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.