Protein Engineering Canada Conference: A Review

Last month, leaders in the fields of protein engineering and synthetic biology met at the campus of the University of British Columbia in Vancouver, Canada, for the 3rd Protein Engineering Canada (PEC) Conference 2018. The PEC Conference encourages the exchange of ideas and aims to build collaborations in an engaging atmosphere by bringing together students, young investigators, and protein engineering experts. SGI-DNA was a proud contributor to the conference and hosted a lunch seminar entitled “Emerging Technologies: Library Construction using the BioXp™ 3200 System,” presented by Steve Riedmuller, Sr. Director of Field Application Science.

In the well-received seminar, Riedmuller discussed advantages of using the BioXp System for library construction. The BioXp System is an automated benchtop genomic workstation that builds synthetic linear DNA fragments with powerful applications for synthetic biology. DNA libraries are vital for drug discovery, and scientists working in protein engineering, discovery biology, structural biology, synthetic biology, antibody engineering, and enzyme engineering can benefit from BioXp™ Libraries to quickly determine an optimized targeted gene. The BioXp System offers a wide array of library types, including degenerate, variant, and scanning libraries. Riedmuller described the capabilities of the BioXp System and presented a case study demonstrating how the BioXp System drastically accelerated library production when used in conjunction with Gibson Assembly® cloning for the direct transfection of synthetic constructs. Additional details of rapid library construction can be found in this Application Note.

Riedmuller also discussed how the BioXp System can be used for accelerated cancer vaccine production. Introducing genes into eukaryotic cells provides a foundation for cancer vaccines. The premise behind cancer vaccines is that the presence of neoepitopes (“new” peptides that are the result of mutations in cancer cells only) can be used as a target to trigger a patient’s own immune response to specifically attack and destroy cancer cells. One of the technical hurdles of these types of personalized medicines is the time required to prepare an individualized treatment, in this case, a cancer vaccine. Using the BioXp System to build epitopes based on the genetic profile of a patient’s tumor cells dramatically reduces the turnaround time required for creating personalized cancer vaccines, which is critically important to patients facing a terminal diagnosis and recently discussed in this Seattle Times article. 

The BioXp System offers a high throughput solution for protein engineering and synthetic biology applications. In the hands of the scientists in attendance at the PEC Conference (and readers of this blog), this instrument offers new opportunities for faster and more expansive research and discovery.

For more information about the products and applications mentioned in this blog post, please see:

BioXp 3200 System – sgidna.com/bxp3200
Gibson Assembly Reagents - sgidna.com/reagents.html

Meet SGI-DNA: Rahul Gautam, Customer Success

You’re familiar with our synthetic biology solutions and services. Now we’d like to introduce you to the faces behind our innovative products, the people dedicated to building next-generation synthetic biology tools to advance your research.

First off in this new “Meet SGI-DNA” blog series is Rahul Gautam, Customer Success Manager, Synthetic Biology. Of course, we’re biased, but we think Rahul is pretty extraordinary. He’s one of the most well-liked, personable members of the SGI-DNA team and he’s a dedicated leader, who strives to ensure customer success. We recently sat down with Rahul and asked him a few questions about his experience here at SGI-DNA.

"The more I interact with customers, the more I learn about the true potential of synthetic biology."

What is your background?

I have officially been with SGI-DNA for four years now, but I started my journey, majoring in Biology and minoring in Business Administration and Management at UC San Diego. My goal as a student was to explore the business side of biotechnology. Taking business classes through UCSD’s Rady School of Management was inspiring, offering a perfect blend of science, technology, and business. During my senior year, I took Dr. Stephen Mayfield’s introductory Biofuels course, which highlighted Craig Venter and the amazing work that Synthetic Genomics was doing in the algae biofuels space. A few months later, I came across an opening for a product management internship at SGI-DNA, which had just been launched by Dr. Venter and Dr. Daniel Gibson as a subsidiary of Synthetic Genomics. Joining SGI-DNA at a unique time meant that I could parlay my internship into a full-time position on our Customer Support team.

When did you first become interested in science?

