New Standards. Novel Framework for Custom Peptide Synthesis.

RFC 100: Standard for Synthesis of Customized Peptides by Non-Ribosomal Peptide Synthetases

With this RFC, we introduce a standardized framework for the production of short synthetic peptides by engineering customizable non-ribosomal peptide synthetases (NRPS). The framework consists of the NRPSDesigner, a software tool for the in silico design of user-defined NRPSs, a platform for standardized cloning and expression of NRPSs in different bacterial hosts (please refer to our RFC 99 below) and a peptide labeling and quality control procedure for the easy validation of NRP production.

Our framework seeks to overcome the two major challenges present in the synthetic peptides field today: making peptide production flexible and scalable at the same time. Chemical peptide synthesis is flexible and enables the incorporation of non-proteinogenic amino acids. However, it is relatively expensive and often uneconomical when used for large-scale peptide production. In contrast, producing recombinant peptides may be cheaper, but their composition is mostly restricted to only 22 different building blocks.

RFC 100 combines the advantages of chemical and recombinant peptide synthesis: it enables the incorporation of hundreds of non-proteinogenic amino acids into the synthetic peptides and is at the same time easily scalable and thus applicable for industrial peptide production. Thus it highly facilitates the production of peptide-based antibiotics, detoxifying agents or chelators applicable for recycling of precious metals from electronic waste. We believe, that RFC 100 represents not only a great foundational advance in the synthetic peptide production field but could become the gold standard for recombinant peptide production in the synthetic biology community.

RFC 99: HiCT: High Throughput Protocols For CPE Cloning And Transformation

RFC 99 provides detailed instructions for a rapid and cost efficient cloning and transformation method which allows for the manufacturing of multi-fragment plasmid constructs in a parallelized manner. HiCT thereby combines simplified versions of published and established standard protocols for cloning and transformation, namely Circular Polymerase Extension Cloning (CPEC) [1,2] and high-throughput transformation of CPEC constructs into competent bacterial cells.

We used HiCT successfully for constructing whole libraries of NRPS modules composed of different natural or synthetic NRPS domains (see our project on IndC domain shuffling). For constructs up to 10 kb in size, HiCT was working as efficient as Gibson Assembly in our hands. We thus recommend HiCT for high-throughput assembly of customized NRPS composed of up to three modules (please refer to RFC 100 above) or for shuffling domains within single NRPS modules.

Quan, J., & Tian, J. (2009). Circular polymerase extension cloning of complex gene libraries and pathways. PloS one, 4(7), e6441. doi:10.1371/journal.pone.0006441

Quan, J., & Tian, J. (2011). Circular polymerase extension cloning for high-throughput cloning of complex and combinatorial DNA libraries. Nature protocols, 6(2), 242–51. doi:10.1038/nprot.2010.181

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