The Strand - Synthetic Biology Newsletter, Issue 7
In This Issue
Interview with Dr. Farzad HaerizadehConsidering that genetic engineering is one of the most controversial and misunderstood aspects of algal research, we were interested in getting this scientist's take on this rapidly evolving field...read more >>
NEW Bioinformatics Software Platform Enabling Biodesign and More
The online tool is designed for molecular biology, metabolic engineering, genetic engineering, and synthetic biology professionals who want to rationally design, characterize, and assemble genetic “parts”, optimizing genetic components for a broad range of applications...read more >>
Tips & Tricks
Gene Synthesis or Strings™ DNA Fragments: When to Use What
Gene synthesis is the easiest way to get your gene; it comes fully cloned and 100% sequence-verified. 6 new tips...read more>>
Genome Engineering using TAL Effectors
New tools such as transcription activator-like (TAL) effectors have enabled researchers to rapidly and easily modulate gene expression in many types of…read more>>
Synthetic Biology Resources
Interview with Dr. Farzad HaerizadehConsidering that genetic engineering is one of the most controversial and misunderstood aspects of algal research, we were interested in getting this scientist’s take on this rapidly evolving field, what his work at Life Technologies is like, and his observations on the benefits of genetic engineering in the world of algae.
What first got you involved in algal genetic research?
I got involved with algae research when I came to Life Technologies about two years ago. I had been at Codexis Inc. in the Bay Area, where I had worked on metabolic and pathway engineering, mainly in yeast and E. coli, for the production of biofuels and other bio-based compounds with commercial potential.
It’s exciting to be in algae because it’s a young field with enormous, untapped potential. If you think about how long we have been working with other organisms, it’s much longer—plants, for example, have been bred for something like 10,000 years. Then in the 1960s, the introduction of new cropping systems, crop protection, and tools brought about the “green revolution”, initiated by Norman Borlaug, which significantly increased the amount of calories produced per acre. The revolution in algae is still ahead of us.
Your team at Life Technologies is developing synthetic biology “parts, devices, and chassis” for algae and other microorganisms. Please describe that work further.
At Life Technologies, we are developing toolkits and methods to make it easier for scientists in both academic and industry labs to work with algae. Right now, there are many different strains being used, so everyone has to invent their methods from scratch. We’d like to make things easier, and to that end, launched our GeneArt® Algae Engineering Kits for Chlamydomonas reinhardtii and Synechococcus elongatus. We selected these two organisms to start with because they are frequently used as model systems. Moving forward, we are working to release kits such as ultra transformation reagents for organisms that are currently being used in industrial production such as Chlorella.
Read the complete interview with Dr. Farzad Haerizadeh at Algae Industry Magazine.com.
Send us your story about how Life Technologies is enabling you to make breakthroughs.
NEW Bioinformatics Software Platform Enabling Biodesign and MoreVector NTI® Express Designer bioinformatics software delivers a new synthetic biology design paradigm for sequence analysis and design
An extension of the Vector NTI® software offerings, Express Designer incorporates an integrated gene synthesis service for rapid sequence submission and order placement, effectively building a bridge from DNA sequencing to gene synthesis. Customers can use the Vector NTI® tool to design custom parts and submit them directly to the Life Technologies GeneArt® portal for synthesis.
“Molecular and synthetic biologists can now move from construct design to synthesis faster than ever before,” said Nathan Wood, Vice President of Synthetic Biology at Life Technologies. “This launch represents a true game-changer in the ease and rapidity with which investigators can design and order DNA customized to their needs, as well as the confidence they can have that constructs will perform successfully in their experiments.”
Vector NTI® Express Designer also provides sequence optimization to fine-tune expression levels of cloned genes, enables construction of multiple vectors for compatible and simultaneous function, and generates variants from template DNA parts, devices, and circuits more effectively. In addition to designing and ordering custom components, the software also contains an electronic database or “mini electronic lab notebook” that allows researchers to maintain a searchable record of constructs and experimental results that can be referred to and utilized to optimize constructs and circuits.
