The Strand - Synthetic Biology Newsletter, Issue 11
|Synthetic Biology and Cell Engineering at Life Technologies.
More and more scientists are talking about synthetic biology, but what is synthetic biology, and which technologies stand behind this hot new field of life science? Our scientist Jon Chesnut gives you insight into how we understand synthetic biology and cell engineering. read more
||GeneArt® Strings™ DNA Fragments—now up to 3 kb in one fragment|
|Also find out about our new GeneArt® Elements™ for vector construction and combinatorial parts assembly|
Before now, the use of TAL technology was largely reported in eukaryotic hosts. Read the latest using bacteria read more
|Did you know?
You have choices when it comes to obtaining your gene of interest. read more
GeneArt® Combinatorial Libraries Featuring TRIM Technology
|Upcoming Events and Synthetic Biology Resources read more|
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Tips & Tricks
How to choose the best miRNA/siRNAs for your project
RNAi is an excellent tool for transient knockdown of gene expression, and many research labs already use this technique. We offer more than 2,000 different predesigned miRNAs and more than 200,000 siRNAs. But which one is best for your experiments?
The video guides for our miRNA and siRNA search tools will show you a simple and fast way to choose the best miRNA/siRNA for your project, helping to ensure that your research maintains its momentum.
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Have tips and tricks you use in your lab? Send us your tips
Synthetic Biology and Cell Engineering
|The field of synthetic biology can be described as the pursuit of engineering living systems for new or enhanced applications. The field represents a rapidly growing area and a new approach to the application and study of life sciences. Here, several traditionally separate disciplines converge to create intellectual interfaces at which rapid innovation occurs.
The fields of electrical engineering, biology, and informatics are coming together to allow the development of cutting-edge research tools that will enable applications in multiple areas. For instance, drug development research could be made more efficient and cost-effective through the creation of high-content reporter cell lines and genomically engineered disease models.
Our energy and manufacturing needs could be better addressed by the engineering of microbes and plants for more efficient production of biofuels and industrial chemicals from basic feedstocks. Food production could benefit, as well, through engineering tolerance to drought, herbicides, and saline conditions. In total, the field of synthetic biology is an intersection of powerful technologies that will help lead us into a new future. One aspect of synthetic biology involves the specific engineering of the cellular genome to modify the behavior of a particular system. We currently focus on eukaryotic cells (mammalian cells and algae) for this purpose. Historically, deletion or insertion of genetic material has been achieved in a random manner, necessitating large and laborious screening efforts in order to identify and isolate the appropriate cell population for the intended purpose. In recent years, new tools have been developed that allow specific delivery of various modifications to essentially any address in the cellular genome. Specifically, the TAL effector plant pathogen proteins and the CRISPR/Cas9 bacterial immune response system represent the state of the art in these tools.
Now we can link these delivery agents to functionalities such as nucleases to create double-stranded DNA breaks in whatever gene we desire. These breaks are then repaired by the host cell machinery, often leaving a “scar” in the form of a deletion or insertion. If this scar appears in the coding sequence of a gene, it can be used to introduce an in-frame stop codon. This represents an easy way to knock out genes specifically and efficiently in order to study loss-of-function effects. We offer GeneArt® Precision TAL nucleases in the form of paired TAL effectors, each fused with a monomeric domain of the FokI nuclease. When the TAL nucleases bind flanking the desired cut site, the FokI domains dimerize and create a double-strand break. CRISPRs, on the other hand, are guided to the desired cut site by a single-stranded RNA molecule that is designed specifically for each locus. If the aim of the experiment is to repair or induce a change in the genome or add new genetic elements, a donor DNA fragment can be added along with the editing tools. This fragment needs only approximately 500 base pairs of homology to each side of the break to efficiently insert itself into the genome. If the goal is not to completely knock out a gene but to modify the level of gene expression in a more transient manner, GeneArt® Precision TAL effectors and GeneArt® CRISPR can be used to specifically deliver transcriptional activators or repressors that allow fine control of gene expression, enabling study of cellular metabolism at a level never before possible. We and others have shown that these tools, when directed to various promoter regions, can increase endogenous gene expression upwards of 30-fold and repress activity nearly 100%, either transiently or more stably, as a result of epigenetic modification. The targeting rules for these tools have not been completely determined, but significant work is being done to enable these as reliable tools to modulate expression in endogenous cellular pathways. Besides offering the tools to specifically edit the genome and modulate gene expression, we offer a suite of supporting tools that cover the cell engineering workflow. In addition to Ion Torrent™ next-generation genome sequencing and transcriptome profiling systems, TaqMan® qPCR kits, and an array of cell culture and analysis tools, we have developed systems specifically designed for enrichment of correctly engineered cells to reduce the number of clones need for screening, as well as an easy-to-use system to screen candidate pools and colonies for locus-specific engineering. Our surrogate reporter systems enable identification and enrichment of edited cells by either flow cytometry or bead-based selection.
