Codon-Restrained Method for Both Eliminating and Creating Intragenic Bacterial Promoters

Future
applications of synthetic biology will require refactored
genetic sequences devoid of internal regulatory elements within coding
sequences. These regulatory elements include cryptic and intragenic
promoters, which may constitute up to a third of the predicted Escherichia coli promoters. The promoter activity
is dependent on the structural interaction of core bases with a σ
factor. Rational engineering can be used to alter key promoter element
nucleotides interacting with σ factors and eliminate downstream
transcriptional activity. In this paper, we present codon-restrained
promoter silencing (CORPSE), a system for removing intragenic promoters.
CORPSE exploits the DNA-σ factor structural relationship to
disrupt σ70 promoters embedded within gene coding
sequences with a minimum of synonymous codon changes. Additionally,
we present an inverted CORPSE system, iCORPSE, which can create highly
active promoters within a gene sequence while not perturbing the function
of the modified gene.