequencing Capacity

300 Million

Annual Synthesis Capacity

Gen9 Proprietary Information and Technology

Agenda

1. The Synthetic Biology Opportunity

2. The Gen9 Advantage

3. Working with Gen9:

o Custom Gene Synthesis

o Variant Libraries

6

Genes Pathways Genomes

Gen9 Proprietary Information and Technology

The Gen9 Advantage: Synthesizing the Future

7

Drew

Endy

Stanford

Bio-engineering

George

Church

Harvard

Genetics

Joe

Jacobson

MIT

Fabrication

Founded 2009

First commercial product 2012

35+ Employees

Located in Cambridge, MA, USA

Founders Investors

Gen9 Proprietary Information and Technology

The Gen9 Advantage: Chip to Gene Process

8

• Standard synthesis

• Microliter scale

• Plate at a time

• Humans/robots

• Complex process

• 10s at once

• Manual QC

• Variable yield

• High Cost

Gen9

Others

Chip Design

Oligo Synthesis

Gene Assembly

Quality Control

• Parallel Chip synthesis

• 105 oligos synthesized simultaneously

• Pooled Assembly

• Enzymatic Error Correction

• 1,000s parallel synthesis

• Next-Gen Sequence

Verification

• Low $/bp cost

Gen9 Proprietary Information and Technology

The Gen9 Advantage: The BioFab® Platform

Highly Parallel, Scalable, Automated Process

Shipment Design Services

Error Correction

On-Chip Synthesis

DNA Assembly

Sequence

Verification Clonal

Selection

CAD FAB

9 Gen9 Proprietary Information and Technology

The Gen9 Advantage: Design Services

• Difficult Sequences – Removal of high or low GC%, restriction enzyme sites, homopolymers and repeats

• Variant Libraries – Rational design from amino acid

variants and

• Safety & Security – Usage of IGSC harmonized screening

• Back Translation to any codon frequency table requested

• NEW - Online design and ordering portal with best-in-class

DNA editing tools and optimized for parallel sequence

processing

10

GCG◦GGC◦CCC◦CCC◦CCC

Ala Gly Pro Pro Pro GCA◦GGT◦CCA◦CCC◦CCT

Ala Gly Pro Pro Pro

Gen9 Proprietary Information and Technology

Screenshot seq analysis

11

Review Your Sequence Analysis Results

Optimize!

The Gen9 Advantage: Oligos on a Chip

A.P. Blanchard, R.J. Kaiser, L.E. Hood. Biosensors and Bioelectronics, Volume 11, 1996

The Starting material for the BioFab process is an eluate pool from Agilent OLS chip

Gen9 Proprietary Information and Technology 12

The Gen9 Advantage: DNA Assembly

13

Scalable gene synthesis by selective amplification of DNA pools from high-fidelity microchips

Kosuri S, et al. Nat Biotechnol. 2010 Dec; 28(12):1295

Pooled DNA Assembly of Chip-Synthesized Oligos

Gen9 Proprietary Information and Technology

The Gen9 Advantage: Chip to Gene Assembly

14

Kosuri S., et al. Nature Biotechnology, 2010

Truncated

Product

Gen9 Proprietary Information and Technology

Error Type Error-enriched Untreated Error-depleted Error-depleted 2x

Single Deletion 65 37 1 1

Multiple

Deletion

25 4 6 0

Single Insertion 7 3 0 0

Multiple

Insertion

0 0 0 0

Substitution 15 19 3 3

Other 1 0 0 0

The Gen9 Advantage: Error Filtering

15

Protein-mediated error correction for de novo DNA Synthesis. Carr PA, Park JS, Lee Y-J, Yu T, Zhang S, Jacobson JM. Nucleic Acids Research, 2004, Vol. 32, No. 20

e162

MutS treatment selectively filters out error laden DNA molecules

MutS treated

Gen9 Proprietary Information and Technology

The Gen9 Advantage:

Improving Fidelity for Longer Constructs

16

Better error rates results in less bacterial colony ‘picks’ to ensure a chance of

identifying a ‘perfect’ construct

1:600

1:1400

1:10,000

Carr PA, Park JS, Lee Y-J, Yu T, Zhang S, Jacobson JM. NAR, 2004

Gen9 Proprietary Information and Technology

The Gen9 Advantage: Sequence Verification

17

CCCGCATGGAAGCGGGCCTGGTGG Actual Sequence

Sanger Sequencing

NGS Sequencing

CCCGCATGGAAGCGGGCCTGGTGG

NGS redundancy results in more accurate base calling

Gen9 Proprietary Information and Technology

Agenda

1. The Synthetic Biology Opportunity

2. The Gen9 Advantage

3. Working with Gen9:

o Custom Gene Synthesis

o Variant Libraries

18

Genes Pathways Genomes

Gen9 Proprietary Information and Technology

Working with Gen9

• Gen9’s extensive portfolio of GeneBit, GeneByte, and GeneByte Plus

products

o From 400bp to as large as 10 kb

o Custom vector cloning available

o Turnaround time in as little as 20 business days

• Utilizes Gen9’s manufacturing and customer support infrastructure

• Industry-leading, volume-based construct pricing on a per-base basis

19

linear vector

GeneBit (0.4kb-1.0kb) or Clonal, Sequence Perfect

GeneByte (1.0kb-4.0kb) or

GeneByte Plus (4.0kb-10.0kb)

Clonal, Sequence Perfect

Clonal, Sequence Perfect

Affordable, Sequence-Verified, Clonal Constructs

Gen9 Proprietary Information and Technology

Working with Gen9: Variant Libraries

20

Product – Variant Library

Rationally Designed Combinatorial Variant DNA library of 108 overall diversity

Single-Chain Variable Fragment

260 AA

3 2 3 2 2 3 3 2 2 2 2 2 2 2 2 9 3 4 2 3

Overall Diversity is 3x2x3x2x2x3x3x2x2x2x2x2x2x2x2x9x3x4x2x3 ≅ 108

First Steps – Back translate and parse into manageable oligonucleotide pools

Gen9 Proprietary Information and Technology

Working with Gen9: Variant Libraries

21

We parsed the DNA sequence into 7 oligo pools, equaling 5,496 different oligo sequences

18 2 2 1 288 5184 1

Final Steps – Error filtering, pooled assembly and quality check for fidelity and representation

InnovationCollaboration

Dedication Inte

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genesbase pairs

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GeneByte

DNA

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Variant

BioFab

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service

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Gen9 DNA =

Dedication, Innovation, Collaboration & Integrity

22 Gen9 Proprietary Information and Technology