Modeling

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Overview

Our main goal is to make a reproducible biofilm. The strength of the biofilm is determined by the degree of intercellular connectivity through curli fibers. With modeling, it is possible to determine which factors have a strong influence on the intercellular connectivity. For instance, one could argue that a higher csgB nucleator production would lead to more curli fibers and therefore an improved connectivity. But if the csgA production would be limiting, solely short curli chains would be formed possibly preventing the cells to even interconnect.

Generic placeholder image — Fig. 1: Illustration depicting the cells interconnecting by curli fibers. The csgA protein is secreted by the cells to the extracellular space. A curli can be formed when the csgA reacts with the nucleator protein csgB, located at the outer membrane of the cells.

The modeling can be divided in 4 sections. In section 1, the csgA production rate, intracellular csgA concentration and csgB membrane concentration are determined. As our csgA production is controlled under the induction of rhamnose, all rates and concentrations are calculated for two levels of induction (0.2% (w/v) and 0.5% (w/v)) rhamnose. In section 2, the rates and concentrations are used in a grid model which is able to make an estimate on the characteristic time of curli formation. In section 3 the characteristic time for curli formation is used to predict the strength of the biofilm. Finally, in section 4, an application in MATLAB is presented able to calculate the printing time of a certain figure or shape with our Biolinker printer. To go to either of these sections, click on one of the buttons below or select your section of interest in the modeling submenu!

Gene Modeling

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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.

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