This is accomplished using a transiently expressed reporter system specifically designed with the intended locus sequence. Once the candidate clones have been identified, our GeneArt® Genomic Cleavage Detection Kit allows rapid measurement of the efficiency of the initial editing event as well as the percentage of modified loci in the enriched population. All together, our tool set enables researchers to choose from a set of optimized, validated technology systems designed to work together to help them answer important scientific questions faster and with less effort. We are complementing our efforts in cell engineering with strong programs in synthetic DNA production and assembly to create the payload content, which will be critical for future applications. We are also working to create a bioinformatics “ecosystem” that will enable researchers to work with digital data from multiple sources and make predictions and model experiments as well as design and order the appropriate tools easily. All together, we are working toward a broad, integrated cell engineering workflow solution that brings together the power of cutting-edge science from multiple disciplines to a single source for the researcher of the future.
GeneArt® Strings™ DNA Fragments—now up to 3 kb in one fragment
Also find out about our new GeneArt® Elements for vector construction and combinatorial parts assembly
GeneArt® Strings™ DNA Fragments were introduced last year, but now they are even bigger and better! Bigger because Strings™ Fragments can now be ordered up to 3 kb in length (increased from the previous 1 kb maximum). And better because we introduced an error-correction step in the manufacturing process, which helps improve the sequence fidelity and reduces screening effort.
GeneArt® Strings™ DNA Fragments are:
- Custom-made linear double-stranded DNA fragments up to 3,000 bp in length
- Assembled from synthetic oligonucleotides using the same high-quality process developed for GeneArt® gene synthesis
- Produced typically within 5 business days (up to 1 kb) or 8 business days (1–3 kb) (shipping times vary depending on location)
- Ready for cloning in your lab by the method of your choice; you get at least 200 ng DNA
- Available through online ordering and optimization using the GeneArt® portal
Not happy with your expression vector? If you would like to optimize not only your coding sequence but your whole expression construct or assemble an entirely new vector, our two new GeneArt® Elements™ services offer solutions for you.
- With GeneArt® Elements™ Vector Construction, choose from predefined DNA parts in our repository (e.g., promoters, terminators, ribosomal binding sites, open reading frames) or your custom vector sequences to build your specific construct. The intuitive CAD-like software within the GeneArt® portal will assist you with your vector design and ordering.
- With GeneArt® Elements™ Combinatorial Parts Assembly, combine predefined DNA parts in various combinations (e.g., promoter 1–5, ORF 1–2, terminator 1–3, etc.) to build a diverse set of larger constructs. All proposed part combinations can be created, allowing you to build a multitude of constructs, for example, to test new metabolic pathways, expression cassettes, or any other complex circuit to identify the best construct for your application.
Learn more about GeneArt® Elements™ Vector Construction
Learn more about GeneArt® Elements™ Combinatorial Parts Assembly
Application of synthetic TALE repressors in bacteriaThe use of transcription activator-like effectors (TALEs) is getting more and more popular in today’s research labs. TALEs have their origin in Xanthomonas bacteria, where the proteins alter plant host gene expression by binding to selected regulatory gene sequences and thus facilitating better susceptibility to infection. Understanding the specialized architecture of the DNA-binding domain of TALEs makes it possible to predict the DNA-binding sequence, and that knowledge can be used to synthesize TALEs to bind to any DNA sequence.
