The spacing between the dl binding sites and the twi class E boxes present in the twi regulatory fragment is 6bp, the natural spacing of dl and twi sites in the rho NEE.

A mutated twi proximal element (PE) in which i. the core sequences of the low affinity TD4 and TD5 dl binding sites have been mutated to the optimal sequence to create high affinity sites and ii. two copies of an E box from the rho promoter (with core sequence CATATG) have been inserted just upstream of the TD4 and TD5 sites, followed by twi sequence from -180 to +160bp relative to the transcription start site, are fused upstream of lacZ.