News and Events

The Gibson Assembly^® method was first developed by Dr. Dan Gibson and his colleagues at the J. Craig Venter Institute. With Dr. Gibson’s expertise, they successfully synthesized a 583kb Mycoplasma genitalium genome in 2008 and a 1.2 million bp Mycoplasma mycoides genome in 2010, the largest synthetic genome to date. The team overcame the limitations of traditional cloning by developing the Gibson Assembly^® method, which allows for seamless DNA assembly without the presence of restriction enzyme sites. Since the likelihood of unwanted restriction sites increases with the length of the sequence, large genomic sequences could not be assembled prior to this method.

Assembly in Stages

The Gibson Assembly^® reaction relies on a mix of enzymes (exonuclease, polymerase, and ligase) that anneal overlapping ends between DNA fragments in an isothermal reaction. The team built the synthetic genome in several stages: 1000 bp cassettes were assembled from overlapping synthetic oligos, which were recombined in sets of 10 to produce 10,000 bp assemblies. The 10kb assemblies were further recombined to produce 100kb assemblies. From there, these fragments formed the complete 1.2 million base pair genome.

Creation of a Bacterial Cell Controlled by a Chemically Synthesized Genome

Dr. Gibson joined Synthetic Genomics Inc. in 2011 after more than 6 years in JCVI. He brought the synthetic genomic tools developed at JCVI to SGI, leading to the development of automated DNA synthesis, assembly, and error correction technology used today at SGI-DNA.

1 Step Away From a Perfect Clone

The Gibson Assembly^® method has been developed into a high fidelity 1 step reagent kit by Dan Gibson’s team at Synthetic Genomics Inc. SGI’s Gibson Assembly^® HiFi 1 Step Kit is now available for purchase through SGI-DNA.

Vibhu Gupta, Senior Product Manager at SGI, says "the main advantage of the new formulation compared to other commercial kits is its ability to accurately assemble multiple fragments with greater than 90% cloning efficiency." The team's experimental results show that the high fidelity formulation ensures assembly junctions are error free, resulting in more perfect clones and lower sequencing costs. The kit is also capable of assembling large genome sized fragments up to 100kb.

Commercial licensing is now available to companies interested in assembling synthetic plasmids, pathways and genomes that result in commercial products.

SGI-DNA uses the Gibson Assembly^® method today to assemble large and complex DNA constructs, delivering synthetic genomes and pathways to its commercial customers. The Gibson Assembly^® technology will be prominently featured in SGI’s future product launches. Stay tuned for more news.

The International Genetically Engineered Machine (iGEM) Foundation is dedicated to advancing synthetic biology education and collaboration within the scientific community. In 2012, iGEM spun out of MIT and became a non-profit organization based in Cambridge, MA. iGEM fosters scientific research and education by organizing competitions for students worldwide. Last weekend, the Giant Jamboree brought over 2500 students and researchers to Cambridge to compete in the largest synthetic biology competition in the world.

Spin the wheel to win SGI-DNA prizes.
This Harvard student is one of our grand prize
winners, he won an Apple TV!

As a leader in synthetic biology, Synthetic Genomics works closely with iGEM to provide gold level sponsorship and biosecurity support. Using SGI's Archetype software, Toby Richardson screens iGEM's synthetic genes against regulated pathogenic sequences for every competition. These guidelines are implemented for every gene order submitted to SGI-DNA as well. As a member of the International Gene Synthesis Consortium (IGSC), we enforce strict international biosecurity guidelines for every gene synthesized, thus promoting the beneficial application of gene synthesis technology.

Team UCSD and SGI-DNA at iGEM's Giant Jamboree

SGI is also proud to sponsor the only UC San Diego team to compete in the Giant Jamboree this year. The UCSD team consisted of a variety of graduate and undergraduate students specializing in Bioinformatics, Cell & Molecular Biology, Bioengineering, Mathematics, Biochemistry, and Computer Science. The team developed a web tool called SBiDer: Synthetic Biocircuit Developer, that can bypass the months required to design, build and test a genetic circuit. SBiDer leverages existing devices to construct a database that can explore circuit designs. Users can search for a displayed circuit's literature reference, characterization data, and images through a powerful visual interface.

It was impressive to see the creativity and applications behind each synthetic biology project at this year's Giant Jamboree. The accomplishments of these students are sure to lead to advancements in medicine, food & nutrition, environment, energy, software, and more. It was a great pleasure to meet and educate the next generation of synthetic biology scientists!

LA JOLLA, Calif., Nov. 20, 2014 /PRNewswire/ -- SGI-DNA announces the launch of the Gibson Assembly® HiFi 1 Step Kit which allows researchers to assemble multiple, large DNA fragments in a simple one hour, one-step reaction.

