Expanding the Genetic Code of an Animal

Greiss, Sebastian; Chin, Jason W.

doi:10.1021/ja2054034

Cited by 233 publications

(222 citation statements)

References 61 publications

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“…ND, no data. mammalian culture cells, and plants (58)(59)(60)(61)(62). Similar translational switches can also be constructed using these pairs and can be easily implemented with applicable organisms.…”

Section: Discussionmentioning

confidence: 99%

High-Yield, Zero-Leakage Expression System with a Translational Switch Using Site-Specific Unnatural Amino Acid Incorporation

Minaba

Kato

2014

Appl Environ Microbiol

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Synthetic biologists construct complex biological circuits by combinations of various genetic parts. Many genetic parts that are orthogonal to one another and are independent of existing cellular processes would be ideal for use in synthetic biology. However, our toolbox is still limited with respect to the bacterium Escherichia coli, which is important for both research and industrial use. The site-specific incorporation of unnatural amino acids is a technique that incorporates unnatural amino acids into proteins using a modified exogenous aminoacyl-tRNA synthetase/tRNA pair that is orthogonal to any native pairs in a host and is independent from other cellular functions. Focusing on the orthogonality and independency that are suitable for the genetic parts, we designed novel AND gate and translational switches using the unnatural amino acid 3-iodo-L-tyrosine incorporation system in E. coli. A translational switch was turned on after addition of 3-iodo-L-tyrosine in the culture medium within minutes and allowed tuning of switchability and translational efficiency. As an application, we also constructed a gene expression system that produced large amounts of proteins under induction conditions and exhibited zero-leakage expression under repression conditions. Similar translational switches are expected to be applicable also for eukaryotes such as yeasts, nematodes, insects, mammalian cells, and plants.

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Section: Discussionmentioning

confidence: 99%

High-Yield, Zero-Leakage Expression System with a Translational Switch Using Site-Specific Unnatural Amino Acid Incorporation

Minaba

Kato

2014

Appl Environ Microbiol

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“…Yet, limitations of the technology remain: most importantly these include the need for more widespread access to the required plasmids and the global applicability towards all protein systems. However, recent impressive advances in the field such as amber suppression in living animals 197,198 , and significant increases in suppression efficiency by knockout of the gene for RF1 in E. coli 199 are beginning to open up the possibility of amber-stop codon suppression becoming an indispensable tool for the incorporation of unnatural 'tags' into proteins to undertake site-selective modification. The creation of an 'amber-free' E. coli variant is also farsighted in this regard 200 .…”

Section: Review Nature Communications | Doi: 101038/ncomms5740mentioning

confidence: 99%

Selective chemical protein modification

2014

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“…Two articles expand on the DNA-based protocols by incorporating the use of positive and negative selections to edit the genome Dickinson et al 2013), similar to the protocols developed for targeted transgene insertion by Mos1 (Frøkjaer-Jensen et al 2012). Chen et al (2013) generated templated modifications in the unc-4, unc-5, and ben-1 genes by using the antibiotic hygromycin for positive selection (Greiss and Chin 2011) and fluorescent markers together with a heat-shock-inducible toxin gene peel-1 for negative selection (Seidel et al 2011). The results presented by Dickinson et al (2013) stand out for having most fully realized the potential of the CRISPR-Cas9 system for à la carte genome editing.…”

Section: Dna Injectionmentioning

confidence: 99%

Exciting Prospects for Precise Engineering of Caenorhabditis elegans Genomes with CRISPR/Cas9

Frøkjær-Jensen

2013

Genetics

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With remarkable speed, the CRISPR-Cas9 nuclease has become the genome-editing tool of choice for essentially all genetically tractable organisms. Targeting specific DNA sequences is conceptually simple because the Cas9 nuclease can be guided by a single, short RNA (sgRNA) to introduce double-strand DNA breaks (DSBs) at precise locations. Here I contrast and highlight protocols recently developed by eight different research groups, six of which are published in GENETICS, to modify the Caenorhabditis elegans genome using CRISPR/Cas9. This reverse engineering tool levels the playing field for experimental geneticists.Progress in science depends on new techniques, new discoveries and new ideas, probably in that order.Sydney Brenner (Robertson 1980, from the symposium

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Expanding the Genetic Code of an Animal

Abstract: Genetic code expansion, for the site-specific incorporation of unnatural amino acids into proteins, is currently limited to cultured cells and unicellular organisms. Here we expand the genetic code of a multicellular animal, the nematode Caenorhabditis elegans.

Cited by 233 publications

References 61 publications

High-Yield, Zero-Leakage Expression System with a Translational Switch Using Site-Specific Unnatural Amino Acid Incorporation

High-Yield, Zero-Leakage Expression System with a Translational Switch Using Site-Specific Unnatural Amino Acid Incorporation

Selective chemical protein modification

Exciting Prospects for Precise Engineering of Caenorhabditis elegans Genomes with CRISPR/Cas9

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