3/22/2023 0 Comments Step seq clone![]() ![]() Additional in vivo methods require known sequence data and are based on Rec/ET recombineering, (12) such as linear–linear-(LLHR) (13) and linear–circular-homologous recombination (LCHR), (14) exonuclease combined with RecET recombination (ExoCET), (15) and Cas9-assisted targeting of chromosome segments (CATCH). Traditionally, the most common method has been the capture of large-insert genomic libraries via the packaging of partially digested genomic DNA into cosmid-, fosmid- or BAC-based (bacterial artificial chromosomes) libraries, which still has the advantage of not requiring genome sequence data. (11) To experimentally validate the ever-increasing database of BGCs, a range of in vitro and in vivo methods for cloning and BGC capturing have been developed. (10) Synthetic biology uses molecular engineering techniques to manipulate natural product pathways and can subsequently be coupled with heterologous expression. In an attempt to activate BGCs, two broad methodologies that involve either untargeted altering of the physical bacterial culture environment ( e.g., different medium (9)) or synthetic biology can be employed. The complementation of DiPaC with SLIC depicts a time and cost-efficient method for simple capture and expression of new natural product pathways. As a proof of principle, the majority of the cyanobacterial hapalosin gene cluster was cloned as a single piece (23 kb PCR product) using this approach, and predicted transcriptional terminators were removed by simultaneous pathway refactoring, leading to successful heterologous expression. ![]() ![]() Here, we have further streamlined DiPaC by reducing cloning time and reagent costs by utilizing T4 DNA polymerase (sequence- and ligation-independent cloning, SLIC) for gene cluster capture. Direct pathway cloning (DiPaC) is an emerging synthetic biology strategy that utilizes long-amplification PCR and HiFi DNA assembly for the capture and expression of natural product biosynthetic gene clusters. New biosynthetic gene cluster capturing methods to efficiently clone and heterologously express natural product pathways have thus been developed. The need for new pharmacological lead structures, especially against drug resistances, has led to a surge in natural product research and discovery. ![]()
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