RBS	RPUmax	RPUmin	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]

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

Here we have characterized its transcriptional strength as a function of aTc induction (ng/ml) for different RBSs. Three different induction curves were obtained and are reported in figure:

The estimated parameters of the Hill curves described in the figures are summarized in the table below:

RBS	αpTet [(AUr/min)/cell]	δpTet [-]	ηpTet [-]	kpTet [nM]
BBa_B0030	230.67 [3.7]	0.028 [91.61]	4.61 [23.73]	8.75 [4.16]
BBa_B0031	ND	ND	ND	ND
BBa_B0032	55.77 [12]	1.53E-11 [>>100]	4.98 [57.62]	7.26 [14.98]
BBa_B0034	120 [5.95]	0.085 [40.6]	24.85 [47.6]	9 [5.43]

The operative parameters are summarized in the table below:

RBS	RPUmax	RPUmin	Switch point [ng/ml]	Linear boundaries [MIN; MAX] [ng/ml]
B0030	1.53	~0	7.95	[4.66;11.99]
B0031	ND	ND	ND	ND
B0032	3.16	~0	6.7	[4.45;10.05]
B0034	2.73	0.23	8.96	[8.27;9.71]

Characterization of enzymes AiiA and LuxI

LuxI has been characterized through the Biosensor BBa_T9002 (see modelling section) using the p_Tet-RBSx-LuxI-TT measurement systems.
The parameters V_max, k_M,LuxI and α_RBSx were estimated with a simultaneous fitting of the data collected as described in measurement section for the four measurement parts p_Tet-RBSx-LuxI-TT assayed by BBa_T9002 biosensor section.

The provided parameters k_M and V_max represent the enzymatic activity of LuxI, described by our model. They must not be confused with the operative parameters of the Michaelis-Menten relation. These synthetic parameters have a great importance, since they can be used in more complicated models in order to predict the behavior of complex circuits.

The AiiA enzyme activity has been characterized under the regulation of p_tet promoter, assaying its enzymatic activity.

The parameters k_cat, k_M,AiiA and α_RBSx would have been estimated with a simultaneous fitting of the data collected as described in measurement section for the four measurement parts p_Tet-RBSx-AiiA-TT assayed by BBa_T9002 biosensor section. Unfortunately, their estimation revealed difficult.
In the first experiments with the measurement system p_Tet-RBSx-AiiA-TT in LOW-COPY at pH=7 no degradation of HSL was observed. The collected data are shown in the figure below. HSL degradation is identical in the measurement system and in the negative control after 21 hours.

Experiments on these parts gave us the opportunity to characterize only the activity of the enzyme in E. COLI TOP10 in high copy number plasmid, providing only some information about the order of magnitude of the model parameters, which has been designed to work in E. COLI MGZ1 in low copy number plasmid.

Characterization of the efficiency of RBSs from the community collection

RBSs were used for the fine tuning of CTRL+E. Different experimental conditions were assayed.

Estimated efficiencies in pSB4C5 plasmid with -RBSx-mRFP-TT coding sequence under the control of the specified promoter:



Estimated efficiencies in pSB4C5 plasmid with pTet-RBSx-GeneX-TT, with GeneX=mRFP, AiiA or LuxI:

Identification of bacterial growth parameters

The bacterial growth curve has been modelled as a logistic curve and is represented by the following equation:

where μ represents the growth rate of the cells (E. coli MGZ1 in M9 supplemented medium) and N_max represents the maximum number of cells in the well. For a detailed description of the parameters, see modelling section. For details on parameters identification, see identification section. The growth curves in all the performed experiments are measured in O.D.₆₀₀. Since the N species in the model is expressed in cell number, a conversion factor has been estimated. The conversion factor K_O.D.toC.F.U. has been estimated as follows.

