Phage-assisted continous evolution is based on the generation of selection phages, which have geneIII replaced by the gene of interest. For the generation of your own selection phage, we are offering a golden-gate cloning standard being based on the extraordinary potential of the type IIs restriction enzyme BsaI. For insertion of your gene of interest into the phage backbone, amplification with primers containing the following overhangs is required: Following the successfully performed amplification with these primers, a golden gate reaction (refering to the golden gate assembly protocol) with the BioBricks BBa_K2398021 and BBa_K2398022 should be performed. GoldenGate cloning usually delivers good results. In the following of this reaction, the assembled plasmid should be transformed into electrocompetent S1059 or S2208 cells. These strains, established by the liu lab, carry a plasmid (pJC175e) that provides geneIII under a psp-promoter. This promoter gets activated by phage infection, and ensures that geneIII is not present in the uninfected cell. For further information about the principle of phage propagation, have a look at our RFC (hier Link einfügen) As an alternative to the assembly with two phage backbone fragments, the golden gate assembly reaction can be also performed using only the BBa_K2398023 BioBrick. In this case, amplification of the gene of interest has to be implemented with other overhangs: If you want to produce your selection phage on your own by using another cloning method, please keep in mind that the terminator of geneVIII may be immediately followed by the RBS of the evolving gene. Futhermore, the last 180 bp of geneIII should not be removed, because they contain the promoter for the following operon of phage genes. In that case, we provide the surrunding sequences of the gene of interest below: