Part:BBa_K201001:Experience

To experimentally and mathematically characterize the device and its sensitive to the inducer, we performed both a static and a dynamic GFP analysis after IPTG induction. Dh5alpha cells were co-transformed with the BBa_K201001 on a high copy number plasmid (pSB1A2) and BBa_K201002 on a low copy number plasmid (pSB3K3).

Static response. Cells were inoculated in 5 ml of M9 medium with 0, 10, 20, 40, 60, 80, 100 uM IPTG, respectively. After O/N growth at 37° (about 12 h) samples were collected and slides prepared for microscope analysis. Images were then analyzed with the [http://2009.igem.org/Team:Bologna/Software VIFluoR software]. To obtain a significant representation of bacterium fluorescence, it was necessary to acquire several images, each one reporting a sufficient number of bacterial cells. VIFluoR operates the image segmentation and then recognises the bacterial cells yielding the mean fluorescence per bacterium as the output. The experimental data (Fig. 1) were used to identify, by the [http://2009.igem.org/Team:Bologna/Modeling mathematical model], the operator binding affinity for the repressor LacI (K= 1.7 nM).

Fig.1. Experimental data (blue lines) of the static induction after 0, 10, 20, 40, 60, 80, 100 uM IPTG induction of the system. Tha data were fitted by the model (green line) to identify the operator-repressor binding affinity (K= 1.7 nM)

After model identification, we computed the curve for the LacI repressed GFP generator (LacI inverter)(Fig.2).

Dynamic response. Dh5alpha cells were inoculated in the morning (9 a.m.) in 5 ml of M9 medium with no IPTG. After daily growth (about 8 h) the culture was diluited to an OD=0.1. To perform the induction analysis, the culture was splitted in two. A half was induced with 100 uM IPTG and the other was grown in control medium. 200 ul of each sample were used to fill plate wells (six wells each). Cells were grown into a fluorimeter (Tecan M200) O/N (about 12h) at 37°. In the morning growth and fluorescence data were analyzed (Fig. 3 and 4, respectively) and fitted by the model (Fig. 5).

Fig.3. Growth curve for the uninduced (black line) and induced (100 uM IPTG, red line)system.

Fig.4. Absolute fluorescence curve for the uninduced (black line) and induced (red line, 100 uM IPTG)system.

Fig.5. Model fitting of the experimental data. Experimental data (blue lines) were fitted by the model considering a constant (red line) or a varying (green line) amount of RNA polymerase

As shown in Fig. 5, experimental data were fitted by the model considering both a constant and a varying amount of the RNA polymerase enzyme. A good fitting can only be obtained if a varying Polymerase is consider. This can be explained considering the GFP expression varies depending on the growth phase.

Fig. 6 shows LacI promoter repression as predicted by the model simulation.

Fig.6. LacI promoter repression as predicted by the model.

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