Science has always been a big part of my life. Both of my parents were scientists. My father worked at The Scripps Research Institute and my mother was at Invitrogen when it was still a small startup in the early ‘90s. Science consistently made its way into discussions at the dinner table or when my parents’ scientist friends came to visit. Growing up the ‘90’s also provided me with a dynamic environment of scientific programming on the Discovery Channel, National Geographic, the BBC as well as the cutting edge scientific programming on PBS. To this day, I still watch Nova and Nature every week.

In a nutshell, what do you do at SGI-DNA?

I do anything and everything that helps our customers succeed with our services and products. Whether helping a customer place their first order or managing complex projects, I strive to make our customers happy. In a nutshell, I act as the liaison between the scientific acumen of SGI-DNA and our customers. I’m the Magic Johnson of SGI-DNA. <laughter> Kidding aside, I think it’s a good analogy. I’m like a point guard, distributing the proverbial ball amongst my teammates to best support the needs of our customers. My objective is to ensure customer success and build customer relationships that enhance the user experience.

What do you find most interesting about what you do? What do you find to be the most rewarding?

Not to sound cliché but making our customers happy motivates me every day. Our customers are doing cutting-edge research across the industrial synthetic biology spectrum from textiles to cancer immunotherapy and novel personalized medicine therapies. Knowing that SGI-DNA technologies are fueling the research that could provide new, better treatments for patients is motivating and exciting. The more I interact with customers, the more I learn about the true potential of synthetic biology.

What do you think are the most exciting possibilities for synthetic biology?

Without a doubt new approaches to combating human disease. Synthetic biology is helping drive the advancements we are already seeing in personalized medicine. The impact of synthetic biology on the future of cancer research will be significant over the next decade. Improving the overall human experience through synthetic biology is happening on many levels— human disease, nutrition, and through the development of novel chemistry. It’s both fascinating and rewarding to be part of SGI-DNA, a company focused on synthetic biology advancements through its products and services.

What are your interests and hobbies?

I have an endless list of interests and hobbies. I love sports, especially football and basketball. I’m also interested in history and political science. I often joke that my secondary career choice would be as an Indologist (the study of Indian literature and history). As my mother always told me, “You should never have an excuse to be bored.” I guess I have taken that to heart and am completely curious about the world around me. Through my work, I get to interact with many people doing unique and interesting things, both inside and outside the company. This is an incredibly exciting time to be working in synthetic biology, the next great frontier of scientific advancement.

Automated Gene Synthesis for Personalized Medicines

This week, leading researchers and professionals will gather for the annual American Society of Gene and Cell Therapy meeting in Chicago. At this highly anticipated event, researchers will present research and clinical data with the latest advancements in genetic and cellular therapies. SGI-DNA is excited to participate in this prominent and influential gathering. Sr. Product Manager, Dr. Michael Sturges will present: “cGMP Synthetic Gene Synthesis to Enable Precision Medicine” (poster #320) on Wednesday, May 16 at 5:30 pm.  

Personalized medicines (or precision medicines) constituted 1 out of every 4 drugs approved by the US FDA over the past 4 years¹ and last year was no exception with 35% of FDA-approved “new molecular entities” classified as personalized medicines. Several concomitant factors have contributed to the growth of this burgeoning field, including the revolutionary improvements in genome sequencing technology, including advances in biomedical informatics and data analytics.

Human monoclonal antibody (mAb)-based targeted therapy toward various cancers constitutes a large portion of personalized medicines. Cancer immunotherapy often targets immune checkpoint proteins with the goal of increased T cell production to harness endogenous immune activity against cancer cells. One example is programmed death-ligand 1 (PD-L1), which is upregulated in certain cancers, causing a reduction in T cell activity, thereby contributing to uncontrolled tumor cell growth. Two fully human mAb therapeutics, avelumab and durvalumab, that target programmed death-ligand 1 (PD-L1), were approved last year. Bavencio (avelumab) was approved for the treatment of metastatic Merkel cell carcinoma and Imfinzi (durvalumab) was approved for the treatment of advanced urothelial carcinoma. Both of these therapeutics are considered personalized medicines since the decision to administer these agents can be based on PD-L1 expression levels in the tumors of patients.

