Genome-scale promoter engineering by coselection MAGE

Abstract

The use of marker coselection in combination with multiplex automated genome engineering (MAGE) is reported to improve the efficiency of engineered changes in bacterial genomes. The authors use the method to insert twelve 20-base-pair T7 promoters to control indirubin and indigo production. Multiplex automated genome engineering (MAGE) uses short oligonucleotides to scarlessly modify genomes; however, insertions >10 bases are still inefficient but can be improved substantially by selection of highly modified chromosomes. Here we describe 'coselection' MAGE (CoS-MAGE) to optimize biosynthesis of aromatic amino acid derivatives by combinatorially inserting multiple T7 promoters simultaneously into 12 genomic operons. Promoter libraries can be quickly generated to study gain-of-function epistatic interactions in gene networks.