Part:BBa_K516214:Experience

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••••• UNIPV-Pavia iGEM 2011

NB: unless differently specified, all tests were performed in E. coli MGZ1 in M9 supplemented medium at 37°C in low copy plasmid pSB4C5. Characterized with: BBa_K516210 pTet-RBS30-LuxI BBa_K516211 pTet-RBS31-LuxI BBa_K516212 pTet-RBS32-LuxI Though these parts don't have a transcriptional terminator, they have been characterized in low copy plasmid pSB4C5, that contains the BBa_B0054 terminator. This choice is motivated by the need to reproduce the exact experimental context of the final circuit, as described in solution section. LuxI has been characterized in terms of enzymatic activity under the regulation of pTet promoter. KM,LuxI and Vmax parameters representing its activity have been estimated and the promoter strength (represented by a synthetic parameter αpTet for every pTet-RBS combination) at full induction (100 ng/ml) has been estimated too with a simultaneous fitting of the available data. LuxI has been characterized through the Biosensor BBa_T9002 (see BBa_T9002 experience page). The HSL synthesis rate has been evaluated according to the model equations, properly adjusted. In particular, the ODE system describing the behavior of this measurement circuit is reported here: Since the measurement systems are only assayed in the exponential growth phase, in the equation (3) N < Nmax is assumed. The parameters Nmax, μ and γHSL (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 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 through BBa_T9002 biosensor - experience page. 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 ND=Not Detected The collected data have been used to identify the parameters of our model. Despite the data-poor context, the model predictions fit the experimental data, thus demonstrating that the equation that models the HSL synthesis by LuxI is a good approximation of real processes. 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.

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