Accelerate your Research with Synthetic DNA and Hands-free Cloning

Brought to you in collaboration with Lucigen and SGI-DNA Introduction

Julie K. Robinson

Synthetic Genomics, SGI-DNA

Sr. Product Manager, Synthetic Biology The Potential of Synthetic Biology

C O P Y R I G H T © 2 0 1 7 S Y N T H E T I C G E N O M I C S , I N C. | 2 Synthetic Genomics Collaborative Efforts Advance

C O P Y R I G H T © 2 0 1 7 S Y N T H E T I C G E N O M I C S , I N C. | 3 Advancing Genomic Research Solutions, SGI-DNA

Reagents Instruments

● Gibson Assembly® enables rapid assembly ● BioXp™ 3200 – an automated, benchtop of multiple DNA fragments genomics workstation ● Vmax electrocompetent cells for protein ● Create genes, genetic elements and complex expression genetic sequences ● Uses electronically transmitted sequence to build genes Services Bioinformatics

● Synthesize custom DNA of varying size ● Expert bioinformatics support, which and complexity includes whole genome annotation, gene expression analysis, and other types of ● Utilizes patented Gibson Assembly® sequence design method and error correction technology ● Archetype® software enables ‘omics-based ● Cell engineering services understanding of data to enable researchers ● NGS and plasmid preparation services to discover, analyze and build genes

C O P Y R I G H T © 2 0 1 7 S Y N T H E T I C G E N O M I C S , I N C. | 4 Common Genomic applications Applications of DNA Cloning

Study of Genomes & Transgenic Gene Expression Gene Therapy Organisms

Biopharmaceutical Recombinant Protein Cell Engineering Research Production

C O P Y R I G H T © 2 0 1 7 S Y N T H E T I C G E N O M I C S , I N C. | 6 Synthetic Biology Applications: Gene and Genome Modification

Genome Modification

Synthetic Biology provides precision in genome modification

C O P Y R I G H T © 2 0 1 7 S Y N T H E T I C G E N O M I C S , I N C. | 7 Synthetic Biology Applications : Variant Library Construction

Traditional Library Construction

Prep DNA Mutagenesis Clone Transform Screen

Synthetic Library Construction

Design Clone Transform Screen

Synthetic variant libraries allow a systematic method providing a greater pool of possible variants

C O P Y R I G H T © 2 0 1 7 S Y N T H E T I C G E N O M I C S , I N C. | 8 Cloning and the BioXp™ System The Gibson Assembly® Method

Largest Synthetic Construct to date 1.2 Mbp Mycoplasma Genome

Developed in 2009 by Dr. Daniel Gibson to construct the first synthetic cell.

Gibson Assembly® method has become a mainstay in many synthetic biology labs due to its ease-of-use, robustness, and flexibility.

C O P Y R I G H T © 2 0 1 7 S Y N T H E T I C G E N O M I C S , I N C. | 10 Gibson Assembly® Cloning: Faster & More Efficient Than Traditional Cloning

C O P Y R I G H T © 2 0 1 7 S Y N T H E T I C G E N O M I C S , I N C. | 11 BioXp™ 3200 System Overview BioXp™ Overview

The BioXp™ system is the world’s first DNA printer. This personal genomic work station provides rapid hands-free applications from building synthetic DNA, directly cloning these fragments in an overnight run and constructs libraries for Next Generation Sequencing all in a 2 x 2 foot space on your benchtop.

The BioXp™ system is evolving to provide additional capabilities along the genomic workflow.

BioXp™ current applications: • BioXp Tiles – builds 400 bp to 1800 bp DNA fragments • pUCGA Cloning – direct assembly & cloning into a subcloning vector • Custom Cloning – direct assembly cloning into customer vector • NGS Library Construction Kit – builds libraries for Illumina Next Generation Sequencing platform

BioXp™ 3200 System; a true genomic workstation to accelerate genomic discovery.

C O P Y R I G H T © 2 0 1 7 S Y N T H E T I C G E N O M I C S , I N C. | 13 BioXp™ System Benefits

• Each run offers up to 32 reactions

• Hands-free assembly and cloning of 400 bp to 1.8 kb DNA fragments

• Provides workflow control in obtaining DNA fragments and clones

• Compatible with multiple cloning techniques

• Streamlines genomic processing

• Faster turnaround time than conventional methods

• Simple to use – Loading the deck and starting a run takes <5 minutes

The BioXp™ benefits focus to automate to help scientists accelerate genomic discovery

C O P Y R I G H T © 2 0 1 7 S Y N T H E T I C G E N O M I C S , I N C. | 14 BioXp™ Product Types