While these medicines are considered “personalized,” these therapies are still based on predictive group outcomes. An entirely new type of personalized medicine is emerging, however, that tailors a specific treatment to an individual patient. One novel therapy includes targeting neoantigens or peptides found only on the surface of cancer cells for cancer vaccines. Although none have been approved to date, several neoantigen therapeutics are in development or early stage clinical trials. All of these neoantigen-based therapeutics rely on upstream DNA synthesis for development and production of the customized therapeutic.

As this relatively recent class of DNA-based pharmacological agents continues to emerge in research, development, and in clinical trials, the infrastructure to support these therapeutics must be established and validated. One of the biggest challenges in customized, personalized neoantigen treatment plans is the turnaround time required. In order to create a custom treatment, a patient’s DNA must be sequenced and analyzed to allow for the identification of the targeted neoantigens. Production of the customized therapeutics must then be in accordance with cGMP (current good manufacturing practice). Traditional methods can take several months, a time that is incompatible with successful intervention.

To address this need for the development and rapid production of customized, personalized medicines, SGI-DNA established a cGMP production laboratory for synthetic DNA constructs.

The addition of the BioXp 3200 System –a fully automated, genomic workstation that rapidly generates high-quality linear double-stranded DNA fragments and clones them into a vector using the Gibson Assembly^â method—will accelerate and streamline customized construct production. The qualification of the BioXp System for inclusion in the cGMP manufacturing is in progress.

To learn more about SGI-DNA instrumentation and the deployment of the cGMP suite, please visit us at the American Society of Gene and Cell Therapy Annual Meeting in Chicago, during the Oligonucleotide Therapeutics I Poster Session (#320) on Wednesday, May 16 at 5:30 pm.

References

1.        www.personalizedmedicinecoalition.org/Resources/Personalized_Medicine_at_FDA_An_Annual_Research_Report

A Year in Perspective: Nine Landmark Announcements Made by SGI-DNA and Synthetic Genomics in 2017

2017 has been another milestone year, witnessing the expansion of therapeutics-based platforms & services offered by Synthetic Genomics (SGI) and SGI-DNA. Some highlights from the past year include:

Paving the Way for Rapid Precision Medicine

• This past June, SGI-DNA added custom, on-demand current good manufacturingpractice (cGMP) gene synthesis, custom cloning, and DNA scale-up for clinical applications to the genomic services offering. The inaugural use of this state of the art cGMP laboratory is for Advaxis, a clinical-stage biotechnology company, and its upcoming Phase 1 clinical trial of ADXS-NEO, a personalized, neoantigen-targeted cancer immunotherapy.

The BioXp & Vmax Goes to Europe!

• The BioXp™ 3200 System made its European debut at the VIB Research Institute in Belgium where it is in use in the Thomas Jacobs laboratory for the development and optimization of plant genome editing using CRISPR/Cas systems. Since that initial placement, multiple additional BioXp™ instruments are now in use across Europe.  
• Vmax™ Express cells for recombinant protein expression and growth media are now available in electrocompetent and chemically competent formats directly from European-based distributors. This past September, SGI-DNA entered into key partnership distribution agreements with BioCat and Cambridge Bioscience to make this next generation protein expression system readily available to researchers thoughout Europe. 

Make Way for RNA Replicon!

• Replicon technology is a self-amplifying tunable RNA platform with the potential for use in the development of vaccines and therapeutics. As part of the Duke University-led DARPA Pandemic Prevention Platform program aimed at establishing a system capable of halting viral pandemics within 60 days, SGI is positioned for the rapid, automated assembly of large antibody genes. 
• In collaboration with Ceva Santé Animale, a leader in livestock health, SGI will utilize its next-generation synthetic RNA replicon platform to develop livestock vaccines.
• SGI entered into a collaboration with Arcturus Therapeutics, a leading RNA medicines company, to develop self-amplifying RNA-based vaccines and therapeutics for human and animal health. The collaboration relies on Arcturus’ LUNAR™ nanoparticle delivery system and SGI’s RNA replicon platform, with the ultimate objective of producing more efficacious vaccines and therapeutics.

