The Strand - Synthetic Biology Newsletter, Issue 8
|Using GeneArt® Precision TALs to Challenge Traditional Japanese Silkworm Research
Investigating the genes controlling juvenile hormones and moving forward to genome editing…
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|Gene Optimization and Synthesis for Streamlined Workflows and Expression Enhancement
Today’s de novo gene synthesis technologies offer easy, fast, and affordable access to nearly any desired gene sequence. A viable alternative to the cumbersome cloning and mutagenesis steps…
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Tips & Tricks
First of all, what’s the difference between the GeneArt® Seamless PLUS and the original Seamless Cloning & Assembly Kits?…
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Vector NTI® Express Designer Software
Unleash Your Evolutionary Imagination With Directed Evolution
Synthetic Biology Resources
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Patent Protection for TAL Effector Technology Now Enables Licensing Path
CARLSBAD, Calif., May 13, 2013 /PRNewswire/—Life Technologies Corporation (NASDAQ: LIFE) announced today the issuance of a new patent covering nucleic acids encoding Transcription Activator–Like Effector Nucleases (TALENS) fusion proteins and the formal launch of an associated sublicensing program.
Using GeneArt® Precision TALs to Challenge Traditional Japanese Silkworm Research
Takaaki Daimon, Senior Researcher, National Institute of Agrobiological Sciences, Division of Insect Sciences
Investigating the genes controlling juvenile hormones and moving forward to genome editing
“My research model is primarily silkworms. They are related to moths and are included among lepidopterous insects. I have been conducting research on silkworm molting and how silkworm metamorphosis is controlled,” said Mr. Takaaki Daimon. In March 2012, he analyzed mutated “two-molt” silkworms and reported genes that control metamorphosis from larvae to pupae . Silkworm larvae grow in size with each molting, and larvae typically molt four times before metamorphosing into pupae. The mutated two-molt silkworms, however, only molt two to three times and become pupae while still small, resulting in a very small imago (adult stage silkworm). Mr. Daimon discovered a mutation in the CYP15C1 gene, which leads to small imago that lack the ability to synthesize juvenile hormones.
The “Long-Cherished Dream” of Silkworm Researchers
“The CYP15C1 gene is expressed as epoxy phenoloxidase, which is involved in the biosynthesis of juvenile hormones. We attempted to develop our research but there was a very big problem in silkworm research,” says Mr. Daimon. “RNAi was mostly ineffective, which placed functional analysis of the genes in our organism outside of our reach. Though transgenics could be made, it takes time and there are many restrictions. Gene knock-out experiments were the long-cherished dream of silkworm researchers.”
New Silkworm Research Pioneered by TALEN-Engineered Nucleases
“The first technology to appear was ZFN (zinc finger nucleases), and because other researches struggled with it and gave up, saying that synthesis and verification is difficult, I tried GeneArt® Precision TALs custom services. It eliminates tedious preparations such as propagating plasmids and purchasing special restriction enzymes, and offers delivery in a short period of time. When I gave it a try, I understood that it was a technology that was well-suited to silkworms,” said Mr. Daimon. “In my system, RNA is injected directly into a fertilized egg and the occurrence of mutations at the targeted location is confirmed using a Cel-1 mutagenic detection assay, and the mutations in the germ cells occurred at a high rate of efficiency, about 10 to 50%. I was really surprised by the results. The rate of mutations for zebrafish and frogs are high but TAL was quite effective for silkworms as well.” After using the GeneArt® Precision TALs custom services, Mr. Daimon moved the insert to a T7 expression vector and used the mMESSAGE mMACHINE® T7 Ultra Kit to prepare mRNA. “From now on, instead of just knock-out, I would like to experiment with various genome editing, including knock-in as well as functional changes to clarify interesting insect phenomena. Silkworms are highly useful for silk production and it seems that it would be possible to knock-in useful genes to make silkworms produce a large amount of proteins,” said Mr. Daimon.
Silkworm rearing is a long-held tradition in Japan, a nation that uses and maintains 400 varieties of silkworms. By adding new technology to the accumulation of this existing knowledge, it is possible that silkworms will be used in production not only for clothing but also for agriculture and the production of raw pharmaceutical materials.
Figure 1. On the left is a normal four-year-old silkworm. On the right is a silkworm with one juvenile hormone biosynthesis enzyme knocked out with TAL. Even with its small three-year-old body, it produces silk and has already become a pupa.
- Daimon T, et. al. (2012) Precocious metamorphosis in the juvenile hormone-deficient mutant of the silkworm, Bombyx mori. PLoS Genetics 8(3):e1002486.