BioXp™ Tile BioXp™ pUCGA Cloning BioXp™ Custom Cloning BioXp™ NGS Library Prep

• Linear ds DNA fragment • Cloned DNA fragment • Cloned DNA fragment • 8 rxn, 16 rxn formats • Customer sequence • Customer sequence • Customer sequence • Genomic or Plasmid DNA • No modifications • pUCGA Amp • Customer vector • 5 hour prep time • Pool of DNA • Pool of Clones • Pool of Clones • Illumina compatible • TAT 3-5 days • TAT 3-5 days • TAT 3- 5 days • $40 - $50 / RXN • 0.11 – 0.14 bp • 0.15 bp – 0.18 bp • Intro price 0.15 bp Downstream: Cloning options: Downstream: Downstream: • Illumina sequencing • Any cloning method • Transform • Transform • Customer designs DNA • Confirm Full length • Confirm Full length • Sequence Verify • Sequence Verify • Subclone into vector of choice • Ready to use

BioXp™ products add flexibility to streamline tedious benchwork.

C O P Y R I G H T © 2 0 1 7 S Y N T H E T I C G E N O M I C S , I N C. | 15 How do I engage with the BioXp? BioXp™ 3200 System Workflow

Submit the sequences you’d like to build. Day 0

SGIDNA reviews the sequence, designs the Day 4/5 oligos and creates your custom kit.

The BioXp kit is shipped to you in 3 – 5 days. You can load the instrument and start the job

in about 10 minutes to run overnight. Day 5/6

Your DNA fragments or cloning reaction is ready for your downstream workflow. Day 5/6

C O P Y R I G H T © 2 0 1 7 S Y N T H E T I C G E N O M I C S , I N C. | 17 Modular BioXp™ Deck

Key Components: • Thermocycler DNA purification • Chilled locations • 8-Well strip holder • DNA purification

BioXp™ deck provides great flexibility and simplicity with a modular design

C O P Y R I G H T © 2 0 1 7 S Y N T H E T I C G E N O M I C S , I N C. | 18 Customized BioXp™ Product Map

Page 1 Outlines Project details, location of Tiles or clones in the Recovery plate

Green – high likelihood of successful assembly Red – more challenging construct to assemble

Page 2 Reagent components included in kit Match correct sizes of materials

Module A – store at +4C Module B/C – store at -20C

Product map outlines sequences and included reagents.

C O P Y R I G H T © 2 0 1 7 S Y N T H E T I C G E N O M I C S , I N C. | 19 BioXp™ Tiles Current BioXp™ Compatibility Guidelines

BioXp™ 3200 DNA Construct Specifications

Size Ranges 400 bp to 1800 bp DNA Fragments GC Rich Complexity 400 bp to 1 kb, 20 – 70% GC > 1 kb to 1.8 kb, 40 – 60% GC Composition A, T, C or G residues only

Sequence Complexity Low sequence complexity (i.e. tandem repeats etc) Sequence analysis will determine best approach for assembly Fidelity Error corrected to minimize the occurrence of mutations Yield Approximate yield: 200 ng of purified, linear dsDNA PCR product. Actual yield varies per construct. Cloning results in a completed cloning reaction ready to transform. Format 96-well plate format in final volume of 45 µl in TE.

Sequences outside of compatibility range can be built by SGI-DNA Synthesis Team.

C O P Y R I G H T © 2 0 1 7 S Y N T H E T I C G E N O M I C S , I N C. | 21 BioXp™ system builds a range of DNA fragments

C ------40% GC ------C ------45% GC ------

Construct Size (bp) control 1416 1 430 2 660 3 890 C ------55% GC ------C ------60% GC------4 1120 5 1350 6 1580 7 1810 ladder 2-log

C O P Y R I G H T © 2 0 1 7 S Y N T H E T I C G E N O M I C S , I N C. | 22 BioXp™ Cloning BioXp™ Custom Cloning Overview

The BioXp™ 3200 is the first and only automated cloning system that allows scientists to build synthetic genes and clone them directly into their vector of choice hands-free in their lab.

.Eliminates subcloning

.Up to 32 custom cloning reaction sizes into 1 – 4 vectors in a single run

.Job yields both tile and cloned product

.Single button processing (approx. 18 hours)

.Supports DNA samples with low and high 400 bp – 1 kb, GC (20 -70%) ; >1 kb - 1.8 kb, GC (40 - 60%)

.Protocols, QC methods provided on the the BioXp™ Custom Cloning web page

C O P Y R I G H T © 2 0 1 7 S Y N T H E T I C G E N O M I C S , I N C. | 24 Using the Gibson Assembly® Method to Clone BioXp™ Tiles

Gibson Assembly® Cloning is: • Simple • Efficient and fast • Seamless • Not dependent on restriction enzyme digestion

Combines the strength of Gibson Assembly® cloning with the power of synthetic biology

C O P Y R I G H T © 2 0 1 7 S Y N T H E T I C G E N O M I C S , I N C. | 25 BioXp™ Custom Cloning Workflow

Increased flexibility for customer vectors

The BioXp™ Custom Cloning produces DNA fragments that are cloned directly into the vector loaded onto the BioXp™ deck. Customer: ● Design Gibson Assembly based cloning strategies ● Prepare the vector – linearization ● Run a QC, quantitate the vector ● Order the BioXp™ Custom Cloning product on the portal ● Load the BioXp™ with reagents and your vector (up to 4) ● Collect clones, transform, plates, picks ● Determine full length clones, sequences Full 32 reactions per run 3 – 5 day delivery of reagents 6 – 8 days to Error Free Clones

C O P Y R I G H T © 2 0 1 7 S Y N T H E T I C G E N O M I C S , I N C. | 26 High Cloning Efficiency of BioXp™ Clones

To demonstrate the cloning efficiency of the BioXp capabilities, we utilized the pUCGA 1.0 vector, and DNA fragments built and cloned using the BioXp Cloning module.

The cloning efficiency was calculated.

As expected, the smaller fragments cloned at a higher rate with an overall average of 75% efficiency in a hands-free process.

BioXp™ Clones offer hands-free, high efficiency cloning.

C O P Y R I G H T © 2 0 1 7 S Y N T H E T I C G E N O M I C S , I N C. | 27 Finding Error Free Clone in 6 - 8 days

C O P Y R I G H T © 2 0 1 7 S Y N T H E T I C G E N O M I C S , I N C. | 28 Choosing your Competent Cell Gibson Assembly® Transformation Requirements

High efficiency competent cells maximize your success • Avoid experimental repeats • Increase colony counts

Two strains recommended for BioXp Cloning, Gibson Assembly Cloning :  TransforMax™ EPI300™ Electrocompetent E. coli  E. cloni® 10G Chemically Competent E. coli

Failed transformations can result from low-efficiency competent cells • Few or no colonies = “Did my cloning reaction work?” • Troubleshooting = additional time, reagent costs

C O P Y R I G H T © 2 0 1 7 S Y N T H E T I C G E N O M I C S , I N C. | 30 Recommended competent Cells for Gibson Assembly® Choosing your strain

Blue/white Plasmid Transformation T1 Phage Custom Strain recA- endA- Colony Copy Efficiency (TE) Resistant Formats Screening Control* TransforMax ≥1 x 1010 cfu/µg √ √ √ √ √ EPI300 E. cloni 10G ≥1 x 109 cfu/µg √ √ √ √ √

*Inducible copy control of plasmids containing the oriV origin of replication (Bac, fosmid vectors)

• Optimized genotypes for plasmid preparation

• recA minus: minimizes unwanted recombination Customer-defined • endA minus: high quality DNA preps dispense volumes and plates with Custom Comp Cell Services • Flexible formats: 96-well plates, large or small aliquots • http://www.lucigen.com/Custom-Competent-Cells-/

C O P Y R I G H T © 2 0 1 7 S Y N T H E T I C G E N O M I C S , I N C. | 31 Competent Cells for Gibson Assembly® Choosing your transformation method E. cloni® 10G Chemically Competent Cells: • Easy to work with, lower cost per reaction • No specialized equipment required • Larger DNA volumes can be transformed • Generally lower efficiency than electrocomp cells • Amenable to high-throughput cloning

TransforMax™ EPI300™ Electrocompetent Cells: • Shorter protocol, amenable to automation • Electroporator required, requires cuvettes • Lower DNA volumes (<3 µL) • Higher transformation efficiency • Can accept large ( up to 145 kb) plasmids

http://lucigen.com/samplecells.html

C O P Y R I G H T © 2 0 1 7 S Y N T H E T I C G E N O M I C S , I N C. | 32 Comparison of Gibson Assembly® using E. cloni® 10G and EPI300™ cells

Transformation efficiency of assembly reaction cannot be calculated.

High transformation efficiency of Gibson Assembly reactions with E. cloni 10G and Epi300 cells

C O P Y R I G H T © 2 0 1 7 S Y N T H E T I C G E N O M I C S , I N C. | 33 FREE Lucigen Competent Cell Samples

E. cloni® 10G Chemically Competent Cells TransforMax™ EPI300 Electrocompetent Cells http://lucigen.com/samplecells.html

Request your FREE sample today for improved

C O P Y R I G H T © 2 0 1 7 S Y N T H E T I C G E N O M I C S , I N C. | 34 Resources: Application notes

Visit sgidna.com/resources

C O P Y R I G H T © 2 0 1 7 S Y N T H E T I C G E N O M I C S , I N C. | 35 BioXp™ NGS Library Construction Kit BioXp™ NGS Library Construction Kit

The BioXp NGS library construction kit provides Illumina-compatible NGS chemistry offers preparation of genomic and plasmid DNA next generation sequencing. This system is optimized for a common workflow at scales appropriate for non-HTP users with no need for user optimization.