This is the first high fidelity kit on the market using the Gibson Assembly® method. Researchers can now assemble up to 5 fragments, including large constructs (up to 100 kb), in a fast, reliable reaction.

"With the Gibson Assembly® HiFi 1 Step Kit researchers can obtain a perfect clone in their initial attempt," says Vibhu Gupta, Senior Product Manager at SGI-DNA. "The kit provides a proprietary enzyme formulation for Gibson Assembly® that results in significantly less errors at cloning junctions which is a common challenge for researchers in a variety of disciplines."

A researcher from the University of California, San Diego, Erna van Niekerk, PhD., comments, "I have tried it with a construct and ran a parallel experiment with a competing kit from another vendor. I picked 3 colonies from the Gibson Assembly® HiFi 1 Step Kit and 3 colonies from the competing product. Three of 3 Gibson clones gave me the right insert. Zero of 3 from competing kit gave me the right insert. Of the 3 Gibson clones: 2 of 3 had the correct sequence both forward and reverse sequencing confirmation and 1 of 3 had an extra nucleotide."

The Gibson Assembly® method was developed in 2009 by Dan Gibson, Ph.D., and his colleagues at the J. Craig Venter Institute during the team's quest to construct the first synthetic cell. It uses a one-step, isothermal approach to enable the rapid assembly of oligonucleotides and multiple DNA fragments. Since its introduction to the life science community, the Gibson Assembly® method has become a mainstay in many synthetic biology laboratories and has attracted interest from both the academic and commercial life sciences community due to its ease-of-use, robustness and flexibility. This Gibson Assembly® kit provides researchers SGI's first commercially available option utilizing the Gibson Assembly® method.

The Gibson Assembly® HiFi 1 Step Kit is available in 3 sizes: 5 reactions (starter kit), 10 reactions and 50 reactions.

For more information on the Gibson Assembly® HiFi 1 Step Kit, please visit www.sgidna.com

All products are intended for research use only. Not intended for diagnostic uses.

The Gibson Assembly® method is also available under commercial license. For more information contact us at info@sgidna.com

About SGI-DNA

SGI-DNA, a wholly owned subsidiary of Synthetic Genomics, Inc (SGI), is responsible for all commercial aspects of SGI's synthetic DNA business and focuses on strategic business relationships with both academic and commercial researchers. Building on the scientific advancements and breakthroughs from leading scientists such as J. Craig Venter, Ham Smith, Clyde Hutchison, Dan Gibsonand their teams, SGI-DNA utilizes unique and proprietary DNA technologies to produce complex synthetic genes and reagents. SGI-DNA also offers a comprehensive suite of genomic services, including whole genome sequencing, library design, and other bioinformatics services.

SGI-DNA Technical contact: Vibhu Gupta, Senior Product Manager, 858.433.2288; vgupta@syntheticgenomics.com

Media contact: Heather Kowalski, hkowalski@syntheticgenomics.com; 858-361-0466

Logo - http://photos.prnewswire.com/prnh/20141120/160071LOGO

SOURCE Synthetic Genomics Inc.

RELATED LINKS
http://www.syntheticgenomics.com

SGI-DNA was delighted to join other synthetic biology enthusiasts at SynBioBeta San Francisco last week, where Craig unveiled the latest advances from Synthetic Genomics during his powerful keynote speech.

Engineering Pig Genomes
Among the most provocative endeavors presented by Craig was SGI's collaboration with United Therapeutics to transplant engineered pig organs into humans. With SGI's expertise in DNA design, synthesis, and genome editing, our scientists are rewriting the pig genome to deliver engineered cells for United Therapeutics to generate pigs embryos with humanized lungs. Although the project is still in its early stages, researchers have shown that baboons can survive with xenotransplanted lungs for up to one year by changing just a few genes.

SGI and United Therapeutics Collaborate to Humanize Pigs for Organ Transplants

Synthetic Flu Vaccines
Another notable program at SGI is the development of synthetic flu vaccines with Novartis. When China's CDC made the latest H7N9 viral genes available online on April 2013, the research teams quickly applied synthetic genomic tools and technologies to generate initial vaccine seeds in just 6 days. This was the first real time demonstration of pandemic preparedness utilizing synthetic gene assembly, dramatically improving the pandemic response by increasing the speed and overall yield of vaccines during production. Clinical trial results reported by Novartis were positive, with 85% of subjects immunologically protected after receiving the second dose of cell culture vaccine (combined with synthetic adjuvant MF59). The vaccine is now in large scale production.

Influenza Genome Sequencing Project Locations

In Craig's vision for vaccine production, hospitals and pharmacies around the world will simply download the latest viral genome and convert it to a vaccine for distribution, via our Digital to Biological Converter.

The Future of DNA Assembly
Input - digital genetic code
Output - vaccine & other therapeutics

DNA on Demand
For now, the advances made by instrumentation engineers at Synthetic Genomics will be demonstrated through the BioXp 3200, the first automated DNA assembly instrument of its kind. More details to follow...

Assemble up to 32 constructs (plus control) with the BioXp 3200

"Scientists have to be optimistic about their ability to change the world in the face of people who resist technology" -J. Craig Venter

Seasons Greetings! Our product development team has kindly provided these tips to maximize the efficiency of your Gibson Assembly® reactions with SGI's Gibson Assembly® HiFi 1 Step Kit. If you have any other detailed questions please contact techservices@sgidna.com, we are happy to work with you!

Recommendations for Designing Homologous Overlap Regions
The optimal length of the overlap region depends on the number and length of the fragments in the assembly reaction. Longer overlap regions will result in higher efficiency for large fragment assemblies. You may need to optimize PCR amplification reactions when using PCR primers with long homologous overlap regions.

Vector Selection
We recommend using a high copy number vector such as pUC19 for small insert(s) (< 10 kb) or non-toxic gene(s). For large insert(s) or toxic gene(s), use a low copy number or inductive vector such as pBAC.

Your DNA Concentration Does Matter!
Make sure to use DNA samples with A260/280 > 1.8. For optimal results, use DNA at a concentration ≥ 40 ng/μl. If the amount of DNA is limited, the assembly reaction may be performed using DNA at concentrations between 20–40 ng/μl with reduced efficiency. Do not use DNA at concentrations < 20 ng/μl. Always use a high fidelity PCR polymerase when amplifying your DNA.

Don't Forget to Purify Your DNA
For vector DNA prepared by PCR, make sure to gel-extract the linear product to minimize vector background. Purify DNA inserts by column or ethanol purification to remove restriction or PCR enzymes that could interfere with the Gibson Assembly® method. If your PCR reaction or synthetic assembly yields non-specific fragments, we recommend purifying the fragment of interest using gel extraction or another size fractionation method.

Running Your Reaction

Use a vector: fragment ratio of 1:1 to 1:5, depending on the size of the insert. For a fragment that is < 1 kb, we recommend using a 5 fold excess. Our HiFi kit has been optimized for a reaction volume of 5 μL substrate DNA and 5 μL of GA 1 Step Master Mix (2X).

Transformation Tips
Transformation of the Gibson Assembly® product yields the most clones with high efficiency electrocompetent cells of 1010 cfu/μg of pUC19 plasmid. We recommend using Epi300™ electrocompetent cells (Epicentre® EC300-105).

Dilute the assembled product 1:5 with nuclease free water before transformation. Note that the excess salt in the assembly reaction may decrease the transformation efficiency if not diluted.

Always plate two plates (one low and one high volume). Although the efficiency of the assembly reaction may decrease with increasing number of insert fragments, the cloning efficiency should remain the same. Fewer observed colonies may not be indicative of an inefficient transformation.

We acknowledge that some of you may run a "dirty" experiment every now and then, but we suggest you follow these tips to get the most successful reactions out of your Gibson Assembly® HiFi 1 Step Kit. For more help with your Gibson Assembly® kits, please contact techservices@sgidna.com We hope you have a productive and happy new year!

LA JOLLA, Calif., Jan. 21, 2015 /PRNewswire/ -- SGI-DNA announces the launch of the Gibson Assembly® Ultra Kit which allows researchers to perform complex, large, and multiple fragment assemblies in a simple, two-step reaction.

"With the Gibson Assembly® Ultra Kit, researchers can assemble up to 15 fragments at one time in less than two hours," says Vibhu Gupta, Senior Product Manager at Synthetic Genomics, Inc. "The kit provides a proprietary enzyme formulation for Gibson Assembly®, allowing researchers to assemble fragments ranging from 100 bp to 100 kb.""Our goal is to provide researchers with cutting-edge, easy to use, and seamless technology that takes synthetic biology to a whole new level."

This Gibson Assembly® Ultra Kit is the second kit in SGI's growing Gibson Assembly® portfolio utilizing the Gibson Assembly® method. The first kit launched was the Gibson Assembly® HiFi 1 Step Kit, a high fidelity formulation that significantly reduces errors/mutations at the cloning junctions, enabling researchers to assemble up to 5 fragments in a one hour, one-step reaction.

The Gibson Assembly® method was developed in 2009 by Dan Gibson, Ph.D., and his colleagues at the J. Craig Venter Institute during the team's quest to construct the first synthetic cell. Since its introduction to the life science community, the Gibson Assembly® method has become a mainstay in many synthetic biology laboratories and has attracted interest from both the academic and commercial life sciences community due to its ease-of-use, robustness, and flexibility.