Doubling Algae Biofuel Production

• After announcing their continued partnership at the beginning of the year, SGI and ExxonMobil reported in Nature Biotechnology, the engineering and development of an algae stain that more than doubles oil production compared to the native strain¹. This breakthrough announcement brings the teams one step closer toward realizing the goal of commercializing algae as a viable alternative fuel source.

Notable Publications

• The Pamela Silver laboratory at Harvard University, in collaboration with SGI scientists, utilized the personal genomic workstation, the BioXp™ 3200 System, as well as commercial DNA synthesis, to construct the largest genome for a cumulative bacterial recoding project to-date². This large undertaking (over 1500 codon replacements across 176 genes) paves the way toward creating a completely recoded organism for downstream therapeutic applications.
• Kent Boles and Krishna Kannan and their SGI teams built the Digital-To-Biological Converter (DBC) prototype, an instrument that is capable of on-demand construction of DNA, RNA, proteins, viral particles, and pharmaceuticals from DNA sequence information³. Ultimately, their invention could revolutionize the implementation of personalized medicine.

This has been an exciting year of innovation and advancement for SGI and SGI-DNA. We’ve continued to listen to our customers and broaden the scope of unique solutions to enable and accelerate scientific discovery. As 2017 draws to a close, we look forward to next year, brimming with the anticipation of more discovery and collaboration. 

Happy Holidays & Happy New Year from the SGI-DNA team! We wish you continued success and advancement in 2018.

References

1. Ajjawi I, Verruto J, Aqui M, Soriaga LB, Coppersmith J, Kwok K, Peach L, Orchard E, Kalb R, Xu W, Carlson TJ. Lipid production in Nannochloropsis gaditana is doubled by decreasing expression of a single transcriptional regulator. Nature biotechnology. 2017 Jul 1;35(7):647-52.
2. Yu Heng Lau, Finn Stirling, James Kuo, Michiel A. P. Karrenbelt, Yujia A. Chan, Adam Riesselman, Connor A. Horton, Elena Schäfer, David Lips, Matthew T. Weinstock, Daniel G. Gibson, Jeffrey C. Way, Pamela A. Silver; Large-scale recoding of a bacterial genome by iterative recombineering of synthetic DNA. Nucleic Acids Res 2017 gkx415. doi: 10.1093/nar/gkx415.
3. Boles KS, Kannan K, Gill J, Felderman M, Gouvis H, Hubby B, Kamrud KI, Venter JC, Gibson DG. Digital-to-biological converter for on-demand production of biologics. Nature Biotechnology. 2017 May 29.

Case Study: Automating DNA Assembly at the Synthetic Biology Center, MIT

Jake Becraft, Graduate Student in Ron Weiss Laboratory at MIT

“I work with repetitive DNA sequences that are incompatible with routine DNA fragment synthesis services. The BioXp™ System allows me to quickly build these complex fragments, with high fidelity, at my bench.”

Jake Becraft used the BioXp™ System to build complex DNA fragments needed for his RNA binding study. 

Background

The Weiss lab at Massachusetts Institute of Technology (MIT) has successfully created a variety of biological circuits involving RNA binding proteins. To investigate the specific binding properties of these RNA binding proteins, graduate student Jake Becraft wanted to create a library of variants. 

Problem

The DNA encoding the proteins Jake studies is complex, containing repetitive regions that make them difficult or expensive to synthesize commercially. 

Solution

Build the DNA variants in the lab using the BioXp™ System.

Results

The BioXp™ System successfully built 10 library variants. Following downstream assembly of the fragments with a plasmid backbone and two additional constant regions, Jake obtained all the error-free clones needed to perform his RNA binding studies.

Learn more about Jake’s BioXp success story

Aimee from SGI-DNA sat down with Jake Becraft and asked him to share more about his project and experience operating the BioXp System. 

Aimee: Can you explain the project you are working on, and how you used the BioXp System?

