期刊
ACS SYNTHETIC BIOLOGY
卷 5, 期 6, 页码 452-458出版社
AMER CHEMICAL SOC
DOI: 10.1021/acssynbio.5b00219
关键词
bioengineering; genomics; multiplex automated genome engineering; MAGE; synthetic biology; automation; software
资金
- National Science Foundation [1253856]
- National Institutes of Health [T32GM007499, T32GM007223]
- Gruber Foundation
- Direct For Biological Sciences
- Div Of Molecular and Cellular Bioscience [1253856] Funding Source: National Science Foundation
Genome engineering technologies now enable precise manipulation of organism genotype, but can be limited in scalability by their design requirements. Here we describe Merlin (http://merlincad.org), an open-source web-based tool to assist biologists in designing experiments using multiplex automated genome engineering (MAGE). Merlin provides methods to generate pools of single-stranded DNA oligonucleotides (oligos) for MAGE experiments by performing free energy calculation and BLAST scoring on a sliding window spanning the targeted site. These oligos are designed not only to improve recombination efficiency, but also to minimize off target interactions. The application further assists experiment planning by reporting predicted allelic replacement rates after multiple MAGE cycles, and enables rapid result validation by generating primer sequences for multiplexed allele-specific colony PCR. Here we describe the Merlin oligo and primer design procedures and validate their functionality compared to OptMAGE by eliminating seven AvrII restriction sites from the Escherichia coli genome.
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