BBa_J23101 is the reference standard promoter for the computation of RPUs. As discussed in 'data analysis' section, RPUs are relative units for the evaluation of promoter strength, based on a mathematical model of the transcription and the translation of a reporter gene.

The RPUs are supposed to be indepedent on the experimental setup, provided that the reference standard BBa_J23101 must be assayed in the same experimental condition of the studied promoter. It means that if the studied promoter is in a low copy number plasmid and drives the expression of a reporter protein P, J23101 must be assembled in the same vector upstream of the same reporter P. This approach is in accordance with the philosophy of synthetic biology, based on the concept of 'modularity' of the components. According to this approach, the assembly of basic well characterized modules to build complex circuits allows the prediction of the circuit behavior starting from the knowledge on the basic parts.

Salis et al. [Nat Biotec, 2009] stated that 'Identical ribosome binding site sequences in different genetic contexts can result in different protein expression levels' and again 'It is likely that this absence of modularity is caused by the formation of strong secondary structures between the RBS-containing RNA sequence and one protein coding sequence but not another.'

For this reason, RPUs might not be reliable when comparing the same promoter with different RBSs because of the un-modularity of the RBS. In order to asses what's the effect of RBS 'un-modularity' on RPUs reliability, we have built a set of four constitutive promoters (BBa_J23101) followed by one of the four RBSs tested. These parts were used to evaluate RBS efficiency. Data were collected and analyzed as described in 'Measurements' and 'Data analysis' sections. RPUs and Synthesis rate per cell [AUr] were computed and results are summarized in the table below.

NB: in the RPU computation, the J23101-RBS34-mRFP-TT construct has been considered as the reference standard. With this assumption, RPUs are identical to the estimated RBS efficiency.

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.

The evaluation of RBS efficiency can be performed in a very intuitive fashion:

• 1. select the RBSs you want to study
• 2. assemble them in a Promoter - XX - Coding sequence circuit

This simple measurement system allows the quantification of RBS efficiency depending on the whole measurement system (i.e.: promoter and encoded gene). Today it has not still been completely validated the hypothesis that every functional module in a genetic circuit maintains its behavior when assembled in a complex circuits, even if many researchers implicitly accept this hypothesis when performing characterization experiments.

To rationally assess the impact that this hypothesis has on the genetic circuit design and fine tuning, several measurement systems were built to evaluate the dependance of RBS modularity from the promoter or the coding sequence separately.

In particular, in order to investigate if RBS efficiency depends on the promoter, the same coding devices (RBSx-RFP-TT) were assembled downstream of different promoters (J23101, pTet, pLux). Measuring the system output and evaluating the RBS efficiency. The results are summarized in the table below:

^* The RBS efficiency for inducible devices expressing mRFP was estimated as the ratio of the AUCs (Area under the curve) of the induction curve of the system with the studied RBS and the B0034 reference: AUC_{P, RBSx}/AUC_{P, B0034}

^** The RBS efficiency for constitutive promoters expressing mRFP was computed as the ratio between S_{cellP, RBSx}/S_{cellP, B0034}

^*** The RBS efficiency for pTet promoter driving the expression of AiiA enzyme was computed as the ratio α_{pTet, RBSx}/α_{pTet, B0034} estimated for the measurement system pTet-RBSx-AiiA-TT. α_pTet was estimated as described here. pTet was tested at full induction (100 ng/ml).

^**** The RBS efficiency for promoters driving the expression of LuxI was computed as the ratio α_{pTet, RBSx}/α_{pTet, B0034} estimated from the measurement systems pTet-RBSx-LuxI. α_pTet was estimated as described here. pTet was tested at full induction (100 ng/ml).

From this table, it is evident that, whilst α_pLux assumes significantly different values for different RBSs, η_pLux and k_pLux assume very similar values. This result shows that RBS variation modulates the amplitude of Hill function, not affecting the shape of the curve. The four induction curves result to be the same Hill function modulated in amplitude by a parameter, that is the estimated RBS efficiency for this measurement system.

These results are quite encouraging, because suggest that, given the non-modular behavior of RBS dpending on the encoded gene, the RBS has a modular behaviour respect to the promoter.

The operative parameters are summarized in the table below:

The protocols for the characterization of pTet promoter are reported in the pTet measurement section.

This promoter is widely studied and characterized usually using the strong RBS BBa_B0034. Here we have characterized its transcriptional strength as a function of aTc induction (ng/ul) for different RBSs. Four different induction curves were obtained and are reported in figure:

α parameter (representing the maximum trascriptional rate in the studied range of induction) varies as expected with the RBS variation and also the δ and η parameters are quite different among the RBS variations.

The k_pTet parameter is quite constant among the RBS variations, thus suggesting that in this case the RBS variation doesn't substantially affect the switch point of the Hill curve, even if the amplitude and the maximum slope are not quite maintained (for the η parameter, maybe fitting problems).

The operative parameters are summarized in the table below: