Team:UNIPV-Pavia/Project/Motivation2

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UNIPV TEAM 2011

Results


Contents

NB: unless differently specified, all tests were performed in E. coli MGZ1 in M9 supplemented medium at 37°C. For the cloning of the parts, E. coli TOP10 was used.

Parts assembly

All the parts have been cloned with success. The part name, plasmids and quality controls are reported in the Freezer section.

Characterization of basic modules

Characterization of promoters pTet and pLux

Inducible and constitutive promoters were assembled upstream of different 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 (Scell) 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:

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

    • RPUmin 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 of pLux 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%].

RBS RPUmax RPUmin Switch point [nM] Linear boundaries [MIN; MAX] [nM]
B00304.280.201.08[0.36; 3.27]
B00314.930.550.25[0.03; 2.30]
B00329.490.020.47[0.07; 3.07]
B003421.530.510.53[0.08; 3.77]

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

Here we have characterized its transcriptional strength as a function of aTc induction (ng/ul) 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]
Data are provided as average [CV%]

The operative parameters are summarized in the table below:

RBS RPUmax RPUmin Switch point [ng/ml] Linear boundaries [MIN; MAX] [ng/ml]
B00301.53~07.95[4.66;11.99]
B0031NDNDNDND
B00323.16~06.7[4.45;10.05]
B00342.730.238.96[8.27;9.71]

Characterization of enzymes AiiA and LuxI

LuxI has been characterized through the Biosensor BBa_T9002 (see modelling section) using the pTet-RBSx-LuxI-TT measurement systems.
The parameters Vmax, kM,LuxI and αRBSx 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 87 8.5 ND 252

The provided parameters kM and Vmax 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 ptet promoter, assaying its enzymatic activity.

The parameters kcat, kM,AiiA and αRBSx 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 difficult.
In the first experiments with the measurement system pTet-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:

RBS effpLux effpTet effJ23101 Declared efficiency
B00300.401.68142.450,6
B00310.01ND0.040,07
B00320.190.41930.400,3
B00341111


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

RBS effmRFP effAiiA effLuxI Declared efficiency
B00301.720.530.450,6
B00310.030.830.0280,07
B00320.370.50N.D.0.3
B00341111

Laboratory for Biomedical Informatics | Centre of Tissutal Engineering

University of Pavia
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