The Gibson Assembly® Ultra Kit is available in 3 sizes: 5 reactions (starter kit), 10 reactions and 50 reactions. Mastermixes are sold separately.

For more information on the Gibson Assembly® Ultra Kit, please visit www.sgidna.com/ultra_kit

For more information on the Gibson Assembly® HiFi 1 Step Kit, please visit www.sgidna.com/hifi_kit

All products are for research use only and are not intended for diagnostic uses.

The Gibson Assembly® method is also available under commercial license. For more information contact us at info@sgidna.com

About SGI-DNA

Technical Contact
Vibhu Gupta: vgupta@syntheticgenomics.com, 858.433.2288

Media Contact
Heather Kowalski: hkowalski@syntheticgenomics.com, 858-361-0466

Logo - http://photos.prnewswire.com/prnh/20141120/160071LOGO

SOURCE Synthetic Genomics Inc.

Traditional restriction digestion cloning can often be tedious and ineffective. Watch the video below to learn about a fast and efficient way to clone with SGI-DNA's Gibson Assembly® HiFi 1 Step Kit. Assemble 5 DNA fragments with confidence with our 1 hour, 1 step protocol - without the use of restriction enzymes.

With SGI-DNA's Gibson Assembly® HiFi 1 Step Kit, you are just one step away from your perfect clone.

https://sgidna.com/gibson_videos.html

A New Hope: The BioXp

Dr. Matthew Lux holds the 3D printed Imperial Death Star.

Not long ago in a laboratory not so far away…

Dr. Matthew Lux, an informatics expert, started researching DNA’s storage capabilities. In 2013, Lux submitted a proposal to the Edgewood Chemical Biological Center (ECBC)’s In-house Laboratory Independent Research Program (ILIR). Now, the DNA CODE has risen from translated binary code and can now reside in bacteria, the last resource for information storage.

With the support of SGI-DNA’s BioXp, Dr. Lux leads a brave new line of research to restore peace and accelerate discovery to the galaxy. The US Army’s ECBC has sent its most daring informatics expert on a not-so-secret mission, to explore the potential of microbial memory and encode plans for the imperial death star. It is a time of hope for gene synthesis.

Download the ECBC Article Featuring Dr. Lux’s Research (pdf).

BioXp 3200 System, New #DNAMYWAY Bags
and DNA on TAP Glasses at #SLAS2016

We're excited to be gearing up for the Society of Laboratory Automation and Screening 2016 conference in San Diego next week, here is what we have scheduled:

Monday

10:00 a.m.-6:30 p.m.: Exhibit Hall Booth #701 featuring live BioXp™ System demos and new #DNAMYWAY bags (see image)
3:00-3:45: Room 16B: Press Briefing on the launch of the BioXp™ System cloning module (press release) featuring the world premiere of the BioXp™ System video

Tuesday

9:30 a.m.-6:00 p.m.: Exhibit Hall Booth #701 featuring live BioXp™ System demos
2:00 p.m.-2:30 p.m.: Steve Riedmuller, SGI-DNA Senior Director, Field Applications, will be at the SLAS member center presenting his participation in a publication on a synthetic yeast artificial chromosome
6:00 p.m.-9:00 p.m. DNA on TAP, 275 Fifth Ave, network and enjoy free food and drink with real BioXp™ System users and get a free DNA on TAP pint glass!

Wednesday

9:00-1:00: Exhibit Hall Booth #701 featuring live BioXp™ System demos

See you in San Diego next week, be sure to follow us on Twitter for live updates!

SGI-DNA's recently published Gibson Assembly^® Cloning Guide contains 40 pages filled with protocols, tips, and FAQs to help molecular biologists use this revolutionary technology. We also provide you perspective and an understanding of the decades of work in recombinant DNA and molecular biology that led to the discovery and development of Gibson Assembly.

As part of our new #DNAMYWAY blog series, we'll provide information on advances in synthetic biology, updates on new protocols, emerging applications as well as other interesting information such as the DNA cloning timeline below. This historical view on key discoveries that have advanced our ability to study the function of DNA can be found in the Gibson Assembly^®Cloning Guide.

Each milestone in the diagram below is represented by a vertical bar, and if you've seen the design of the BioXp™ 3200 System, these bars will look familiar to you, they represent DNA fragments being joined. We've linked each milestone on the timeline to the respective publication(s) so you can learn about the events leading up to the 2009 publication on Gibson Assembly® by SGI's own Dan Gibson. Hover over the milestone with your mouse and left click, the publication will open in a new tab.

