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UNIPV-Pavia iGEM 2011

Characterized with:

BBa_K516330 pLambda-RBS30-LuxR-T-p_Lux-RBS30-mRFP-TT
BBa_K516331 pLambda-RBS30-LuxR-T-p_Lux-RBS31-mRFP-TT
BBa_K516332 pLambda-RBS30-LuxR-T-p_Lux-RBS32-mRFP-TT
BBa_K516334 pLambda-RBS30-LuxR-T-p_Lux-RBS34-mRFP-TT

The inducible device BBa_K081022 was assembled upstream of different mRFP coding sequences, containing an RBS from the Community collection.

The assembled RBSs are:

BioBrick code	Declared efficiency
BBa_B0030	0,6
BBa_B0031	0,07
BBa_B0032	0,3
BBa_B0034	1

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.

The Hill function relating the induction to the S_cell is reported below:

S_cell=α * ( δ + (1-δ)/(1+(K/Induction)^η) )

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 estimated parameters for the Hill functions are summarized in the table below. For more details on parameter estimation, see the model section.

RBS	α_{p_Lux} [(AUr/min)/cell]	δ_{p_Lux} [-]	η_{p_Lux} [-]	k_{p_Lux} [ng/ml]
BBa_B0030	438 [10]	0.05 [>100]	2 [47]	1.88 [27]
BBa_B0031	9.8 [7]	0.11 [57]	1.2 [29]	1.5 [26]
BBa_B0032	206 [3]	0 [>>100]	1.36 [10]	1.87 [9]
BBa_B0034	1105 [6]	0.02 [>100]	1.33 [19]	2.34 [18]

Data are provided as average [CV%].

From this table, it is evident that, whilst α_{p_Lux} 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:

RBS	RPU_max	RPU_min	Switch point [nM]	Linear boundaries [MIN; MAX] [nM]
B0030	4.28	0.20	1.08	[0.36; 3.27]
B0031	4.93	0.55	0.25	[0.03; 2.30]
B0032	9.49	0.02	0.47	[0.07; 3.07]
B0034	21.53	0.51	0.53	[0.08; 3.77]

These operative parameters provide useful information on the behavior of this 3OC6-HSL inducible device. RPU_max assumes very different values in terms of RPUs. This can't be explained by RBS modulation, since RPUs have been evaluated by normalizing S_cell of p_Lux-RBSx for the one of J23101-RBSx. It is evident that some nonlinear effect on maximum strength, maybe due to saturation phenomena on protein expression, occur. The same RPUs should be observed for every RBS, since the normalization by the standard reference used for RPUs computation should eliminate the RBS contribute. Here different RPUs are observed, maybe due to nonlinear RBS behavior or to saturation phenomena occurring with this very strong promoter. The switch point and linear boundaries are quite constant in all the cases, showing that the linear region of this system is not affected by RBS changes.

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