Abstract
Recombineering and multiplex automated genome engineering (MAGE) offer the possibility to rapidly modify multiple genomic or plasmid sites at high efficiencies. This enables efficient creation of genetic variants including both single mutants with specifically targeted modifications as well as combinatorial cell libraries. Manual design of oligonucleotides for these approaches can be tedious, time-consuming, and may not be practical for larger projects targeting many genomic sites. At present, the change from a desired phenotype (e.g. altered expression of a specific protein) to a designed MAGE oligo, which confers the corresponding genetic change, is performed manually. To address these challenges, we have developed the MAGE Oligo Design Tool (MODEST). This web-based tool allows designing of MAGE oligos for (i) tuning translation rates by modifying the ribosomal binding site, (ii) generating translational gene knockouts and (iii) introducing other coding or non-coding mutations, including amino acid substitutions, insertions, deletions and point mutations. The tool automatically designs oligos based on desired genotypic or phenotypic changes defined by the user, which can be used for high efficiency recombineering and MAGE. MODEST is available for free and is open to all users at http://modest.biosustain.dtu.dk.
Highlights
Recombination-mediated genetic engineering is readily applied for introduction of genetic variation in various organisms
The tool automatically designs oligos based on desired genotypic or phenotypic changes defined by the user, which can be used for high efficiency recombineering and multiplex automated genome engineering (MAGE)
MAGE Oligo Design Tool (MODEST) provides a platform for automatic design of oligos for MAGE and recombineering and is designed for the ease of use for researchers without programming experience
Summary
Recombination-mediated genetic engineering (recombineering) is readily applied for introduction of genetic variation in various organisms. The use of short, single-stranded oligos has been shown to allow genomic modifications at high efficiencies in Escherichia coli [1,2,3] and Saccharomyces cerevisiae [4], thereby abolishing the need for a selectable marker [1] This allows rapid generation of mutants with specific changes and enables modification of many genomic sites at once, e.g., by multiplex automated genome engineering (MAGE) [1,5]. Designing oligos for recombineering and MAGE can be tedious, timeconsuming, and often leads to sub-optimal oligos in terms of recombination efficiency This can be a significant hindrance for implementation of multiplex genome engineering and makes larger scale projects, involving modifications of many targets, impractical or even impossible (e.g. for several thousand oligos). Sawitzke et al [6] points out that the MMR system can be avoided by adding multiple specific changes in an oligo, and we are planning to develop an algorithm with this functionality in a future update
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