Wednesday, September 27, 2017

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.

Wednesday, August 2, 2017

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.

Tuesday, June 27, 2017

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 BioXp3200 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


Thursday, March 9, 2017

Transformation of Gibson Assembly Constructs

With spring right around the corner, we're in a time of transformation. So, it seems like a fitting time to discuss another kind of transformation -- transformation of Gibson Assembly® constructs. As all researchers know, transformation is a critical step in all cloning and assembly reactions. Here, we’d like to take a moment to address some of the ways you can maximize success.

Tips for Transformation

Since Gibson Assembly® cloning has the capability to assemble multiple fragments simultaneously resulting in complex assemblies, it is especially important to use high efficiency competent cells for transformation. Electroporation yields high transformation efficiencies, and it is often the preferred method for labs carrying out the most complex assembly reactions. For labs that do not have access to electroporation equipment or for more routine assemblies, transformation with high efficiency chemically competent cells can also be used with success.

High Cloning Efficiencies


As shown in the image above, we achieve cloning efficiencies of over 90% when assembling 2, 5, or 6 fragments with the Gibson Assembly Ultra kit, followed by electroporation into TransforMax™ EPI300™ Electrocompetent E. coli (Epicentre® Cat. No. EC300110). We have a long history of performing electroporation with EPI300 cells, and they offer a useful advantage of compatibility with large, inducible clones. 

But what about other transformation options?

Comparing Competent Cells

We have previously demonstrated that Gibson Assembly constructs can be successfully transformed into a wide variety of competent cells. The results of those studies can be found in the Application Note “Gibson Assembly® HiFi 1 Step and Ultra Kits are Compatible with Multiple Electrocompetent and Chemically Competent Cells”.

A list of the different types of competent cells, their respective transformation conditions, and observed transformation efficiencies is shown in the following table.




As you can see, Gibson Assembly
® cloning is compatible with a wide range of competent cells, yielding baseline transformation efficiencies in the 108 and 109 range.

Lucigen E.cloni® 10G Cells As A Low-Cost Alternative

Recently, we performed a side-by-side transformation comparison using EPI300™ cells and chemically competent E. cloni® 10G cells (Lucigen Cat. No. 60107). The results of that study can be found in an Application Note entitled “High-efficiency, low-cost transformation of Gibson Assembly constructs”. In that study, we showed that 10G cells offer a low-cost alternative for high efficiency transformation, yielding more transformants with 10G chemical transformation than EPI300™ electroporation. 




Gibson Assembly constructs can be successfully transformed into a wide variety of competent cells. For detailed protocols, please refer to our Gibson Assembly
® HiFi 1-Step or Ultra User Guides and Application Notes. Learn more about Gibson Assembly products at sgidna.com.

Tuesday, February 7, 2017

Change Your Workflow With These 7 Innovative Products

With 2017 fully under way, researchers around the world are taking a hard look at their ambitious milestones. How can we improve our processes? How can we meet our time-sensitive deadlines?

Offering a multitude of solutions, SGI-DNA seeks to address those questions with the following list of new and innovative products.

7 Innovative Products From SGI-DNA That Could Change Your Workflow

 

Cell Lines  

  • Vmax™ Express enables the production of significantly more recombinant protein (up to 4X), a full day faster than E.coli protein expression systems.
  • Syn2.0™ Minimal Synthetic Cells are engineered cells with a genome smaller than that of any autonomously replicating cell found in nature. These cells have many potential applications, notably as a platform for investigating core functions of living organisms. Syn2.0 Minimal Synthetic Cells have a genome of approximately 576 kb and a doubling time of about 92 minutes.
  • Syn3.0™ Minimal Synthetic Cells, like Syn2.0, have many potential applications as well. Syn3.0 Minimal Synthetic Cells have a genome of approximately 531 kb and a doubling time of about 180 minutes.

BioXp System Applications

  • The Custom Cloning Module on the BioXp™ System allows you to clone up to 32 unique, synthetic genes directly into your vector-of-choice in an automated, hands-free, overnight run, eliminating the need for sub-cloning.
  • The BioXp™ NGS Library Construction Kit automates and simplifies next-generation sequencing libraries for up to 8 or 16 samples. Built for Illumina sequencing platforms, it enables hands-free construction of barcoded libraries in under 5 hours.
  • The Bio360™ Minifuge is compact and economical microcentrifuge for tubes and strips, is the ideal companion to the BioXp System.

Reagents

  • XactEdit™ Cas9 Nuclease kits and enzymes allow you to perform targeted guide RNA-directed double-stranded DNA cleavage. For added flexibility, the enzyme is available in normal and concentrated formats and the corollary kit includes a positive control, making it an ideal choice for first-time users.

What's Next for SGI-DNA?

In 2017, we're looking forward to several new products and their exciting applications, especially with regards to the Vmax Express product line. In the meantime, if you would like to know how to get #DNAMyWay download your copy of the Gibson Assembly® Cloning Guide today.

