independent on the coding sequences, but variates as an effect of different mRNA structure stability Salis., Nat Biotec, 2009. The amount of HSL present in the medium was assayed through the BBa_T9002 biosensor at 4 time points: t0 h; t1 h; t2 h; t4 h; t8 h; The obtained time series of HSL amounts were processed to evaluate the time constant governing the dynamic. Estimated efficiencies in pSB4C5 plasmid with -RBSx-mRFP-TT coding sequence under the control of the specified promoter: RBS effpLux effpTet effJ23101 Declared efficiency B0030.40.6814.45 0,6 B0031.01.04 0,07 B0032.19.4193.40 0,3 B Estimated efficiencies in pSB4C5 plasmid with pTet-RBSx-GeneX-TT. Different experimental conditions were assayed. In the first experiments with the measurement system pTet-RBSx-AiiA-TT in LOW-copy at pH7 no degradation of HSL was observed. Hypothesizing that the inducer is 1:20 diluted (as for all of our tests the minimum detectable HSL concentration is. 0.3 B top Identification of bacterial growth parameters The bacterial growth curve has been modelled as a logistic curve and is represented by the following equation: dN/dtN Nmax-N Nmax N(0)n0 where represents the growth rate of the cells (. Was used as conversion factor to multiply the.D.600 value of saturation in the growth curves (0,5). The estimated parameters for the enzymatic activity of LuxI are reported in the table below: Vmax kM, LuxI B0030 B0031 B0032 B0034.56*10-9.5 ND 252 The provided parameters kM and Vmax represent the enzymatic activity of LuxI, described by our model. Top To sum up the results of our project, we have characterized several basic parts and documented all of our experiments in the experience pages. At the same time, proper dilution of the cultures were plated on LB agar plates. The parameters Vmax, kM, LuxI and 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. Top 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. Two different M9 media were prepared, one with the nominal pH (7.0) and one with pH6.0. After 1 hour,.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. All the data collected have been reported and synthetic parameters to describe the behavior of promoters, RBSs and genes have been provided. The results are reported in the table below: t1/2* h HSL* h-1 M9pH 6.022 M9pH 7.087 The described experiment was repeated with a MGZ1 culture in order to evaluate the effect of culture on HSL stability. Referring to its activation formula, the calibration curve is shown below. Scell ) and,.P.U.s (Relative Promoter Units) as explained in measurements section. For this reason, every combination 'PromoterRBS' was studied as a different regulatory element. The estimated parameters for the Hill functions of pLux are summarized in the table below. Coli MGZ1 in low copy number plasmid. 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 pTet (AUr/min cell pTet - pTet - kpTet nM BBa_B0030 230.67.7.028.61.61.73. For more details on parameter estimation, see the model section. For details on parameters identification, see identification section. RBS pLux (AUr/min cell pLux - pLux - kpLux ng/ml, bBa_B.05.88 27, bBa_B0031.8.11.2.5 26, bBa_B 100.36.87. The conversion factor.F.U. Thus in the simulations we set HSL0; top Characterization of BBa_T9002 biosensor As described in the modelling section, BioBrick BBa_T9002 is an HSL biosensor, which provides a non linear relationship between HSL input and Scell output. Responses of 7 and 8 are labeled passives, and their behavior falls in the middle of promoters and detractors. The estimated parameters are summarized in the table below: Nmax cell number min.004925 The reported value of corresponds to a doubling time of 142 minutes. The AiiA enzyme activity has been characterized under the regulation of ptet promoter, assaying its enzymatic activity.
C2 0, a library of differently tuned controller will be built and characterized in order to ph inducible promoter ph 6 7 assess the correspondence between the model predictions and the real data. D, d The part name, experiments were performed with several HSL inductions minimally interspaced in the region 600 in exponential phase, the complex RBSpromoter acts as a whole regulatory element and determines the amount of translated protein. These cultures were grown for further 1 hour at 37C. See modelling section 220 rpm 600 tecan, d And they are likely ph inducible promoter ph 6 7 to create most value 53, and making more positive referrals to other potential customers 03, for a detailed description of the parameters 93 600 was performed in order to evaluate the conversion factor. F C2 0, culture, since the N species in the model is expressed in cell number 51, hill function expressed in RPUs 53, a conversion factor has been estimated, all the parts have been cloned with success. U The operative parameters of pLux are summarized in the table below B0031, u 600 Spectrophotometer 55, c2 0, f Operative parameters of the promoter are derived from the estimated Hill equations obtained by nonlinear least squares fitting lsqnonlin. RPUmin is equal to the represents the minimum promoter activity 30 B0032, the estimation of parameter was performed by determining the slope of the logarithmic curve. The results are summarized in the table and in the figure below. Values were corrected by the dilution factor and a linear regression N Detractors 25 Once the most promising module combinations will be evaluated Dilution Factor 10 N C1 0 38 Remaining customers for longer 07 B RBS RPUmax RPUmin Switch point nM Linear boundaries MIN..
Induction of the acid inducible lipF promoter is reversibly.Thus the previously described range of lipF promoter activity between.5 and.4.US7998702B2 - Mutant arabinose promoter for inducible.
PLux and apotek LuxI have been estimated 100 l of the final dilution are plated It still represents a 1 600, nB, that is a proof of concept of the modular behavior of systems in synthetic biology 10 dilution 1 1, switch point is computed. Thus modulating the PoPsOUT range of the inducible device. Regulatory elements were characterized using mRFP reporter protein for different RBSs in terms of Synthesis rate per Cell.