A Brief History of Molecular Cloning and Synthetic Genomics

If you want to learn more about using Gibson Assembly® in your lab, review any of the 1200+ articles that reference the 2009 publication . These labs have said goodbye to traditional cloning methods and have chosen Gibson Assembly. Now they can add from 1 to 15 fragments into the vector of their choice a single round of cloning without the need for restriction digestion or subcloning. Get started by requesting your copy of the free Gibson Assembly® Cloning Guide and you'll be well on your way to getting #DNAMYWAY.

Gibson Assembly® Design Strategies 101: Primer Design & Homologous Overlaps

To help you create fragments with appropriately designed overlaps, SGI-DNA has gathered some helpful primer design strategies to keep in mind when using PCR to generate DNA fragments for your own Gibson Assembly® cloning reactions.

Tip #1: Design ends with 20-80 bp homologous overlaps

Remember when designing your homologous overlap that the length varies based on fragment length and number. Use the wrong fragment length, and you risk your DNA fragment not assembling properly.

The table below shows the overlap length needed based on fragment length and number.

Tip #2: Consider the lengths and the numbers of fragments

The optimal length of the overlap region depends on the number and length of the fragments in the assembly reaction. Longer overlap regions will result in higher efficiency when working with multiple fragments or longer fragments.

Tip #3: Examine the sequence of your overlaps

Avoid tandem repeats, homopolymers, high secondary structure, and extremely high or low GC content. All of these can result in an efficient annealing of your overlapping regions. If you encounter these regions, you may want to consider selecting a different region.

Tip #4: Design PCR conditions appropriate for the Tm of your primers and fragment size

To get a single amplification product when generating overlaps by PCR, it may be necessary to optimize PCR amplification reactions when using PCR primers with long homologous overlap regions.

Tip #5: Add useful sequences to your overlaps when needed

Adding a restriction enzyme site to primers between the overlap region and the sequence-specific segment enables subsequent release of the insert from the vector. In this case, be certain that the restriction enzyme site introduced in the primers is not also present within the insert.

Want to make your primer design even easier? Check out the Gibson Assembly Primer® Tool.

Download the Gibson Assembly® Cloning Guide for additional tips and tricks for performing Gibson Assembly® cloning.

“Imagine any lab building anything they need with no human intervention beyond loading the machine.” –Katie Lyons

Synthetic Genomics (SGI) Scientist, Katie Lyons, recently spoke at the 2016 Synthetic Biology: Engineering, Evolution, and Design (SEED) conference in Chicago. For those who were not able to make it to hear the talk, here is a summary of her presentation.

Historically, life science researchers have been completely dependent on a template. If they were lucky, researchers could get a template from a culture collection or from a fellow researcher; otherwise, they would go bioprospecting to the ends of the earth (or Mars) for their samples. With recent advancements in synthetic biology, all you need today is a sequence. Next generation sequencing has enabled a wave of information to be made readily available.

Lyons quipped “Now you may need to curb your enthusiasm when you design your experiments.” She cited three major milestones for synthetic biology from J. Craig Venter Institute (JCVI) and SGI labs: first synthetic genome of Mycoplasma genitalium; the first self-replicating cell – Mycoplasma mycoides; and the first minimal bacterial genome.

“All of this was done by people with great hands, spending a lot of time doing the work,” said Lyons.

All of these advancements led to a need to automate key processes. We have reached a point where machines, such as the BioXp™ 3200 System, can do this work instead: build oligos to genes overnight.

Lyons spoke about the key considerations for building genes in today’s world of automation. The BioXp™ System’s analytical tools are designed to make decisions based on the sequence characteristics and, while the machine itself is capable of complex processes. It yields predictable and reliable results, and it is user friendly, so that any lab working with DNA would be able to use the machine. But most importantly, the machine is adaptable, and new protocols are being continually modified and improved upon.

What happens inside the box? Oligonucleotide assembly, error-correction, to purification.

Since its launch in 2015, SGI-DNA has added cloned plasmids to the product offering. Lyons announced the newly expanded complexity feature, which means that the machine is now able to build a wider range of sequence types: 20-70% GC content for 400 bp to 1 kb and 40-60% GC content for 1-1.8 kb.

Next on Lyons’s to-do-list: working on higher fidelity, improved target yields, larger construct size, and expanded downstream capabilities.

Giving a real world example of how powerful this process can be, she spoke about the H7N9 influenza outbreak in 2013. Rather than wait for the virus to make it to the US from China, researchers at JCVI used the sequence information that was available online and began to synthesize the genes de novo. Gene synthesis took less than 24 hours and they had viral seeds well over a week before CDC received wild type virus from China. Lyons calls this rapid response a game changer. This process allowed for the vaccine to be stockpiled in December before the second wave of the virus hit. Now with the BioXp™ 3200 System, this process can become even more efficient.

