For an inducible device, the RBS variation has the purpose to stretch the induction curve, thus modulating its PoPs-OUT range.

The complex RBS-promoter acts as a whole regulatory element and determines the amount of translated protein. RBSs have been reported to have an un-modular behavior, since the translational efficiency is not independent on the coding sequences, but variates as an effect of different mRNA structure stability [Salis et al., Nat Biotec, 2009]. It is not possible to separate the effects of the sole promoter and of the sole RBS on the total amount/activity of gene product (in this case study, mRFP).

For this reason, every combination 'Promoter+RBS' was studied as a different regulatory element. Regulatory elements were characterized using mRFP reporter protein for different RBSs in terms of Synthesis rate per Cell (S_cell) and R.P.U.s (Relative Promoter Units) as explained in measurements section.

Operative parameters of the promoter are derived from the estimated Hill equations obtained by nonlinear least squares fitting (lsqnonlin Matlab routine) of the Hill function expressed in RPUs:

• RPU_max is equal to the α and represents the maximum promoter activity

• RPU_min is equal to the α * δ represents the minimum promoter activity

• Switch point is computed as the abscissa of the inflection point of the Hill curve and it is representative of the position of linear region

• Linearity boundaries are determined as the intersection between the tangent line to the inflection point and the upper and lower horizontal boundaries of the Hill curve.