csgA production rate

The csgA production rate is defined as the production of csgA per second per cell. To be able to calculate this number, the following unknown parameters need to be characterized:

I. Activity of the promoter (with different levels of rhamnose induction).

II. Export rate of csgA to the extracellular space.

If these parameters are known, a model for the csgA production can be formulated and the export of csgA to the extracellular space can be calculated.

The activity of the promoter, denoted as PoPs, can be calculated by a kinetic experiment with GFP. For this experiment, we made a construct with GFPmut3 (modified GFP variant with stronger fluorescent signal) and csgA under the same rhamnose promoter. The constructs used for this experiment are depicted in Fig. 2.

As csgA and GFP are under the same promoter, the fluorescence of GFP can be related to the production of csgA. It is presumed that the promoter activity is independent of the addition of the GFPmut3 gene.

As already mentioned in the section “Kinetic experiment” under “Project”, both the fluorescence and OD600 were measured. It is important to also measure the cell density, as we are interested in the fluorescence per cell per second. To calculate how much fluorescence corresponds to a certain amount of GFP (in nanogram), a calibration curve has been constructed with wtGFP. As the ratio of emission maxima for GFPmut3 relative to wtGFP is 21 at an excitation of 488nm (Brendan et. al), the correct mass amount of GFP mut3 could be determined.

In Fig. 3, the OD600 is plotted for each of the samples in the kinetic experiment. As can be seen from Fig. 3, there is some difference in OD600 between the samples. Therefore, the fluorescence signal normalized by the number of cells as shown in Fig. 4:

In Fig. 3, the OD600 is plotted for each of the samples in the kinetic experiment. As can be seen from Fig. 3, there is some difference in OD600 between the samples. Therefore, the fluorescence signal normalized by the number of cells as shown in Fig. 4:

As can be seen from Fig. 4, GFPmut3 only appears if the culture has been induced by rhamnose. Furthermore, it can be seen that a higher induction level of rhamnose leads to an increase in GFPmut3 and thus fluorescence. Finally, as the fluorescence signal is normalized by the cell density, one can make statements about the activity of the rhamnose promoter. The promoter seems to not be active right after induction, but more after 3 or 4 hours. This is in accordance with data from literature (Wegerer et. al), in which a low amount of fluorescence with a rhamnose promoter was observed after 2 hours of induction.

The calibration line of fluorescence versus mass amount GFPmut3 is given in Fig. 5.

corresponding function of the GFPmut3 calibration line with mass_GFP in ng is:

With the calibration line, which was made with the exact same settings as the kinetic experiment, the mass amount of GFPmut3 per cell can be calculated. With Eq. 1, the fluorescence signal was converted to mass amount of GFPmut3 for rhamnose induction of 0.2% (w/v) and 0.5% (w/v).

After this conversion, the units are still in nanogram per OD600. As the desired unit is molecules per cells, the following dimension analysis has been performed:

The result of this unit conversion is shown in Fig. 6:

The GFPmut3 steady state concentrations are thus:

As the data is now converted to molecules per cell, it is possible to construct a model with the purpose of predicting the same steady state levels as in the performed experiments. The only unknown remaining in this model is the promoter activity, which can be varied to correctly fit the data.

The model that was made is a mathematical model of ordinary differential equations (ODE’s) describing the GFP mRNA concentration (Eq. 2), immature GFP protein concentration (Eq. 3) and mature GFP protein concentration (Eq. 4) in Table 1. All concentrations are modeled per cell per second. The immature protein is the nonfluorescent GFP, the mature protein is the fluorescent protein (which will be fitted to the data).