New parts

J23101x series

1. BBa_K516130 (wiki name: J101-E5 ) J101-RBS30-mRFP-TT
2. BBa_K516131 (wiki name: J101-31 ) J101-RBS31-mRFP-TT
3. BBa_K516132 (wiki name: J101-E7 ) J101-RBS32-mRFP-TT

Data are provided as average [Standard error].

J23101 promoter with RBS Scell R.P.U.s RBS30 122 [13.23] 2.56 [0.27] RBS31 2 [0.25] 0.04 [0.01] RBS32 20 [1.7] 0.40 [0.03] RBS34 50 [1.87] 1 [0.02]

LuxI expression cassette driven by aTc-inducible pTet promoter

• BBa_K516210 (wiki name: E13 ) pTet-RBS30-LuxI
• BBa_K516211 (wiki name: E14 ) pTet-RBS31-LuxI
• BBa_K516212 (wiki name: E15 ) pTet-RBS32-LuxI
• BBa_K516214 (wiki name: E16 ) pTet-RBS34-LuxI

AiiA expression cassette driven by aTc-inducible pTet promoter

• BBa_K516220 (wiki name: E24 ) pTet-RBS30-AiiA-TT
• BBa_K516221 (wiki name: E25 ) pTet-RBS31-AiiA-TT
• BBa_K516222 (wiki name: E26 ) pTet-RBS32-AiiA-TT
• BBa_K516224 (wiki name: E27 ) pTet-RBS34-AiiA-TT

Existing parts

Notes for promoter characterization

Inducible and constitutive promoters were assembled upstream of different coding sequences containing an RBS from the Community collection.

The assembled RBSs are:

RBSs

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]. In addition, it is not possible to separate the effects of the sole promoter and of the sole RBS on the total amount of mRFP produced. For this reason, every combination 'Promoter+RBS' was studied as a different regulatory element.

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

• 3. measure the output of the circuits and calculate the RBS efficiency as the ratio of the output relative to the output of the circuit with the standard RBS.

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:

RBS	effpLux*	effpTet*	effJ23101**	Declared efficiency
B0030	0.40	1.6814	2.45	0,6
B0031	0.01	ND	0.04	0,07
B0032	0.19	0.4193	0.40	0,3
B0034	1	1	1	1

On the other end, to investigate the dependance of RBS modularity on the coding sequence, the same regulatory elements (pTet-RBSx) were assembled upstream of different encoded gene (mRFP, AiiA and LuxI). RBS efficiency was assessed and the results are summarized in the table below:

RBS	effmRFP**	effAiiA***	effLuxI****	Declared efficiency
B0030	1.72	0.53	0.45	0,6
B0031	0.03	0.83	0.028	0,07
B0032	0.37	0.50	N.D.	0.3
B0034	1	1	1	1

^* 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).

The parts we used to characterize the RBSs are listed here:

• mRFP expression with different promoters
• J23101
• BBa_K516130 J101-RBS30-RFP-TT
• BBa_K516131 J101-RBS31-mRFP-TT
• BBa_K516132 J101-RBS32-mRFP-TT
• BBa_J23101 J101-RBS34-mRFP-TT
• pTet
• BBa_K516230 pTet-RBS30-mRFP-TT
• BBa_K516231 pTet-RBS31-mRFP-TT
• BBa_K516232 pTet-RBS32-mRFP-TT
• BBa_I13521 pTet-RBS34-mRFP-TT
• pLux
• BBa_K516330 pLambda-RBS30-LuxR-T-pLux-RBS30-mRFP-TT
• BBa_K516331 pLambda-RBS30-LuxR-T-pLux-RBS31-mRFP-TT
• BBa_K516332 pLambda-RBS30-LuxR-T-pLux-RBS32-mRFP-TT
• BBa_K516334 pLambda-RBS30-LuxR-T-pLux-RBS34-mRFP-TT
• pTet driving the expression of different genes
• mRFP
• BBa_K516230 pTet-RBS30-mRFP-TT
• BBa_K516231 pTet-RBS31-mRFP-TT
• BBa_K516232 pTet-RBS32-mRFP-TT
• BBa_I13521 pTet-RBS34-mRFP-TT
• AiiA
• BBa_K516220 pTet-RBS30-AiiA-TT
• BBa_K516221 pTet-RBS31-AiiA-TT
• BBa_K516222 pTet-RBS32-AiiA-TT
• BBa_K516224 pTet-RBS34-AiiA-TT
• LuxI
• BBa_K516210 pTet-RBS30-LuxI
• BBa_K516211 pTet-RBS31-LuxI
• BBa_K516212 pTet-RBS32-LuxI
• BBa_K516214 pTet-RBS34-LuxI

pLux promoter

• BBa_K516330 (wiki name: E17 ) pLambda-RBS30-LuxR-T-pLux-RBS30-mRFP-TT
• BBa_K516331 (wiki name: E18 ) pLambda-RBS30-LuxR-T-pLux-RBS31-mRFP-TT
• BBa_K516332 (wiki name: E19 ) pLambda-RBS30-LuxR-T-pLux-RBS32-mRFP-TT
• BBa_K516334 (wiki name: E20 ) pLambda-RBS30-LuxR-T-pLux-RBS34-mRFP-TT

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

RBS	αpLux [(AUr/min)/cell]	δpLux [-]	ηpLux [-]	kpLux [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_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:

