Large DNA fragments cloning and manipulating
Explogen offers arranged cosmid library construction at a highly competitive price. Typically, our genomic libraries have an average size of inserted DNA 35-43 kb, which is sufficient for cloning large chromosomal regions, for example natural products biosynthetic gene clusters. We are using in-house developed vectors (pEXG) that could be easily adapted for different tasks and organisms. Also these vectors allow for re-assembly of several DNA fragments into a single clone up to 120 kb in size. The assembly vectors are designed to stably maintain large DNA fragments whilst simultaneously facilitating their purification in high yield.Targeted cloning
As an alternative, the improved TAR (Transformation-associated recombination) cloning can be offered when a particular region of genome is desired. The use of our own vectors allows precise, direct cloning and stable maintenance of DNA fragments up to 80 kb in size. Explogen TAR vectors are employing direct selection of clones with the insert of interest, significantly shortening time of cloning and increasing its efficiency. EXG modified TAR cloning procedure allows direct selection of inset-carrying clones.
Showcase 1:
Massive cloning of biosynthetic gene clusters from Streptomyces: Cosmid library of Streptomyces sp. was constructed, end-sequenced and mapped to the genome. The cosmid library covers the entire genome resulting in direct access to 15 biosynthetic gene clusters ready for expression in heterologous hosts.
Showcase 2:
Direct cloning of daptomycin biosynthetic gene cluster: 67 kb DNA fragment covering the entire daptomycin biosynthetic gene cluster was directly cloned from the chromosome of Streptomyces filamentosus in just two weeks using EXG developed TAR technology. The cluster was expressed in Streptomyces chassis strain resulting in efficient production of daptomycin.
DNA cloning services
- - Customized cosmid vector adapted for species of interest and for the purpose of the project (expression, gene deletion, etc)
- - End-sequencing and mapping.
- - When needed, genome sequencing, assembly and analysis.
- - Cost and time efficient service.
- - Large insert size ranging from 35 to 43 kb.
- - Assembly of several clones into large construct up to 120 kb.
- - Customized high-capacity vector adapted for species of interest and for the purpose of the project (expression, gene deletion, etc).
- - Fast, efficient and precise cloning of desired region of the genome.
- - Cost-effective.
- - From 10 to 80 kb.
- - Custom clone modification by deletion, regulatory elements knocking-in or replacement, introduction of SNPs.
- - Introduction of clone into desired strain for expression (model chassis strains are available).
- - Complete engineering project of different complexity: from design to the final strain verification.