Pierre Galka, Elisabet Jamez, Gilles Joachim and Patrice Soumillion
Institute of life sciences, Université Catholique de Louvain, Belgium
We propose a method for the creation of genetic libraries encoding randomized cyclic peptides by high efficiency in vitro mutagenesis for bacteria and yeasts. Starting from a plasmid matrix and a set of primers, a 4-hours protocol using standard lab products and basic equipment affords a ready-forelectroporation mix that will provide millions of individual transformants, each one capable of biosynthesizing a different cyclic peptide. Our method greatly improve the time and efficiency of standards protocols. We have developed first a new primer design, which significantly improves the PCR amplification efficiency of whole plasmids from fresh or very simply regenerated DNA matrix. This design method ensures efficient PCR over a fruitless pairing between forward and reverse primers (such as in classical protocols). Several mutagenesis were successfully performed with this primer design in the range going from frame shift by singe base deletion, punctual or multiple mutations, to as large as 24 bases insertion in a variety of plasmid vectors.
Our protocol also uses an intramolecular derivative of the Gibson's assembly method combined to the enzymatic digestion of the non-mutated matrix for ensuring a very high electroporation efficiency and a very low wild type background.
All steps of our process are performed in standardized buffer conditions with any intermediate manipulation assuring not only a significant gain of time but above all, the perfect conservation of genetic diversity during creation of mutagenized libraries of millions of independent clones. We use this protocol for creating libraries of biosynthetic cyclic peptides. By mining these libraries with appropriate screening or selection methods for identifying new bioactive compounds.
Keywords: Cyclic peptides, biosynthesis, peptidic drugs.