GeneArt® Precision TALs enable you to generate custom DNA-binding proteins for accurate DNA targeting and precise genome editing.
Gene Optimization and Synthesis for Streamlined Workflows and Expression Enhancement
Today’s de novo gene synthesis technologies offer easy, fast, and affordable access to nearly any desired gene sequence. A viable alternative to the cumbersome cloning and mutagenesis steps carried out in individual labs, gene synthesis offers researchers maximum design flexibility and makes it possible to receive a 100% correct clone in around five business days. In addition, this technology makes every sequence available for manipulation by scientists and offers the ability to utilize the redundancy of the genetic code to optimize protein coding genes and open reading frames of choice for maximum protein expression.
Life Technologies GeneArt® gene optimization and synthesis services—with a capacity of 5.5 Mbp—enable researchers to simplify their workflow, free internal lab resources for value-added work, and to gain speed in research and development by increasing the success rate based on gene expression projects (Figure 1).
|Figure 1. GeneArt® gene optimization and synthesis technologies can help you achieve expression-optimized constructs in record time.|
Going Beyond Codon Optimization for Better Expression
For synthesized genes, expression success depends largely on the quality of the optimization algorithm. The algorithms in the Life Technologies GeneOptimizer® software suite take into account the major contributors to high-level gene expression, including genetic stability, transcriptional efficacy, and mRNA stability. This helps ensure that the resulting synthesized gene is fully optimized for expression in the host organism. Much more than codon optimization, gene optimization is a true multi-parameter challenge, and the GeneOptimizer® software suite is configured to deliver superior results.
|Figure 2. The GeneOptimizer® software suite goes beyond codon optimization to design expression clones for maximum transcription and translation efficiency in the host organism.|
Success is Demonstrated in the Expression of 50 Optimized Genes
The Life Technologies GeneOptimizer® software suite has been developed over the last 15 years by extensive literature research and parallel lab verification. To systematically test the quality of their algorithm, 50 GeneOptimizer® RNA- and codon-optimized candidate genes representing five classes of human proteins—transcription factors, ribosomal and polymerase subunits, protein kinases, membrane proteins and immunomodulators—have been compared with natural genes in autologous expression in HEK293T cells (Figure 2).
The results show that the GeneOptimizer® algorithm increases the overall expression success rate: 100% of the optimized genes did express, and 12% of the natural genes did not express in HEK293T cells (Figure 3). Furthermore, 86% of the optimized genes exhibited higher expression than their natural counterparts, while 10% expressed at the same level in the autologous system. Only 4% showed lower expression than their natural counterparts. These were mainly genes coding for membrane proteins, and lower expression may be expected in such proteins as cell toxicity caused by overexpression is not uncommon. The full results have been published: Fath et al. (2011), PLoS ONE 6(3):e17596.
Figure 3. Using the GeneOptimizer® software, overall expression success rates were increased and yields were improved in 50 constructs tested.
In addition, gene optimization using the GeneOptimizer® software from Life Technologies is not only a strategy to make expression success rates more predictable and improve expression yields in general, but optimized genes represent a powerful tool in functional genomics as the sequence-modified counterpart can be used for the rescue of an siRNA-mediated knockdown as demonstrated in Fath et al.
GeneOptimizer® Technology Outperforms Other Gene Synthesis Technologies
The protein expression increases offered by GeneOptimizer® technology have not only been proven in comparative studies with natural genes but also when compared with gene optimization algorithms from other suppliers of synthesized genes. In one experiment to test the expression of CLK3 kinase, the GeneOptimizer® optimized gene showed a 3-fold increase in expression compared to the non-optimized gene, while optimized CLK3 produced using algorithms from other companies resulted in protein expression that was lower than the non-optimized gene (Figure 4).
Figure 4. Comparative expression analysis of wild type versus gene-optimized constructs. (A) Western Blot analysis using α-His antibodies. Expression levels of two independent transfections per wild type and optimized construct are displayed. Standardization is based on endogenous α-His protein. (B) Summary: Relative expression levels of wild type versus optimized constructs of three independent transfections are displayed.
A Standard for Gene Synthesis
Gene optimization and gene synthesis by Life Technologies have become widely used by scientists at research institutions worldwide and for numerous pharmaceutical and biotech companies that demand high expression from their biotherapeutic and biochemical production environments.
Learn more about GeneArt® gene optimization and synthesis »
Commonly asked question about next generation of in silico cloning and assembly with Vector NTI® Express Designer Software
|Q: Can I use Lasergene to order (we don’t have access to Vector NTI® software)?
A: Our ordering interface only works with Vector NTI® Express Designer Software. You may download a free trial of this software or you may try to copy and paste the sequence from Lasergene into Vector NTI® Express Designer Software.
Have questions to ask us? Send us your questions »
|First of all, what’s the difference between the GeneArt® Seamless PLUS and the original Seamless Cloning & Assembly Kits? The PLUS is an improved version, designed for optimal performance when compared to the original. Both kits allow cloning up to four DNA fragments simultaneously; however, the PLUS version can accommodate up to 40 kb as opposed to just 13 kb in the original kit.|
- Use the GeneArt® Web-Based Design Tool to make your primer design easy, and always include 15 bp of end homology on your primers.
- Use AccuPrime™ Pfx for achieving the best results during PCR amplification of your DNA fragments of interest. Taq polymerases will work, but with lower efficiency due to the addition of A overhangs.
- Purify your PCR products (you can either gel purify them, or use a PureLink® PCR Purification kit such as Cat. No. K310001).
- Also gel purify the digested vector. If the vector is PCR-amplified, gel purify or treat with DpnI to minimize background from the vector.
- Minimize exposure of both PCR products and vector to UV during gel purification.
- Use a 2:1 insert:vector molar ratio in the Seamless reaction.
- Do not incubate the reaction with the GeneArt® Enzyme Mix for longer than recommended as this can lead to deletions, especially if your inserts are small. Always incubate at room temperature, and add the Enzyme Mix last.
- Immediately return the Enzyme Mix to the freezer after using to minimize loss of activity.
- Use the One Shot® DH10B T1 SA competent cells with the GeneArt® Seamless PLUS kit. These are optimized for cloning large assemblies, and we don’t recommend substitution with other E. coli strains.
Have tips and tricks you use in your lab? Send us your tips »
Unleash Your Evolutionary Imagination With Directed Evolution
Natural evolution is extremely effective at generating molecular components and organisms adapted specific sets of constraints. This is accomplished through the interplay of several processes: first, random variants in the amino acid constituents of proteins are generated by mutations in the genes encoding the protein sequence. Next, the different variants of the proteins exert their effect on specific molecular pathways and ultimately on the whole organism. Most variants will have undetectable or deleterious effects, while a few can improve the fitness of the organism and provide an advantage over competing organisms. This advantage will lead, over subsequent generations, to the individuals carrying the advantageous mutation, becoming over-represented in the population. This process is very effective but it is also extremely slow: the need to preserve the functional stability of the organism results in a very low rate of spontaneous gene mutation.
Scientists have been inspired by nature and have explored new ways to adapt the basic mechanisms of evolution to numerous applications spanning from basic research to drug discovery and food manufacturing. In spite of its powerful simplicity, natural evolution, as forementioned, is an extremely slow process. To achieve any practical utility, its laboratory counterpart had to be accelerated by applying the modern techniques of molecular biology. These methods offer a number of options for the generation of a moderately large collection of mutants of a single protein. These collections, commonly called libraries, are limited in the number of variants that can be generated by the intrinsic limitations of the various methods that can be used: PCR random mutagenesis, oligonucleotide-based site-directed mutagenesis, and gene shuffling can only cover a limited fraction of all the theoretical variants. If the entire collection of possible variants is imagined as a vast surface, these methodologies only allow the exploration of small patches or short disconnected segments in the “variants space”. Most problematic, it can be very difficult and time consuming to focus the systematic exploration of variants on specific protein regions of interest.
To support creating the genetic diversity needed to obtain proteins with the characteristics desired, Life Technologies has introduced the GeneArt® Directed Evolution service. The service offers a powerful combination of simplicity and convenience, with the ability to design the path or area of interest in the “variants space” to help ensure that all relevant protein variants can be efficiently produced and tested. Combinatorial libraries with maximum diversity, saturation mutagenesis at sites of choice, random mutagenesis, and defined deletions are now all within your reach. These methods, which are the results of Life Technologies expertise in synthetic biology, are now empowering scientists in academia as well as industry to efficiently produce a staggering number of protein variants and then identify the ones with the desired properties. GeneArt® Directed Evolution minimizes the barriers to protein libraries generation—how far you will take protein evolution is limited only by the creativity you build into the strategies you employ.
Learn more about the GeneArt® Directed Evolution service »
Related publication: Dai et al., (2008) Probing nucleosome function: a highly versatile library of synthetic histone H3 and H4 mutants. Cell 134(6):1066–1078.
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