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]

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 pLux-RBSx for the one of J23101-RBSx. It is evident that some nonlinear effect, maybe due to non-modularity of RBS when the promoter changes or maybe due to saturation effects on protein expression, occur. The switch point are almost constant showing that the modulation in amplitude of the Hill can't be explained by a linear dependance on the RBS efficiency (in this case, in fact, the same RPUs should be observed for every RBS, since the standard reference used for RPUs computation).

pTet promoter

• BBa_K516230 (wiki name: E21 ) pTet-RBS30-mRFP-TT
• BBa_K516231 (wiki name: E22 ) pTet-RBS31-mRFP-TT
• BBa_K516232 (wiki name: E23 ) pTet-RBS32-mRFP-TT
• BBa_I13521 pTet-RBS34-mRFP-TT

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:

The data collected from the mRFP measurement systems were processed as described in data analysis section. The induction curves were obtained by fitting a Hill function as described in modelling section and the estimated parameters for pTet are reported in the pictures and in table below.

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]

Data are provided as average [CV%]

The measurement system pTet-B0031-mRFP-TT couldn't be assayed because its fluorescence output is under the detectability threshold of our measurement instrument. For this reason, the parameters of the corresponding Hill curve couldn't be estimated and are reported as 'Not Determined' ND.

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:

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]

From these parameters, it is evident that whilst the switch-point is almost maintained for all the RBSs, the linear boundaries are similar for RBS30 and RBS32 but for RBS34 are moved on the right of one order of magnitude.

LuxI gene - BBa_C0061

LuxI has been characterized through the Biosensor BBa_T9002 (see modelling section).
The HSL synthesis rate has been evaluated according to the model equations, properly adjusted. In particular, the ODE system is reported here:

Since the measurement systems are only assayed in the exponential growth phase, in the equation (3) N<max is assumed. The parameters N_max, μ and γ_HSL (see the Results section for more details) are known and summarized in the table below:

Nmax	μ	γHSL
1*109	0.004925	0

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 pTet-RBSx-LuxI-TT assayed by BBa_T9002 biosensor section.

The estimated parameters for the enzymatic activity of LuxI are reported in the table below:

Vmax	kM,LuxI	αB0030	αB0031	αB0032	αB0034
3.56*10-9	6.87*103	252	8.5	ND	87

AiiA gene - BBa_C0060

The activity of AiiA enzyme has been evaluated by testing the measurement systems pTet-RBSx-AiiA-TT. Similar to LuxI, a system of differential equations (referring to model equations) has been derived.

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 pTet-RBSx-AiiA-TT assayed by BBa_T9002 biosensor section. Unfortunately, their estimation revealed impossible.
Firstly several tests were performed, considering the subpart pTet-RBSx-AiiA-TT in LOW-COPY at pH=7. An example of them is shown in the figure below: HSL is degradeted during time as well as a negative control, which wouldn't degradate it even after 21 hours.

So, we decided to investigate the behaviour in HIGH COPY in another E.COLI strain, TOP10, in order to understand if the enzyme AiiA would have been worked well. In this case, a significant difference in degradation between pTet-RBSx-AiiA-TT and the negative control was observed, also just after 7 hours. That was the proof of the good functioning (in HIGH COPY) of the enzyme. Then, we decided to come back to MGZ1 LOW COPY, this time at pH=6. Unfortunately, here the experimental data are not shown because the biosensor T9002 didn't work as usual (calibration curve modified), and so, finally, k_M,AiiA and k_cat were impossible to be estimated.

pSB1C3 plasmid with mRFP between S and P bearing pTet (easy-to-clone)

This vector was designed and realized in order to facilitate the cloning of coding sequences downstream of the strong promoter pTet. This vector was assembled by ligating S-P excided mRFP coding sequence from BBa_J61002 and ligating it in BBa_R0040 cut with S and P. Thus, the resulting vector contains mRFP between S and P. pTet cn be easily excided (E-P) and moved in the desired vector (E-P) and then the desired coding sequence can be easily assembled by digesting S-P the vector and X-P the coding sequence, thus obtaining a final part that is standard10-compatible.

Existing parts: sequence debugging

• BBa_K516021 (wiki name: E10 ) RBS31-AiiA-TT Rebuilt existing part from BBa_I13914 (DNA planning)
• BBa_K516022 (wiki name: E11 ) RBS32-AiiA-TT Rebuilt existing part from BBa_I13912 (DNA planning)
• BBa_K516030 (wiki name: E5 ) RBS30-mRFP-TT Rebuilt existing part from BBa_S04180 (DNA planning)
• BBa_K516032 (wiki name: E7 ) RBS32-mRFP-TT Rebuilt existing part from BBa_ J133000 (DNA planning)
• BBa_K516214 (wiki name: E16 ) pTet-RBS34-LuxI Rebuilt existing part from BBa_S03623 (DNA available, only 2008 kit, inconsistent)
• BBa_K516222 (wiki name: E26 ) pTet-RBS32-AiiA-TT Rebuilt existing part from BBa_ J22071 (2008 only, Bad sequencing)
• BBa_K516224 (wiki name: E27 ) pTet-RBS34-AiiA-TT Rebuilt existing part from BBa_K077047 (Part deleted)
• BBa_K516232 (wiki name: E23 ) pTet-RBS32-mRFP-TT Rebuilt existing part from BBa_I20252 (DNA planning)