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.