Team:TU Delft-Leiden/Modeling/Curli/Gene

Gene Level Modeling

We start with the modeling of the gene expression of proteins involved in the curli formation pathway at the gene level. Proteins that are part of the curli formation pathway are CsgA/B/D/E/F/G [1]. CsgA is the main building block of curli fibrils. When produced, this protein is secreted out of the cell by the CsgEFG complex. In the absence of CsgB, there is no curli formation, since the CsgA proteins remain unpolymerized. CsgB is the starting block of each curli fibril and connects the cell membrane to the first CsgA protein in the curli fibril. Once CsgB is located on the outside of the cell surface, the CsgA can polymerize onto the starting curli fibril.
In the constructs we made in the wet lab, CsgA is continuously being produced and the CsgB gene is placed under the control of a landmine promoter, activated by either TNT or DNT reference to landmine. So, when the cells get induced by TNT or DNT, CsgB protein production will get started and CsgA will already be present in the system, as CsgA is continuously being produced. We first modeled this system by constructing an extensive gene expression model of the curli formation pathway. Subsequently, we simplified this model, so less parameters were needed.

summary of the conclusions

Extensive Gene Level Modeling

A first attempt to model the curli growth was to make a deterministic model of the entire pathway for the curli units. We did this by starting with the transcription of the csgA and csgB genes with production rates K_tlA & K_tlB and degradation rates dmA and dmB. Then the csgA and csgB mRNA is translated with rates K_tsA & K_tsB. This crates an unfolded CsgA_cell and CsgB_cell proteins with degradation rates dA_cell & dB_cell. The CsgB_cell can attach the cell membrane with the help of csgEFG at rate K_Bmem and form CsgB_mem. The CsgA_cell can be transported outward (CsgA_out with degradation rate dB_out) when it interacts with a csgEFG complex on the membrane with rate K_Amem. Finally, the CsgA_out can form curli (with degradation rate dCurl) proportional to CsgB_mem. Translating this into differential equations results in:

$$ d \ mRNA_{csgA} \ / \ dt = K_{tlA} \cdot csgA_{gene} \ - \ dmA \cdot mRNA_{csgA} \tag{0.1} $$ $$ d \ mRNA_{csgB} \ / \ dt = K_{tlB} \cdot csgB_{gene} \ - \ dmB \cdot mRNA_{csgB} \tag{0.2} $$ $$ d \ CsgA_{cell} \ / \ dt = K_{tsA} \cdot mRNA_{csgA} \ - K_{Amem} \cdot CsgA_{cell} \cdot CsgEFG - \ dA_{in} \cdot CsgA_{cell} \tag{0.3} $$ $$ d \ CsgB_{cell} \ / \ dt = K_{tsB} \cdot mRNA_{csgB} \ - \ K_{Bmem} \cdot CsgB_{cell} \cdot CsgEFG - \ dB_{in} \cdot CsgB_{cell} \tag{0.4} $$ $$ d \ CsgB_{membrane} \ / \ dt = K_{Bmem} \cdot CsgB_{cell} \cdot CsgEFG \tag{0.5} $$ $$ d \ CsgA_{out} \ / \ dt = K_{Amem} \cdot CsgB_{cell} \cdot CsgEFG - K_{curli} \cdot CsgB_{membrane} - dCurl CsgA_{out} \tag{0.6} $$ $$ d \ Curli \ / \ dt = K_{curli} \cdot CsgB_{membrane} - d_{curli} \cdot Curli \tag{0.7}$$

This leaves us with an awful lot of constants. Let's start with the simplest: the degeneration rates. The degeneration rates of the mRNA is in the order of 2-5 minutes [1]. Furthermore, the average protein half-life is about 23 hours (it ranges from 12-42 hours). However, for the curli this value cannot be true, since that would mean that most of the curli are already degenerated during biofilm formation (which may take days). We reasoned that the degeneration rates of the proteins is negligible.

Then there is the translation and transcription rates of csgA and csgB. The truth is that these numbers are highly dependent on the strength of the promoter and the RBS. Furthermore, the copy number of the plasmid is also an important factor in the amount of protein produced. We did find that the translation rate by a ribosome is in the order of 20 aminoacids/s.