Before now this technique has predominantly been used for genome engineering in eukaryotic systems such as mammalian cells, yeast, and plants. But it seems that the utility of TALE technology is not limited to eukaryotic systems. Politz et al. described the use of a synthetic TALE construct to repress gene expression in bacteria.
In a recent publication, the researchers from the University of Wisconsin-Madison asked whether a synthetic TAL effector codon optimized for expression in E. coli would be able to repress transcription initiation and/or influence transcription elongation events of multigene operons. In their experiments, the scientists compared the natural LacI repressor to a TALE construct synthesized in the laboratories of Thermo Fisher Scientific, Inc. The synthetic TALE construct, like the native LacI, was designed to bind the bacterial lac operator to inhibit the expression of a plasmid-based fluorescent reporter gene. The authors showed that both constructs had a very strong inhibitory effect on expression of a reporter gene driven by the trc promoter. The same was true for their investigation of transcription elongation of a two-gene reporter operon; that is, both the synthetic TALE and LacI were effectively able to repress expression of nascent elongation to reduce the expression of the second gene in the operon.
In summary, the authors showed that artificial TAL effectors are suitable for regulation of gene expression in prokaryotes. Further studies will be necessary to explore designing artificial TAL effectors to serve not only as repressors but also as activators of transcription. Following this publication, TALEs seem to have potential to play a major role in regulating genome-wide expression not only in eukaryotes but also in prokaryotes.
Read the whole story: Artificial repressors for controlling gene expression in bacteria
Learn more about GeneArt® Precision TALs Products and Services
Recently published your data? Share your data with us and we’ll feature it in our newsletter. Email us
Q. What are the advantages of synthetic libraries over conventional protocols for creating diversity?
A. Conventional protocols for degenerate library creation (e.g., error-prone PCR or the introduction of NNS or NNK codons) incorporate many unwanted mutations. Moreover, methods like DNA shuffling cannot typically be used to achieve recombination of directly adjacent mutations. Synthetic combinatorial libraries, on the other hand, limit the introduction of mutations to defined regions at the precise frequencies requested. In addition, adjacent mutations will be recombined (shuffled) independently of their proximity.
Q. Are there randomization methods that are better than using degenerate codons like NNS or NNK?
A. The TRIM technology provides a superior randomization method, offering users the opportunity to customize the randomization at defined sites. During the chemical synthesis process, preassembled trinucleotide building blocks are used. This gives you the chance to obtain the exact library design you desire by incorporating specific codons one at a time. As a result, the desired amino acids will appear at the frequency you define. Avoiding randomization techniques such as degenerate codons eliminates codon bias and unwanted in-frame stop codons. The use of TRIM oligos provides more control over your design, rationally diminishing the library size and improving your screening success rates.
Q. What parameters affect the price of combinatorial libraries generated using TRIM technology?
A. There are basically two main price drivers when it comes to randomization:
1) The number of randomized codons. The more codons that are randomized, the more costly the library gets.
2) The number of different codon mixes requested. It is more cost-efficient to use the same codon mix multiple times than to have a unique codon mix for every randomized site.
Q. Are there any limitations I have to be aware of while designing a TRIM library?
A. Yes, a randomized stretch of directly adjacent amino acids should not be longer than 20 codons, and the amount of an amino acid that appears at a specified site should not be lower than 1%.
Have questions to ask us? Send us your questions.
Did You Know?
You have choices—the table below outlines three ways to obtain your gene of interest.
|Do-it-yourself gene synthesis, or classical PCR & cloning||Synthetic DNA fragments||Gene synthesis|
|How we can help you||GeneArt® Gene Synthesis Kit
or PCR cloning & reagents
|GeneArt® Strings™ DNA Fragments
Custom-made linear dsDNA fragments up to 3 kb, suitable for any cloning strategy
|GeneArt® Gene Synthesis and Subcloning Service
Full service that delivers validated genes, fully cloned and ready to use with nearly no length restrictions
*This is the number of days for synthesis and to initiate the shipping process, NOT the number of days it will take to receive the delivery. Delivery time varies depending on location.
We want to support your research work by providing meaningful choices that can fit to your individual settings and requirements. If you have further requirements please let us know here.
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