Total synthesis of Escherichia coli with a recoded genome

Nature uses 64 codons to encode the synthesis of proteins from the genome, and chooses 1 sense codon—out of up to 6 synonyms—to encode each amino acid. Synonymous codon choice has diverse and important roles, and many synonymous substitutions are detrimental. Here we demonstrate that the number of codons used to encode the canonical amino acids can be reduced, through the genome-wide substitution of target codons by defined synonyms. We create a variant of Escherichia coli with a four-megabase synthetic genome through a high-fidelity convergent total synthesis. Our synthetic genome implements a defined recoding and refactoring scheme—with simple corrections at just seven positions—to replace every known occurrence of two sense codons and a stop codon in the genome. Thus, we recode 18,214 codons to create an organism with a 61-codon genome; this organism uses 59 codons to encode the 20 amino acids, and enables the deletion of a previously essential transfer RNA

Cardiovascular events and re-stenosis following administration of G-CSF in acute myocardial infarction: Systematic review and meta-analysis

Background: Because of the recently published results of the MAGIC study there is confusion as to whether administration of granulocyte-colony stimulating factor (G-CSF) after acute myocardial infarction (MI) should be regarded as a potentially harmful treatment. This metaanalysis of appropriate clinical studies is intended to show the impact of G-CSF given after MI on aggravated incidence of coronary re-stenosis or progression of coronary lesions. Methods: We used a fixed effects model based on the Mantel-Haenszel method to combine results from the different trials. These studies provided the basis for the current analysis comprising 106 patients of whom 62 were subjected to G-CSF treatment. Results: Minimum lumen diameter (MLD) measured immediately after percutaneous coronary intervention (PCI) was similar in both groups with a diameter stenosis of 12.3% (SD 9.5%) in the G-CSF group and 10.3% (8.5%) in the control group (p = 0.32). At follow-up, both MLD and percentage stenosis were not different between G-CSF-treated and control patients. Subsequently, averaged late lumen loss revealed similar results and no differences between groups (p = 0.11), and neither stent thrombosis nor re-infarction in either group. Conclusions: The current meta-analysis of clinical reports fails to justify an elevated risk for coronary restenosis after PCI in acute MI or adverse events following G-CSF in the setting of MI when used after state of the art treatment in carefully conducted clinical protocols

Adding α,α-disubstituted and β-linked monomers to the genetic code of an organism.

Acknowledgements: This work was supported by the Medical Research Council (MRC), UK (MC_U105181009 and MC_UP_A024_1008) and an ERC Advanced Grant SGCR, all to J.W.C. For the purpose of Open Access, the MRC Laboratory of Molecular Biology has applied a CC BY public copyright licence to any Author Accepted Manuscript (AAM) version arising from this submission. D.L.D. was supported by the Boehringer Ingelheim Fonds and Magdalene College, Cambridge. A.D. was supported by the Swiss Study Foundation. The authors thank S. Oehm and Z. Tnimov for useful discussions; W. Schmied for initial discussions on acylation-based synthetase selection; C. Franco, H. Kramer, T. Morgan and F. Begum at the MRC-LMB mass spectrometry facility and K. Heesom at the proteomics facility of the University of Bristol for performing mass spectrometry; and A. Gautam for assistance and support in using the beamline. We acknowledge the European Synchrotron Radiation Facility (ESRF) for provision of synchrotron radiation facilities.The genetic code of living cells has been reprogrammed to enable the site-specific incorporation of hundreds of non-canonical amino acids into proteins, and the encoded synthesis of non-canonical polymers and macrocyclic peptides and depsipeptides1-3. Current methods for engineering orthogonal aminoacyl-tRNA synthetases to acylate new monomers, as required for the expansion and reprogramming of the genetic code, rely on translational readouts and therefore require the monomers to be ribosomal substrates4-6. Orthogonal synthetases cannot be evolved to acylate orthogonal tRNAs with non-canonical monomers (ncMs) that are poor ribosomal substrates, and ribosomes cannot be evolved to polymerize ncMs that cannot be acylated onto orthogonal tRNAs-this co-dependence creates an evolutionary deadlock that has essentially restricted the scope of translation in living cells to α-L-amino acids and closely related hydroxy acids. Here we break this deadlock by developing tRNA display, which enables direct, rapid and scalable selection for orthogonal synthetases that selectively acylate their cognate orthogonal tRNAs with ncMs in Escherichia coli, independent of whether the ncMs are ribosomal substrates. Using tRNA display, we directly select orthogonal synthetases that specifically acylate their cognate orthogonal tRNA with eight non-canonical amino acids and eight ncMs, including several β-amino acids, α,α-disubstituted-amino acids and β-hydroxy acids. We build on these advances to demonstrate the genetically encoded, site-specific cellular incorporation of β-amino acids and α,α-disubstituted amino acids into a protein, and thereby expand the chemical scope of the genetic code to new classes of monomers

Adding α,α-disubstituted and β-linked monomers to the genetic code of an organism

Acknowledgements: This work was supported by the Medical Research Council (MRC), UK (MC_U105181009 and MC_UP_A024_1008) and an ERC Advanced Grant SGCR, all to J.W.C. For the purpose of Open Access, the MRC Laboratory of Molecular Biology has applied a CC BY public copyright licence to any Author Accepted Manuscript (AAM) version arising from this submission. D.L.D. was supported by the Boehringer Ingelheim Fonds and Magdalene College, Cambridge. A.D. was supported by the Swiss Study Foundation. The authors thank S. Oehm and Z. Tnimov for useful discussions; W. Schmied for initial discussions on acylation-based synthetase selection; C. Franco, H. Kramer, T. Morgan and F. Begum at the MRC-LMB mass spectrometry facility and K. Heesom at the proteomics facility of the University of Bristol for performing mass spectrometry; and A. Gautam for assistance and support in using the beamline. We acknowledge the European Synchrotron Radiation Facility (ESRF) for provision of synchrotron radiation facilities.The genetic code of living cells has been reprogrammed to enable the site-specific incorporation of hundreds of non-canonical amino acids into proteins, and the encoded synthesis of non-canonical polymers and macrocyclic peptides and depsipeptides1–3. Current methods for engineering orthogonal aminoacyl-tRNA synthetases to acylate new monomers, as required for the expansion and reprogramming of the genetic code, rely on translational readouts and therefore require the monomers to be ribosomal substrates4–6. Orthogonal synthetases cannot be evolved to acylate orthogonal tRNAs with non-canonical monomers (ncMs) that are poor ribosomal substrates, and ribosomes cannot be evolved to polymerize ncMs that cannot be acylated onto orthogonal tRNAs—this co-dependence creates an evolutionary deadlock that has essentially restricted the scope of translation in living cells to α-l-amino acids and closely related hydroxy acids. Here we break this deadlock by developing tRNA display, which enables direct, rapid and scalable selection for orthogonal synthetases that selectively acylate their cognate orthogonal tRNAs with ncMs in Escherichia coli, independent of whether the ncMs are ribosomal substrates. Using tRNA display, we directly select orthogonal synthetases that specifically acylate their cognate orthogonal tRNA with eight non-canonical amino acids and eight ncMs, including several β-amino acids, α,α-disubstituted-amino acids and β-hydroxy acids. We build on these advances to demonstrate the genetically encoded, site-specific cellular incorporation of β-amino acids and α,α-disubstituted amino acids into a protein, and thereby expand the chemical scope of the genetic code to new classes of monomers

Adding α,α-disubstituted and β-linked monomers to the genetic code of an organism

Acknowledgements: This work was supported by the Medical Research Council (MRC), UK (MC_U105181009 and MC_UP_A024_1008) and an ERC Advanced Grant SGCR, all to J.W.C. For the purpose of Open Access, the MRC Laboratory of Molecular Biology has applied a CC BY public copyright licence to any Author Accepted Manuscript (AAM) version arising from this submission. D.L.D. was supported by the Boehringer Ingelheim Fonds and Magdalene College, Cambridge. A.D. was supported by the Swiss Study Foundation. The authors thank S. Oehm and Z. Tnimov for useful discussions; W. Schmied for initial discussions on acylation-based synthetase selection; C. Franco, H. Kramer, T. Morgan and F. Begum at the MRC-LMB mass spectrometry facility and K. Heesom at the proteomics facility of the University of Bristol for performing mass spectrometry; and A. Gautam for assistance and support in using the beamline. We acknowledge the European Synchrotron Radiation Facility (ESRF) for provision of synchrotron radiation facilities.The genetic code of living cells has been reprogrammed to enable the site-specific incorporation of hundreds of non-canonical amino acids into proteins, and the encoded synthesis of non-canonical polymers and macrocyclic peptides and depsipeptides1–3. Current methods for engineering orthogonal aminoacyl-tRNA synthetases to acylate new monomers, as required for the expansion and reprogramming of the genetic code, rely on translational readouts and therefore require the monomers to be ribosomal substrates4–6. Orthogonal synthetases cannot be evolved to acylate orthogonal tRNAs with non-canonical monomers (ncMs) that are poor ribosomal substrates, and ribosomes cannot be evolved to polymerize ncMs that cannot be acylated onto orthogonal tRNAs—this co-dependence creates an evolutionary deadlock that has essentially restricted the scope of translation in living cells to α-l-amino acids and closely related hydroxy acids. Here we break this deadlock by developing tRNA display, which enables direct, rapid and scalable selection for orthogonal synthetases that selectively acylate their cognate orthogonal tRNAs with ncMs in Escherichia coli, independent of whether the ncMs are ribosomal substrates. Using tRNA display, we directly select orthogonal synthetases that specifically acylate their cognate orthogonal tRNA with eight non-canonical amino acids and eight ncMs, including several β-amino acids, α,α-disubstituted-amino acids and β-hydroxy acids. We build on these advances to demonstrate the genetically encoded, site-specific cellular incorporation of β-amino acids and α,α-disubstituted amino acids into a protein, and thereby expand the chemical scope of the genetic code to new classes of monomers