[1] Pedersen M, Pedersen S. The functional half-life of an mRNA depends on the ribosome spacing in an early coding region. J Mol Biol. 2011 Mar 18 [2] Maier T, Serrano L. Quantification of mRNA and protein and integration with protein turnover in a bacterium. Mol Syst Biol. 2011 Jul 19 [3] Bremer, H., Dennis, P. P. Modulation of chemical composition and other parameters of the cell by growth rate. Neidhardt, et al. eds. Escherichia coli and Salmonella typhimurium: Cellular and Molecular Biology, 2nd ed. chapter 97, p. 1559, Table 3

to be written, low priority

Simplified Gene Level Modeling

Though the model described above, providing that all rates are known, has a more accurate (though still simplified) representation of the curli assembly system, we have chosen to decrease the complexity further to the bare essentials, as most of the production rates cannot be found in literature. Measuring the accurate rates in the wet lab is, within the scope of this project, infeasible and therefore, we constructed a model that only includes the rate limiting step of the system as this will mostly determine the dynamics of the system.
First of all, we investigated if the diffusion of the CsgA and CsgB proteins to their final destination is the rate limiting step in curli formation. From the literature and the wet lab, we know that the system response to the induction by TNT or DNT is in the order of hours [reference]. If diffusion is the rate limiting step, it would mean that CsgA and CsgB proteins would pile up inside and outside the cell, because it takes a long time for them to travel to their final destination, the end of a growing curli fibril and the outer membrane, respectively. A quick calculation shows that after one second, the displacement of a spherical particle with radius \(r = \ 10 \ nm\) is 6.6 μm due to Brownian motion in liquid water at room temperature using equation 1; many times the bacterial radius! Hence, we conclude that diffusion is not rate limiting [4].

$$ \bar{x}^2 = \ \frac{k_b T t}{3 \pi \eta r} \tag{1}$$

What we do expect to be the rate limiting step for curli formation is the large amount of CsgA and CsgB proteins that have to be produced. Hence, we expect the production rate of one of these proteins to be the rate limiting step. Instead of including the intermediate steps, we have implemented the production of the CsgA and CsgB proteins with one reaction and associated production rate each. These rates have to be measured in the lab. We will use the following system of equations:

$$ \emptyset \xrightarrow{p_{A}} \ CsgA_{free} \tag{2} $$ $$ \emptyset \xrightarrow{p_{B}} \ CsgB \tag{3} $$ $$ CsgA_{free} + \ CsgB \xrightarrow{k} \ CsgA_{curli} + \ CsgB \tag{4} $$

Reactions 2 and 3 represent the production of CsgA and CsgB proteins, respectively. Equation 4 represents the growing of a curli fibril, where a curli fibril reacts with a free CsgA protein to become part of the curli. In reality, this reaction only happens at the end of the curli fibrils. In our model, we assume a homogeneous concentration of all the substances and we cannot discriminate between curli subunits. It is theoretically possible to model the system as an infinite amount of possible reactions that can take place to increase a curli fibril with length i to length i+1 at rate k [7]. However, we are merely interested in the growth rates of the curli, since the distribution of the curli length will follow from the model at the cell level. Therefore, we decided to model the growing of curli at the gene level as reaction 4. We assume that each CsgB protein is the start of a curli fibrils, thus the concentration of CsgB equals the concentration of curli. We can do this, because we showed that the diffusion of CsgA and CsgB proteins to their final destination is not the rate limiting step. Therefore, nearly all the CsgB proteins will be the beginning of a curli fibril in reality and our assumption is valid.
So, in reaction 4 we let a free CsgA protein react with a curli fibril to a CsgA protein that is part of that curli and the curli itself again, as it is immediatily again availible for the next reaction with a free CsgA protein to grow even more. Therefore, curli growth is dependent on the rate k and the concentration of \(CsgA_{free}\) and CsgB.

Writing reactions 2-4 into differential equations results in:

$$ \frac{d}{dt} [CsgA_{free}] = \ p_{A} - \ k [CsgA_{free}][CsgB] \tag{5.1} $$ $$ \frac{d}{dt} [CsgB] = \ p_{B} \tag{5.2} $$ $$ \frac{d}{dt} [CsgA_{curli}] = \ k [CsgA_{free}][CsgB] \tag{5.3} $$

Fortunately, this system can be solved analytically. To do this, we need the initial conditions. Say the CsgB promoter is activated at \(t= \ 0\). At this time there are no curli present, so \([CsgB]|_{t=0} = \ [CsgA_{curli}]|_{t=0}= \ 0\). However, the CsgA promoter is continuously active, so we expect to have an initial concentration \(A_0\) of free CsgA proteins at time \(t= \ 0\).