• NGS chemistry for genomic & plasmid DNA

• Optimized protocol for 10-50ng input

• Single button processing (5 hours)

• Barcodes and purification beads included

• 8 and 16 reaction sizes

• Proven for genomic DNA samples with low and high GC (29-68%)

• Proven for plasmid DNA sample

BioXp™ System offers an automated platform for NGS Library Construction

C O P Y R I G H T © 2 0 1 7 S Y N T H E T I C G E N O M I C S , I N C. | 37 BioXp™ 3200 NGS Library Construction Kit Workflow

Starting with sheared genomic DNA, the BioXp™ 3200 System allows walk away automation of the NGS library construction process, resulting ready to barcoded libraries ready for normalization, pooling and sequencing. This same chemistry is available in a non-automated format for those without access to a BioXp™ system.

clean up

C O P Y R I G H T © 2 0 1 7 S Y N T H E T I C G E N O M I C S , I N C. | 38 Simple Loading of NGS Library Reagents

Sample Plate in carrier

Simple and Rapid Set Up of BioXp™ 3200 for NGS Library Construction.

1) Prep sheared genomic DNA sample to the sample plate 2) NGS library construction reagents 3) load the barcode/output plate and magnetic bead strips 4) Fill the ethanol reservoir 5) Finally add the tips and close the lid and press start.

Buffer1 95ul each well*

Barcode Plate

*Overfill 20ul

C O P Y R I G H T © 2 0 1 7 S Y N T H E T I C G E N O M I C S , I N C. | 39 BioXp™ 3200 NGS Library Construction Results

Average Average % Sample Fragment % Dimer % Library # Reads Sequencing Max Cvg Mapped Size Coverage Bacillus-1 342 2% 92% 1,700,924 96% 43.92 250 Bacillus-2 322 1% 92% 1,623,244 96% 42.34 199 Bacillus-3 324 5% 90% 1,375,190 88% 35.94 489 Bacillus-4 339 1% 92% 1,793,744 98% 48.87 343 Microbe B-1 341 0% 87% 1,743,414 98% 39.51 102 Microbe B-2 328 2% 91% 1,664,782 95% 36.38 105 Microbe B-3 333 3% 90% 1,772,470 94% 36.79 108 Microbe- B-4 333 1% 92% 2,929,992 97% 65.62 158 • Replicate NGS libraries were constructed from Bacillus or Microbe B genomic DNA using the BioXp™ 3200 NGS Library Construction Kit and BioXp™ instrument. Average size distribution of library sequenced and % dimer present in final library, and % library were determined using an Agilent 2100 Bioanalyzer and a High Sensitivity DNA Chip. • Number of reads, % reads mapped to reference genome, average coverage, and maximum coverage were determined using the CLC Genomics Workbench. • Data shown indicates high quality libraries that resulted in reliable sequencing data output.

C O P Y R I G H T © 2 0 1 7 S Y N T H E T I C G E N O M I C S , I N C. | 40 Summary BioXp™ Overview

The BioXp™ system prints the DNA you need and serves as a personal genomic work station. It provides rapid hands-free applications for building synthetic DNA, cloning these fragments into your vector in an overnight run. It also constructs libraries for Next Generation Sequencing all in a 2 x 2 foot space on your benchtop.

The BioXp™ system is continuing to evolve to provide new applications along the genomic workflow. BioXp™ current applications: • BioXp Tile Kit • pUCGA and Custom Cloning Kit • NGS Library Construction Kit BioXp™ upcoming applications: • Build DNA fragments at higher fidelity • Build larger DNA fragments • Building degenerate libraries • Additional Application Notes

BioXp™ 3200 System; a true genomic workstation to accelerate genomic discovery.

C O P Y R I G H T © 2 0 1 7 S Y N T H E T I C G E N O M I C S , I N C. | 42 Imagine the possibilities of the BioXp™ System

The BioXp currently offers a range of applications to accelerate genomic discovery. Ongoing development of new applications are driven by listening to our customers.

Applications under consideration on the BioXp™ System

• Large DNA constructs • Degenerate Libraries • Multi-fragment assemblies • Transformation • In vitro transcription • In vitro translation……..

What application would you like to see developed on the BioXp™ System?

C O P Y R I G H T © 2 0 1 7 S Y N T H E T I C G E N O M I C S , I N C. | 43 Synthetic Biology – where might it take your research?

Thank you for attending!

For more information, visit sgidna.com or lucigen.com

C O P Y R I G H T © 2 0 1 7 S Y N T H E T I C G E N O M I C S , I N C. | 44 C O P Y R I G H T © 2 0 1 6 S Y N T H E T I C G E N O M I C S , I N C.