Monday, December 19, 2016

Gibson Assembly® PBnJ™ Overhang Extension and Sequence Insertion

Using the Gibson Assembly® method to introduce overhangs and insertions


In the Gibson Assembly® Cloning Guide and our last blog post, we introduced a variation of the Gibson Assembly® method that does not rely on the use of homologous overlapping ends for fragment assembly. This technique, Gibson Assembly® PBnJ™ Cloning, has many potential applications. Here, we’d like to focus on two of these, adding DNA overhangs and insertions.

Creating overhangs

To create a 3’ overhang, simply design a primer containing the intended overhang sequence. To protect the primer from chew-back during the assembly process, include four phosphorothioate modifications at the 3’ end of the primer. Creation of a fragment with the 3’ overhang is initiated by combining the single phosphorothioate-modified primer, your corresponding DNA fragment-of-interest, and Gibson Assembly® Ultra Master Mix A. The Gibson Assembly® Ultra procedure yields a DNA fragment containing a 3’ overhang, as shown in the following illustration.


Inserting DNA sequence between fragments during assembly

Insertions can be added during cloning using another variation of this technique. Mutagenesis, promoter or enhancer functional analysis, and large-scale genome modification studies, or even simply adding a short sequence of interest (i.e. gRNA target sequence, barcode, restriction sites, etc.) are all potential applications of Gibson Assembly® PBnJ™ Sequence Insertion Cloning. Gibson Assembly® PBnJ™ Sequence Insertion Cloning adds sequence between adjoining fragments during assembly. 

As shown below, appropriately designed primers are critical to the outcome of the assembly reaction. One primer is designed to contain homology to one of the fragments, and another primer is designed with homology to the other fragment. The insertion sequence must be added to the 3’ end of both primers and is necessary to bridge the fragments to be joined since this is the only region containing homology between the fragments. Both primers are synthesized with at least four phosphorothioate bonds at the 3’ termini to protect them from chew-back during the assembly reaction. The assembly reaction is initiated by combining DNA fragments, appropriate phosphorothioate-modified primers, and Gibson Assembly® Ultra Master Mix A. Following 3’ chew-back mediated by Master Mix A, the reaction undergoes heat inactivation and denaturation, which allows for the annealing of the bridge/insertion region of the primers. Strand extension is then mediated by Ultra Master Mix B resulting in seamless assembly of fragments with an insertion.




Learn more about Gibson Assembly® products at www.sgidna.com/reagents or email us at info@sgidna.com for assistance with Gibson Assembly® PBnJ™ Cloning.

Read the full Gibson Assembly® PBnJ™ Cloning Series.

Thursday, December 1, 2016

How to Perform Gibson Assembly® Cloning With Blunt-Ended Fragments

Introduction

The Gibson Assembly® method is a cloning technology that allows researchers to join DNA fragments, generating seamless constructs into any vector without the need for restriction sites in a single round of cloning. The principle of the Gibson Assembly® method relies on homologous overlap sequence designed into the fragments to be joined. Generating DNA fragments with these homologous regions is accomplished by PCR amplification or DNA synthesis prior to the assembly reaction.

However, what if you have DNA fragments that do not have overlapping regions or cannot be easily amplified by PCR or synthesized? Can you still perform Gibson Assembly® cloning without adding homologous overlaps? Yes, you can. Instead of the standard Gibson Assembly® approach, you can simply use modified oligonucleotides and perform what is known as Gibson Assembly® Primer-Bridge End Joining™ (PBnJ™ Cloning).

Our Gibson Assembly Cloning Guide introduces Gibson Assembly® PBnJ™ Cloning. With the PBnJ™ Cloning approach, researchers can easily clone DNA fragments without overlapping ends. Using simple modification steps, the Gibson Assembly® PBnJ™ Cloning method utilizes single primers or primer pairs with phosphorothioate-modified 3’ ends and the Gibson Assembly® cloning kits, which allows researchers to benefit from both the speed and efficiency of Gibson Assembly® cloning.

What can I do with the Gibson Assembly® PBnJ™ method?
  • Assemble large DNA fragments with non-homologous ends
  • Assemble fragments that are difficult to PCR amplify
  • Assemble parts from a library without introducing PCR-mediated errors
  • Edit (add or delete) sequences at junctions based on primer design
  • Generate unique 3’ overhangs for standard cloning
How does the Gibson Assembly® PBnJ™ method work?

Gibson Assembly® PBnJ™ Cloning relies on the stepwise activities of the Gibson Assembly®Ultra Kit, followed by the Gibson Assembly®HiFi 1-Step Kit. For Gibson Assembly® PBnJ™ Cloning, instead of designing primers to generate homologous overlap regions, a primer pair is used to bridge two adjacent fragments. The primer pairs contain phosphorothioate-modified 3’ ends, which protect the primer from 3’ exonuclease chew back activity during assembly. After template chew back, the primers anneal to the nonoverlapping, single-stranded template sequence, which is later extended and ligated by the 5’ to 3’ polymerase activity of the GA HiFi 1-Step Master Mix. Gibson Assembly® PBnJ™ Cloning can also be adapted to create fragments with 3’overhang extensions or insertions between fragments. Look for more information about these variations next week.

Learn more about Gibson Assembly® products at www.sgidna.com/reagents or email us at info@sgidna.com for assistance with Gibson Assembly® PBnJ™ Cloning.

Read the full Gibson Assembly® PBnJ™ Cloning Series.