Since its launch, the BioXp™ 3200 System has continued to evolve, expanding its capabilities. The world has not seen the end of what this machine is capable of. Lyons concluded her talk by saying, “It does not escape my attention that I am building my replacement… I have 4 machines on my bench that do all my work for me all the time now, and I would not trade them for the world.” With the BioXp™ 3200 instrument, rather than trudging through the weeks of tedious protocols, scientists are now freed to focus their energies on thinking about strategy and the next steps of their experiments.

----

Katie Lyons is a scientist in the DNA Technologies group at Synthetic Genomics (SGI), tirelessly working on developing new features for the BioXp™ 3200 system.

Cloning has evolved since the early 1970s, when John F. Morrow and Herbert Boyer first cloned eukaryotic genes into bacteria. This technology has enabled scientists to discover entire genetic sequences of many species and pharma to develop life-saving drugs, such as insulin, clotting factors, and human growth hormones using cloned genes.

While there are multiple cloning methods now in use, here is a brief compilation and comparison of some commonly used approaches.

Overview of Cloning Methods

Restriction enzyme digest, a classic method, that uses an endonuclease to cut DNA at specific sequences. The enzymes cut double stranded DNA into fragments, allowing for two pieces of DNA can then be fused together during a ligation reaction.
Recombination based cloning or Gateway™ cloning uses recombination sites to insert DNA into a vector. The reaction moves a piece of DNA from one plasmid into another and is done via a single recombination reaction.
TA cloning (PCR fragment cloning) with Taq polymerase leaves a single adenosine (A) overhang on the 3' end of PCR products. This cloning method requires a cloning vector modified to have a T or a U residue on its ends.
Gibson Assembly® Method (isothermal assembly) is a technique that uses overlapping ends to allow for the rapid and seamless insertion of multiple DNA fragments into a plasmid DNA vector.

Cloning Method Comparison Chart

Cloning methods are distinguished by many different features. The chart below details the cloning methods vary by methodology, efficiency, insertion site, ability to clone multiple DNA fragments at a time, vector compatibility, and directionality.

While each method has its benefits for various workflows, the Gibson Assembly® method provides the most comprehensive set of advantages over the traditional cloning methods.

For example, Gibson Assembly® method can be used for simultaneous assembly of multiple inserts and offers substantial time-savings for multiple insert assembly projects. The figure below illustrates how Gibson Assembly® Method is faster and more efficient than traditional approaches.

Advantages of the Gibson Assembly® Method

Faster and more flexible than traditional cloning: 60 to 80 minutes from start to transformation
No need to rely on compatible restriction sites
Can clone into any vector at any site
No need to rely on specific vectors
Clone single or multiple fragments in the same round of cloning
Avoids subcloning
Works for fragments from 100 bp to 100 kb
High cloning efficiencies

>90% using one step method
>95% using two step method

Visit SGI-DNA's webpage to learn more about Gibson Assembly® kits and download an electronic version of SGI-DNA's Gibson Assembly® Guide to learn about all the ways you can get #DNAMYWAY.

Gateway™ is a trademark of Thermo Fisher Scientific.

Gibson Assembly® is a registered trademark of Synthetic Genomics Inc.

Gibson Assembly Assembly US Patent Nos. 7,776,532, 8,435,736, and 8,968,999.

One of the advantages of cloning with the Gibson Assembly® method is that unlike other kits and cloning methods very little starting material is needed. For Gibson Assembly® reactions, we recommend combining insert(s) and vector in a molar ratio of 1:1, using 10–100 ng of each DNA fragment. The only exception is that for combining large DNA fragments over 32 kb in length with the Gibson Assembly® Ultra kit, you may need to increase the amount of DNA up to 300 ng.

Use the formula below to calculate the recommended amounts of starting material for combining DNA fragments with any of our Gibson Assembly® kits or master mixes:

For example:

Assume you have a 2.7 kb vector at a concentration of 25 ng/µL and three DNA fragments with insert sizes of 750 bp, 1.2 kb, and 2 kb. In this example you would want to use the recommended vector to insert ratio of 1:1 for the 1.2 kb and 2 kb insert. For the 750 bp DNA fragment a 1:5 vector to insert ratio should be used as SGI-DNA scientist recommend a 5-fold molar excess when using fragments < 1 kb. Assume for your Gibson Assembly® reaction that you will use 25 ng of vector.