The solution to equation 5.2 is trivial:

$$ [CsgB] = \ p_B t \tag{6}$$

Substituting this into equation 5.1 results in:

$$ \frac{d}{dt} [CsgA_{free}] = \ p_{A} - \ K p_B [CsgA]t \tag{7} $$

It can easily be proven that a first order differential equation of the form

$$ y(t)' + \ f(t)y(t) = \ g(t) $$

has a solution of the form

$$ y(t) = \ e^{-F(t)} \int{g(t) e^{F(t)} dt} + \ y_0 e^{-F(t)} $$

where \(F(t)= \int{f(t) dt}\). In our case, \(f(t) = \ k p_B t\) and \(g(t) = \ p_A\). This yields equation 8.

$$ [CsgA_{free}] = \ p_A e^{\frac{-k \ p_B t^2}{2}} \int{e^{\frac{k \ p_B t^2}{2}} dt} + \ C_{1} e^{\frac{-k \ p_B t^2}{2}} = \ p_A e^{\frac{-k \ p_B t^2}{2}} \int_{0}^{t}{e^{\frac{k \ p_B \tau^2}{2}} d\tau} + \ C_{2} e^{\frac{-k \ p_B t^2}{2}} \tag{8} $$

One with a keen eye may recognize the Dawson function (equation 9):

$$ D_+ (x) = \ e^{-x^2 } \int_{0}^x{e^{y^2} dy} \tag{9} $$

As in our case, \(x^2 = \ k p_B t^2 \) and \(y^2 = k p_B \tau^2 \) and equation 10 obtained.

$$ [CsgA_{free}] = \ \frac{p_A D_+ (t\sqrt{\frac{k \ p_B}{2}})}{\sqrt{\frac{k \ p_B}{2}}} + \ C_{2} e^{\frac{-k \ p_B t^2}{2}} \tag{10}$$

Using the boundary condition \([CsgA_{free}]|_{t=0}= \ A_0\), the expression for the concentration of free CsgA proteins becomes:

$$ [CsgA_{free}] = \ \frac{p_A D_+ (t\sqrt{\frac{k \ p_B}{2}})}{\sqrt{\frac{k \ p_B}{2}}} + \ A_0 e^{\frac{-k \ p_B t^2}{2}} \tag{11}$$

Now, we can fill in equations 11 and 6 into equation 5.3, which gives us equation 12.

$$ \frac{d}{dt} [CsgA_{curli}] = \ k p_B t \left( \frac{p_A D_+ (t\sqrt{\frac{k \ p_B}{2}})}{\sqrt{\frac{k \ p_B}{2}}} + \ A_0 e^{\frac{-k \ p_B t^2}{2}} \right) \tag{12} $$

For the parameters \(p_{A}\), \(p_{B}\), \(k\) and \(A_0\), we have estimated the following values explain:

Table 1: Parameters used to obtain quantitative results from the analytical solution for the curli production.

Parameters	Value	Unit
\(\boldsymbol{p_{A}}\)	\(1.0 \cdot 10^{-10}\)	\(\frac{1}{Ms}\)
\(\boldsymbol{p_{B}}\)	\(1.3 \cdot 10^{-13}\)	\(\frac{M}{s}\)
\(\boldsymbol{k}\)	\(4.0 \cdot 10^{4}\)	\(\frac{1}{Ms}\)
\(\boldsymbol{A_0}\)	\(6.0 \cdot 10^{-6}\)	\(M\)

Plotting equation 12 with the parameter values in table 1 yields the graph shown in figure 1.

Figure 1 shows a steady production of CsgB. \(CsgA_{curli}\) concentration at \(t= \ 0\) is zero as expected, since there is no CsgB at that point. In the next few hours, \(CsgA_{curli}\) concentration peaks. We think that this is due to the high concentration of \(CsgA_{free}\) that is present at \(t= \ 0\). In figure 2, curli growth as function of time is plotted for different initial concentrations of \(CsgA_{free}\).

We conclude the following from figure 2:
Firstly, as expected, curli growth stabilizes to a rate equal to \(p_{A}\) after approximately 2 hours, independent of the initial concentration of \(CsgA_{free}\), \(A_0\).
Secondly, increasing the initial concentration of \(CsgA_{free}\), \(A_0\), increases the height of the peak. Even with zero initial \(CsgA_{free}\) concentration, a small peak can be found at one hour. This is a consequence of \(CsgA_{free}\) build-up when the CsgB concentration is still very small.
Thirdly, during the first two hours, few CsgB proteins are present in the system. We therefore expect that the length of the curli fibrils that started in the first few hours are much longer than the fibrils that started at later times.

References

still has to be made