“The Vector NTI® Express Designer will allow us to tackle difficult design challenges in synthetic biology,” said Chris Voigt, PhD, Associate Professor of Biological Engineering at MIT. “Essentially, the software automates what is typically a hit-or-miss process of designing vectors that can express multiple genes or vectors that can coexist compatibly and interactively in a single host cell, essential steps in the design of complex genetic circuits.” Voigt is a leader in the study of circuit design to create synthetic organisms, a process that requires constructing synthetic multigene pathways.
“The power of the Vector NTI® Express Designer database is that it allows us to track and analyze our experimental results so that we avoid duplicating effort and focus on getting to the answer more quickly,” said Steve Mayfield, PhD, Professor of Molecular Biology at the University of California, San Diego. “We can focus more on what we want our constructs to do and less on how to make them do it.” Mayfield is a pioneer in the genetic design of organisms such as algae for biofuel production.
Vector NTI® Express Designer is built on the Vector NTI® Sequence Analysis and Design platform, and so inherits many of the applications associated with that platform, including: sequence analysis and design, annotation, and illustration; molecular biology data management; open reading frame and restriction enzyme analysis and mapping; primer design; recombinant molecule design, including Gateway® and TOPO® cloning; and GeneArt® seamless assembly cloning and high-order assembly, and in silico gene synthesis.
Built on a trusted history of high-quality sequence analysis and the design tools of Vector NTI® software, Vector NTI® Express Designer enables rational biodesign on the most popular computing architectures, including native Mac OSX 10.6 and higher, Windows XP, Windows 7, and Windows 8 operating systems. Its plug-in architecture allows users to have confidence that additional features and functionality can be added to the platform and deliver more value over time. Its Automatic Updater helps ensure that users will be automatically notified of updates and given the option to download new or updated plug-ins, so that they can always be running the latest, most complete software package available.
Download your free 30-day trial of Vector NTI® Express Designer Software and test this innovative application.
Gene Synthesis or Strings™ DNA Fragments: When to Use What
|Gene synthesis is the easiest way to get your gene; it comes fully cloned and 100% sequence-verified. If you need it inexpensively and are ready to do the cloning work yourself, Strings™ DNA Fragments may be an alternative to full gene synthesis. GeneArt® Strings™ DNA Fragments are custom-made linear, double-stranded DNA fragments, up to 1,000 bp in length. They are synthesized using reliable GeneArt® methodologies and are typically shipped out in 5 business days. They can be optimized and ordered online, and are bulk-sequenced to verify that your desired gene is present in the PCR pool. At least 200 ng of the PCR fragment is delivered dried and ready to use for cloning.|
Situation: You have no template, but only the sequence of your desired gene, or your gene is not amplified by PCR. Depending on your budget, order either a full gene synthesis or a Strings™ DNA Fragment using GeneArt® gene synthesis!
As with gene synthesis, you can order Strings™ DNA Fragments optimized for expression, using the convenient online order portal. Just enter the portal, copy and paste your desired sequence, optimize, and proceed to the cart to place your order.
When you order a Strings™ DNA Fragment, don’t forget to add 5' and 3' sequences that are suited to your cloning method, and add some buffer nucleotides to enable efficient restriction enzyme binding and protection from end deletion.
Even if your gene is longer than 1,000 base pairs, you can benefit from the Strings™ DNA Fragment strategy: just assemble your gene from two or more Strings™ fragments using the GeneArt® Seamless PLUS Cloning and Assembly Kit. Don’t forget to add end sequences to your fragments that support proper homologous recombination.
The GeneArt® Seamless PLUS Cloning and Assembly Kit is a good way to assemble two Strings™ DNA Fragments.
There are two ways to make sure assembly works well:
• Use the design tool to create primers needed for PCR amplification of your Strings™ fragment
• Add 5' and 3' homologous overhangs created by the design tool directly to your Strings™ fragments when you order via the portal This way you can directly use the Strings™ fragments in the seamless reaction—no need to amplify your fragments by PCR to create the homologous overhangs.
Want to know more?
You are invited to join our new webinar, “New Easy Methods for Next Generation Cloning”.
In this presentation we will discuss the GeneArt® Strings™ DNA Fragments and how to create expression plasmids using Strings™ fragments in combination with common, reliable cloning methods. This new methodology saves time and money, allowing scientists to remain focused on results and not on troubleshooting lengthy cloning reactions.
