The Strand - Synthetic Biology Newsletter, Issue 11

  In This Issue

		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
  

Try it out

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 bacteria The 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

Recently published your data? Share your data with us and we’ll feature it in our newsletter. Email us

GeneArt Combinatorial Libraries Featuring TRIM Technology

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.

You have choices—the table below outlines three ways to obtain your gene of interest.

*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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