Membranes provide
a highly selective barrier that defines the boundaries
of any cell while providing an interface for communication and nutrient
uptake. However, despite their central physiological role, our capacity
to study or even engineer the permeation of distinct solutes across
biological membranes remains rudimentary. This especially applies
to Gram-negative bacteria, where the outer and inner membrane impose
two permeation barriers. Addressing this analytical challenge, we
exemplify how the permeability of the Escherichia coli cell envelope can be dissected using a small-molecule-responsive
fluorescent protein sensor. The approach is exemplified for the biotechnologically
relevant macrolide rapamycin, for which we first construct an intensiometric
rapamycin detector (iRapTor) while comprehensively probing key design
principles in the iRapTor scaffold. Specifically, this includes the
scope of minimal copolymeric linkers as a function of topology and
the concomitant need for gate post residues. In a subsequent step,
we apply iRapTors to assess the permeability of the E. coli cell envelope to rapamycin. Despite its lipophilic
character, rapamycin does not readily diffuse across the E. coli envelope but can be enhanced by recombinantly
expressing a nanopore in the outer membrane. Our study thus provides
a blueprint for studying and actuating the permeation of small molecules
across the prokaryotic cell envelope.