To determine how much of each insert are required you can use the formula above to calculate the pmol of vector and use that result to calculate how many nanograms of insert are required to have the appropriate molar ratio of vector to insert. Details on how to perform this calculation are presented below

First, calculate the number of picomoles of your vector

1.55 * [(25 ng/µL)/ 2700 bp] = pmol / µL

0.014352 = pmol / µL.

Next, calculate for each fragment, solving for X.

For a 2 kb fragment:

1.55 * X ng/µL / 2000 bp= 0.014352
0.000775 X ng/µL = 0.14352 pmol /µL
X = 18.52 ng

Repeat these steps for each DNA fragment. You should get 34.7 ng for a 750 bp fragment (remember, this is a 5-fold increase) and 11.1 ng for a 1.2 kb fragment.

Sum for the vector and DNA fragments, the total amount of starting material should equal 89.3 ng.

Now, combine the inserts and vector DNA and initiate the assembly reaction.

For optimal cloning reactions with the HiFi 1-Step and Ultrakits, the starting DNA fragments will be at a minimum concentration of 40 ng/µL. DNA concentrations of 20–25 ng/µL are acceptable, but efficiency may be reduced. For more dilute samples (10–20 ng/µL), we offer the same convenient assembly procedure with consistent and high cloning efficiencies using the Gibson Assembly® HiFi HC 1-Step kit.

The Gibson Assembly® HiFi HC 1-Step kit (HC kit) contains the same components as the HiFi 1-Step kit in a formulation that allows for even more dilute starting material. The introduction of the HC kit to the SGI-DNA Gibson Assembly® suite of high-efficiency assembly products offers users greater flexibility for a wider range of starting DNA concentrations.

With all of our Gibson Assembly® kits and Master Mixes, cloning relies on homologous overlapping ends between adjacent fragments. Once overlapping fragments are combined in the proper ratios and volumes as outlined above, they are combined with Gibson Assembly® reagents and incubated. During incubation, the Gibson Assembly® reagents mediate the generation of compatible ends, annealing, extension, and ligation to create a fully assembled seamless DNA construct.

An overview of joining a single insert with a single vector with the HiFi 1-Step, HiFi HC 1-Step, and Ultra kits is shown below:

Seamless DNA assembly of small amounts of starting material DNA with SGI-DNA Gibson Assembly® kits.

Visit SGI-DNA's web page to learn more about Gibson Assembly® kits and download an electronic version of SGI-DNA's Gibson Assembly® Guide to learn about all the ways you can get #DNAMYWAY.

Gibson Assembly® is a registered trademark of Synthetic Genomics Inc.

Gibson Assembly US Patent Nos. 7,776,532, 8,435,736, and 8,968,999.

This week, we are highlighting the Gibson Assembly® HiFi 1-Step Kit, our quickest and easiest cloning solution. SGI-DNA offers the Gibson Assembly® HiFi 1-Step kit, Ultra kits, and Site-Directed Mutagenesis kits. The Gibson Assembly HiFi kit and Ultra kits are ideal for the assembly of plasmids and BAC constructs. While these kits both enable the cloning of multiple fragments, the numbers of fragments, the recommended sizes of the fragments, and the specifics of the workflow are different. Here, we’ll focus on the features of the HiFi kit.

The Gibson Assembly® HiFi 1-Step method allows for the isothermal assembly of up to 5 unique fragments. As implied, the HiFi 1-step process is performed in a single step.

Gibson Assembly HiFi Protocol

To perform the HiFi 1-Step method, prepare DNA fragments and your vector of choice with overlapping ends. Combine 10-100 ng of each DNA fragment and the vector in equimolar amounts*, then add the HiFi mastermix. After incubating the reaction components at 50oC for 1 hour, the fully ligated constructs are ready to transform into competent cells.

*If insert to be added is >1 kb, a 5-fold molar excess is suggested.

The Gibson Assembly® HiFi 1-Step kit is simple to use and ideal for assembling up to 5 fragments ranging in size from 500 bp to 32 kb. Try our 5 reaction HiFi starter kit today! It's the perfect introduction if you’re new to Gibson Assembly cloning. Larger kit sizes, including bulk pricing, are also available.

Learn more about the Gibson Assembly family of products at sgidna.com

It may seem counterintuitive that a single kit would enable high-efficiency cloning of both very small (~100 bp) and large (100 kb) DNA fragments, but the SGI-DNA Gibson Assembly® Ultra kit accomplishes that and more.

