Optimization Of Direct DNA Cloning And Application Of CcdB Counter-Selection In Red/ET Recombineering

Functional analysis of genome sequences requires methods for cloning DNA of interest. However, existing methods, such as library cloning and screening, are too demanding or inefficient for high-throughput application to the wealth of genomic data delievered by massively parallel sequencing. Rac prophage protein RecE, exonuclease, and its partner RecT, single-stranded DNA binding protein, are able to mediate highly efficient linear-linear homologous recombination (LLHR), which can be used to directly clone DNA fragments from the linearized genome. In the direct DNA cloning based on LLHR, background colonies are mainly caused by intramolecular self-circulation of linear vectors. To reduce the background and improve the ability to clone directly from more complex samples, the LLHR mediated by RecE/RecT was optimized. Using the "double selection" and "two-step recombination" direct cloning strategies developed in this study, ten polyketide synthase and non-ribosomal peptide synthetase gene clusters, each 10-52 kb in length, were all directly cloned into expression vectors from the chromosome of Photorhabdus luminescens. LLHR mediated by RecE/RecT can used not only in direct cloning, but also in synthetic biology to construct complex DNA networks.Red/ET recombineering is a revolutionary method for DNA engineering based on the homologous recombination mediated by Reda/p or RecE/T. Counter seletion is commonly used together with Red/ET recombineering to seamlessly engineering DNA molecules, such as point mutation and module exchange. The ccd operon is a toxin-antitoxin (TA) system existing on the E. coli F plasmid. The ccd TA system includes the CcdB toxin and the Ccd A antitoxin. The target of CcdB is the GyrA subunit of gyrase. It can affect DNA replication and transcription, and lead to cell death. No additional antibiotics or chemicals needed to be supplemented in the CcdB counter selection step. Because counterselection strategies necessarily differ according to the copy number of the target, we describe two variations in this work. For multi-copy targets, we employ two E.coli hosts so that counterselection is exerted by the transformation step that is needed to separate the recombined and unrecombined plasmids. For single copy targets, we put the ccdA gene onto the temperature-sensitive, pSC101 Red expression plasmid so that counterselection is exerted by the standard temperature shift to remove the expression plasmid. Hence this drug-free counterselection method is less work than existing methods and will be useful for genetic engineering not only bacterial artificial chromosomes and plasmids, but also the E. coli chromosome.Spinosad produced by the soil actinomycete Saccharopolyspora spinosa is a promising insect control agent with broad spectrum, high insecticidal activity, and exceptional safety to non-target organisms. However, the capacity of S. spinosa for spinosad production is relatively low. Genetic engineering of S. spinosa is a practical avenue to improve the spinosad production. As homologous recombination is inefficient, we explored the mariner transposon for genetic engineering of S. spinosa in this study. The transposase was expressed in S. spinosa under the control of up-mutated PermE promoter after conjugation, and the mini-transposon including the selection marker flanked by transposase recognition sequences integrated on to the chromosome. Transposon mutagenesis will facilitate not only the strain improvement, but also its regulatory network study of secondary metabolism and its secondary metabolites diversity study.