Jake: I’ve used the BioXp System a number of times. For one of my first projects, I used the BioXp System to create a variant library for investigating a type of RNA binding protein. Specifically, I created a small library of variants looking at switching 7 or 8 amino acids at the N and C terminal to investigate the RNA binding properties of the protein. I was able to take the BioXp fragments and place them directly into my cloning platform.

Aimee: How did the BioXp System enable your project?

Jake: I used the BioXp to generate DNA fragments that contain repetitive sequences. These fragments were too complex to be synthesized by a routine DNA synthesis service. The BioXp System allows me to build highly complex sequences in a short amount of time, at my benchtop, with high fidelity. It is quick easy to use, and requires little training. The BioXp System also saves money since the BioXp synthesis cost is lower than other synthesis providers.

Aimee: How many fragments did you create on the BioXp System?

Jake: For the RNA binding protein library project, I generated ten x 1200 bp DNA variants.

Aimee: What did you do with the BioXp library variants?

Jake: The proteins I study are similar in architecture to transcription activator-like effector nucleases (TALEN proteins), which contain highly conserved domains, including a repeat domain, as well as a variable region. The DNA encoding the domains can be constructed as ~1 kb fragments and assembled using Golden Gate technology. 

For my project, I collected the BioXp fragments and assembled them with two additional constant regions (~1 kb each) and a Golden Gate destination backbone using a hierarchal MoClo (modular cloning) system to create different modular variants. I picked three or four colonies per construct and was able to find the correct sequence even though the DNA I work with is highly repetitive, difficult to clone, and prone to mutation.

Looking ahead

Jake Becraft was one of the first people to realize the benefits of the BioXp System and continues to use the instrument for additional studies and areas of inquiry. Stay tuned for more stories from BioXp users like Jake.

To learn how the BioXp System can advance your research or additional capabilities, visit sgidna.com/bxp3200 or contact info@sgidna.com.

The Digital-to-Biological Converter: From Concept to Reality

In 2013, J. Craig Venter and Dan Gibson, in collaboration with Novartis, published a report in Science employing synthetic biology methods to rapidly accelerate the production of the flu vaccine. The methods described and implemented by the team demonstrated how to reduce flu vaccine production from months to just days. Yet, Venter and Gibson imagined there were ways to accelerate vaccine production even further, and so the idea of the digital-to-biological converter (DBC) was born.

Fast forward to May 2017, the DBC concept is now a reality as Kent Boles and Krishna Kannan and Synthetic Genomics teams worked to make Venter and Gibson’s vision a tangible, working machine. The DBC prototype described in the Nature Biotechnology paper integrates several key Synthetic Genomic technologies into one comprehensive instrument that is capable of building DNA, RNA, proteins, viral particles, and pharmaceuticals from DNA sequence information.

Illustrated above is the DBC and process from submission of DNA sequences to production of viral particles. Source: Nature Biotechnology

Central to the DBC prototype is the same DNA assembly process, including the Gibson Assembly® method and BioXp™ 3200 System, SGI-DNA’s automated instrument that builds DNA fragments and circular plasmids. The DBC adds the upstream capability of analyzing DNA sequence and designing requisite oligonucleotides with Archetype® software and synthesizing the determined oligonucleotides. The instrument then uses the deprotected oligonucleotides for DNA assembly and error-correction. In vitro transcription and translation are additional, integrated downstream processes added to the DBC capabilities. From entering the initial sequence into the DBC user interface, the remainder of the process operates completely hands-free without any human involvement.

Someday soon, after prototype refinement, it is conceivable that DBC instruments could become integral part of hospitals and clinics. After evaluating a patient, a doctor could use the DBC computer interface to request an appropriate treatment, such as insulin or a vaccine, and the instrument could produce the requested medicine on-site. Especially in the case of a pandemic or for rapidly mutating viral infections, such as those caused by influenza A, such a rapid and personalized treatment approach could be much more effective and beneficial than current treatment plans.

Refining and validating the DBC instrument prototype into a commercially available instrument will likely take two to three years, according to Dan Gibson. The full scientific report describing the technology and capabilities of the DBC is listed in the reference below. The DBC has also been featured in several media reports, including a San Diego Union Tribune article.

Reference

1.    Boles KS, Kannan K, Gill J, Felderman M, Gouvis H, Hubby B, Kamrud KI, Venter JC, Gibson DG. Digital-to-biological converter for on-demand production of biologics. Nature Biotechnology. 2017 May 29.

Bacterial genome recoding with the BioXp™ System and Gibson Assembly®

Introduction

Genome-modification technologies, including creating minimal synthetic genomes, gene editing through directed nucleases, such as CRISPR-Cas9 and genome recoding, offer targeted rational design and engineering at the whole organism level. Genome recoding (systematically altering targeted sense codons throughout a genome or defined genome section) is an important emerging synthetic biology field with many potential applications. Objectives of genome recoding include protein engineering with nonstandard amino acids; retooling organisms to generate bio-based pharmaceuticals, nutritionals, or vaccines; redesigning organisms with novel functions; generation of biocontainment methodologies; and creating synthetic organisms to serve as models for elucidating the basic principles of life.

Large-scale genome recoding

Recently, the Silver laboratory at Harvard University published a report describing the largest cumulative bacterial recoding project to date, in which 5% of the Salmonella genome was rewritten. Specifically, within two genomic regions spanning a total of 200 kb, every TTA codon was rewritten to CTA and every TTG codon was rewritten to CTG. These nucleotide changes were silent since all code for leucine. This proof-of-concept report, including the development of a novel method (SIRCAS) to achieve this large-scale feat, sets the stage for Silver’s laboratory to remove every TTA and TTG throughout the entire Salmonella genome. A genetically recoded organism (GRO) devoid of TTA and TTG, and their cognate tRNAs, would not have the ability to translate the missing codons and would be a genetic isolate, unable to properly translate DNA acquired from other cells, viruses, or plasmids. Potential applications of GRO bacteria include creating more effective live vaccines and acting as a living diagnostic and therapeutic agent in the human gut.

Achieving large-scale recoding

To accomplish such a sizeable undertaking, Pam Silver and colleagues purchased 2 to 4 kb synthetic DNA fragments containing the nucleotide changes from commercial vendors, including SGI-DNA. However, a portion of the fragments could not be synthesized commercially. A small fraction of these regions contained highly repetitive DNA which were PCR-amplified and mutagenized from genomic DNA. The remaining fragments were built in-house in the Silver laboratory using the SGI-DNA BioXp™3200 instrument. Unlike commercially synthesized DNA from a now defunct commercial vendor, DNA built on the BioXp system did not have any forbidden restriction enzyme site design constraints and did not contain any extraneous flanking sequences requiring downstream removal. Additionally, DNA built on the BioXp instrument could be conveniently built on site.

After obtaining all requisite DNA constructs spanning the target regions, the 1 to 4 kb DNA fragments were assembled into 10 to 25 kb segments using the Gibson Assembly® method. The 10 to 25 kb segments were then transformed into yeast spheroplasts. Successful assemblies were identified and used for rolling circle amplification and subsequent integration into Salmonella using a new method Silver's group developed called SIRCAS (step-wise integration of rolling circle amplified segments). This project resulted in recoding over 1500 leucine codons within the Salmonella genome and is the first non-E. coli bacterial recoding project as well as the largest bacterial recoding project reported to-date.

This study demonstrates a novel way to achieve large-scale bacterial genome recoding using SGI-DNA BioXp™ System and Gibson Assembly® technologies. Please click here to read the full report.

Reference

Yu Heng Lau, Finn Stirling, James Kuo, Michiel A. P. Karrenbelt, Yujia A. Chan, Adam Riesselman, Connor A. Horton, Elena Schäfer, David Lips, Matthew T. Weinstock, Daniel G. Gibson, Jeffrey C. Way, Pamela A. Silver; Large-scale recoding of a bacterial genome by iterative recombineering of synthetic DNA. Nucleic Acids Res 2017 gkx415. doi: 10.1093/nar/gkx415