Engineered by Daniel Gibson and his team in their pursuit to synthesize a bacterial genome, the Ultra kit has roots in large-scale synthetic biology. What Daniel Gibson and other leading scientists have also discovered, is that the Ultra kit performs exceptionally well in enabling small-scale routine cloning applications quickly and efficiently as well. The Ultra kit mediates the seamless cloning of up to 15 DNA fragments in a two-step reaction in a single tube and can be used with any plasmid or BAC vector. The fragments for assembly can vary widely in size. Standard assembly of a 100 bp fragment with a pUC19 vector yields a circular construct less than 3 kb in size. On the other extreme, multi-stage assembly with the Ultra kit can be used to engineer large constructs 1,000,000 bp in length (1 Mbp).

Gibson Assembly Ultra Protocol

Completed in an 80-minute reaction with less than 6 minutes of hands-on time, constructs assembled with the Ultra kit are double-stranded, fully sealed, and can be used immediately in downstream molecular biology applications. To perform the Gibson Assembly® Ultra cloning method, prepare DNA fragments and your vector of choice with overlapping ends. Combine 10–300 ng (in equimolar amounts*) of each DNA fragment, the vector, and Ultra Master Mix A. After incubating the reaction components through a temperature cycle enabling chew back and annealing, add Ultra Master Mix B to the reaction. Following a 15-minute incubation at 45°C, the fully ligated constructs are ready to transform into competent cells.

*We recommend a 5-fold molar excess of the insert for DNA fragments.

Like the Gibson Assembly® HiFi 1-Step kit, the Ultra kit is available as a 5-reaction starter kit. Larger kit sizes, including bulk pricing, are also available. Whether you are planning a big or a small cloning project, our team of experts can help. Email us at info@sgidna.comfor assistance.

Visit SGI-DNA's web page to learn more about Gibson Assembly® kits and download an electronic version of SGI-DNA's Gibson Assembly® Guide to learn about all the ways you can get #DNAMYWAY.

Gibson Assembly® is a registered trademark of Synthetic Genomics Inc.

Gibson Assembly US Patent Nos. 7,776,532, 8,435,736, and 8,968,999.

Introduction

The Gibson Assembly® methodis 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 Guideintroduces 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/reagentsor email us at info@sgidna.comfor assistance with Gibson Assembly® PBnJ™ Cloning.

Using the Gibson Assembly® method to introduce overhangs and insertions

In the Gibson Assembly® Cloning Guideand 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/reagentsor email us at info@sgidna.comfor assistance with Gibson Assembly® PBnJ™ Cloning.

Read the full Gibson Assembly® PBnJ™ Cloning Series.

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 thanE.coliprotein expression systems.
Syn2.0™ Minimal Synthetic Cellsare 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

TheCustom Cloning Module on the BioXp™ Systemallows 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.
TheBioXp™ 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™ Minifugeis compact and economical microcentrifuge for tubes and strips, is the ideal companion to the BioXp™ System.

Reagents

XactEdit™ Cas9 Nuclease kits and enzymesallow 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 #DNAMyWaydownload your copy of the Gibson Assembly® Cloning Guide today.

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.

Synthetic Genome Assembly in Your Research Lab

Supporting Synthetic Biology Education with iGEM

SGI-DNA Launches Gibson Assembly® HiFi 1 Step Kit for Simple, Seamless and Rapid Construction of Synthetic Genes, Gene Clusters and Genetic Pathways

Highlights from Craig Venter's Keynote at SynBioBeta SF 2014

Gibson Assembly® Tips for More Efficient Reactions

SGI-DNA Launches Gibson Assembly® Ultra Kit for Robust, Seamless and Efficient Construction of Synthetic Genes, Genetic Pathways and Whole Genomes

Efficient, high fidelity cloning in one simple step.

Article 3

A New Hope: The BioXp

SGI-DNA at #SLAS2016: Launch of the BioXp Cloning Module, DNA on TAP

Gibson Assembly®: A Historical Perspective

Gibson Assembly® Cloning: Tips & Tricks for Primer Design

Gibson Assembly® Design Strategies 101: Primer Design & Homologous Overlaps

Automating the Future: The BioXp™ 3200 System

How Does Gibson Assembly® Cloning Compare to Traditional Cloning Methods?

Ideally, How Much DNA Should Be Used For Gibson Assembly® Cloning?

You Can Clone Up To 5 Fragments In a Single Reaction

Gibson Assembly HiFi Protocol

Thinking BIG and small when it comes to DNA cloning

Gibson Assembly Ultra Protocol

How to Perform Gibson Assembly® Cloning With Blunt Ended Fragments

Gibson Assembly® PBnJ™ Overhang Extension and Sequence Insertion

Using the Gibson Assembly® method to introduce overhangs and insertions

Creating overhangs

Inserting DNA sequence between fragments during assembly

Change Your Workflow With These 7 Innovative Products

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

Cell Lines

BioXp™ System Applications

Reagents

What's Next for SGI-DNA?

Transformation of Gibson Assembly Constructs