Reducing metabolic burden in the PACEmid evolver system by remastering high copy phagemid vectors

Orthogonal or non-cross-reacting transcription factors are used in synthetic biology as components of genetic circuits. Brödel et al. (2016) engineered 12 such cIλ transcription factor variants, using a directed evolution ‘PACEmid’ system. The variants operate as dual activator/repressors and expand gene circuit construction possibilities. However, the high-copy phagemid vectors carrying the cIλ variants imposed high metabolic burden upon cells. Here, we ‘remaster’ the phagemid backbones to relieve their burden substantially, exhibited by a recovery in E. coli growth. The remastered phagemids’ ability to function within the PACEmid evolver system is maintained, as is the cIλ transcription factors’ activity within these vectors. The low-burden phagemid versions are more suitable for use in PACEmid experiments and synthetic gene circuits; we have therefore replaced the original high-burden phagemids on the Addgene repository. Our work emphasises the importance of understanding metabolic burden and incorporating it into design steps in future synthetic biology ventures.