• Two cultures C1 and C2 (MGZ1 cells) were grown in 1ml M9 medium till saturation (ON liquid culture, 37°C, 220 rpm).
• Next morning, both C1 and C2 were diluted in M9 medium with a final volume of 1ml with the following dilution factors:
  • 1:1
  • 1:10
  • 1:100
  • 1:1000
  in fresh M9 medium. These cultures were grown for further 1 hour at 37°C, 220 rpm.
• After 1 hour, O.D.₆₀₀ was measured using TECAN microplate reader (don't forget to measure a M9 sample for blanking!)
  NB: from now on, cultures must be placed in ice to stop cell growth.
• At the same time, proper dilution of the cultures were plated on LB agar plates.
  NB: All the dilutions are performed moving 100 μl of culture in previously ice-chilled 900 μl fresch M9. 100 μl of the final dilution are plated (It still represents a 1:10 dilution!)
• Plates were grown overnight and next morning C.F.U. were counted.
• C.F.U. values were corrected by the dilution factor and a linear regression (N vs O.D.₆₀₀) was performed in order to evaluate the conversion factor K_O.D.toC.F.U..
• K_O.D.toC.F.U. was used as conversion factor to multiply the O.D.₆₀₀ value of saturation in the growth curves (~0,5).

The results are summarized in the table and in the figure below:

Culture	O.D.600 TECAN	O.D.600 Spectrophotometer	C.F.U.	Dilution Factor (10^)	N
C1	0,367950002	0,758004416	990	5	990000000
C1	0,044649998	0,091982322	141	6	1410000000
C1	0,004799999	0,009888356	136	5	136000000
C1	0,000549998	0,001133037	20	6	200000000
C2	0,54840003	1,129744917	165	4	16500000
C2	0,058200002	0,119896339	23	5	23000000
C2	0,008700002	0,017922652	251	3	2510000
C2	0,000100002	0,000206011	24	4	2400000

The estimation of μ parameter was performed by determining the slope of the logarithmic curve of O.D.₆₀₀ in exponential phase. Exponential phase was determined by visual inspection as the linear phase of the logarithmic curve of O.D.₆₀₀.

The estimated parameters are summarized in the table below:

Nmax [cell number]	μ [min-1]
1*109	0.004925

The reported value of μ corresponds to a doubling time of 142 minutes.

Estimation of the spontaneous degradation of HSL in M9 medium and in cultures at different pH values

In order to estimate the spontaneous degradation rate of HSL in M9 medium and in a culture of MGZ1 cells as a function of pH, two simple tests have been performed.
Two different M9 media were prepared, one with the nominal pH (7.0) and one with pH=6.0.
These media, now named respectively M9_{pH 7} and M9_{pH 6}, were added with a known concentration of HSL (100 nM) and then incubated at 37°C, 220 rpm (NB: the media were not infected with any culture but the standard growth conditions were reproduced). The amount of HSL present in the medium was assayed through the BBa_T9002 biosensor at 4 time points:

• t=0 h;
• t=1 h;
• t=2 h;
• t=4 h;
• t=8 h;

The obtained time series of HSL amounts were processed to evaluate the time constant governing the dynamic of HSL degradation, supposing an exponential decay. The results are reported in the table below:

	t1/2* [h]	γHSL** [h-1]
M9pH 6	32	0.022
M9pH 7	8	0.087

The described experiment was repeated with a MGZ1 culture in order to evaluate the effect of culture on HSL stability. The estimated values are reported in the table below:

	t1/2* [h]	γHSL** [h-1]
CulturepH 6	∞	0
CulturepH 7	19	0.037

It is evident from the reported data that the spontaneous degradation of HSL is negligible when CTRL+E is implemented in MGZ1 cells grown in M9 medium with pH=6.0 and pH=7.0.
Thus in the simulations we set γ_HSL=0;

Characterization of BBa_T9002 biosensor

Minimum [Scell]	Maximum [Scell]	Switch point [nM]	Lower boundary of linearity [nM]	Upper boundary of linearity [nM]
17.31	739.4	1.39	0.38	5.07

Conclusions

To sum up the results of our project, we have characterized several basic parts and documented all of our experiments in the experience pages. All the data collected have been reported and synthetic parameters to describe the behavior of promoters, RBSs and genes have been provided. Such parameters can be used for the in silico simulation of mathematical models of complex circuits.
For what concerns CTRL+E, that is a proof of concept of the modular behavior of systems in synthetic biology, all the parameters of pTet, pLux and LuxI have been estimated. AiiA parameters revealed difficult to estimate.
Reasonable values for AiiA parameters have been derived from experiments in different contexts. These values were used to simulate the behavior of CTRL+E.
Further experiments are necessary to evaluate the consistence of the derived parameters for AiiA.
Once the most promising module combinations will be evaluated, a library of differently tuned controller will be built and characterized in order to assess the correspondence between the model predictions and the real data.