Have tips and tricks you use in your lab? Send us your tips.
Genome Engineering using TAL Effectors
New tools such as transcription activator-like (TAL) effectors have enabled researchers to rapidly and easily modulate gene expression in many types of cells to create new disease models, optimize new food and fuel sources, and provide new therapeutic platforms. Researchers can assemble designer TAL proteins and create vehicles that target and bind to virtually any spot in the genome of any organism. The ability to target a functional protein to a user-defined DNA sequence is a long-sought goal, with the potential to enable genome engineering and genetic regulation of diverse cell types.
TAL proteins are efficient engineering tools for modifying genomes—nucleases for nicking or cleaving DNA to knock out genes, deleting entire fragments, and facilitating the addition of novel elements into the chromosome. This is currently the most popular application for TAL proteins and has fostered multiple high-profile publications and enabled the production of novel disease models in human embryonic stem cells (hESC) and induced pluripotent stem cells (iPSC).
Also, data are emerging to suggest that TAL nucleases will play a key role in modifying the genomes of crops for creation of more hardy food sources and biofuel platforms. In addition, TAL proteins have been used at Life Technologies and elsewhere to deliver transcriptional activators and repressors to cells, to efficiently modulate expression of specific genes in a transient manner. Once developed, these tools could be used as more specific loss- and gain-of-function tools, potentially more versatile and efficient than RNA interference. Another application under development at Life Technologies is the delivery of epigenetic proteins to modify the epigenetic status, and gene expression, of particular genes and signaling pathways in mammalian cells.
In summary, TAL proteins represent the newest and most exciting elements in the toolbox of genome engineering tools. As such, they have been named the Method of the Year by the journal Science and have been placed in the top 10 scientific breakthroughs of 2012 by Fiscal Times. Arriving at an opportune time, TAL effectors are part of the confluence of disciplines that are creating a new era of functional genomics. Now, for instance, we can create human induced pluripotent stem cell (iPSC) hosts for disease research and more efficient drug discovery. These cells can be produced from diseased or healthy donors, and their genomes can be read and annotated. TAL effectors and gene synthesis can then be employed to write and edit the genomes of these cells to either repair or create mutations. These platforms will find uses in basic research, personalized drug development, and eventually therapy. In the future, TAL effectors and similar tools will be used to engineer a multitude of other organisms for the production of biofuels and industrial chemical development.
Have you recently published your data? Share your data with us and we’ll feature it in our newsletter.
|Q: What are the size limitations on gene synthesis, and how will that change over the next few years?
A: Today, a 10 kb gene can routinely be built as part of the high-throughput production process, whereas such a synthesis would have been very challenging 10 years ago. Standard processes can produce up to 60 kb lengths now, and we can easily synthesize 100 kb lengths in our service labs. Indeed, the major limitations on size come from the use of E. coli as the DNA propagation host. When the size of the synthesized DNA gets significant compared to its own genome, E. coli has difficulties. This can be overcome by switching to other suitable hosts (e.g., yeast), so size limitations will further diminish in the near future. With our gene synthesis and assembly technologies, we are well-positioned to produce megabase genome-sized lengths, and we expect to be routinely building them in the coming years.
Q: How successful are you at synthesizing genes with really high GC content?
A: We have an extremely low failure rate, which is about 1 out of 5,000 or 10,000 genes, and those instances are probably due to gene toxicity and not high GC content. High-GC constructs are more demanding but are nevertheless routinely produced. A special technique is used that takes one week longer, but which is very reliable.
Q: Do you have a minimum sequence size you can make?
A: We have a very short minimum length defined for our processes, but that won’t affect your orders. By building a short DNA molecule with flanking regions that mimic a vector's backbone, we can synthesize an insert of only 1 base pair in length.
Q: I want to do transfection assays. Can you deliver larger amounts that are of the required quality?
A: Yes, we can deliver that. We offer endotoxin-free plasmid preparation that produces plasmids that can be used directly for transfection. One of our most popular solutions is our "ready-to-work" package, which includes cloning into the expression vector you choose, plus delivery of one 1 mg of endotoxin-free DNA in addition to your gene synthesis.
Have questions to ask us? Send us your questions.