Difference between revisions of "Team:Munich/Model"

Detection Limit

One major concern when dealing with the problem of diagnostics on patients is extracting the sample with which detection can actually be performed. Since we wanted our method to be non-invasive, one concern that we needed to deal with is the concentration of pathogens and thus detectable RNA in the patients mucus or other non-invasive sample. First approximations from different papers already showed that virological samples show concentrations no higher than low pM and can even go as low as aM.

Our wetlab experiments indicated that the detection limit of the Cas13a RNase activity is in the range of 10 nM. Using our kinetic data, we estimated the rate constants for the different reactions to create a simple ODE model.
The chemical and differential equations for the model are shown below:

Lysis on Chip

We modelled the lysis process on chip to get an idea of how long lysis would need to take place in order to release enough RNA for downstream amplification. For this, we constructed a very simplistic model for bacterial cell lysis. In this, we estimated the rate constants for cell lysis by common colony PCR protocols which use a 10 minute lysis step at 95 °C for thermolysis. Thus, we considered a half-life of bacteria of 2 minutes at 95 °C. This would result in a lysis efficiency of 96.875%. Starting from this estimation, we considered the rate constant of lysis and thus the half-life using Arrhenius equation as commonly done in the literature:

(7)

(8)

with rate constants k₁ and k₂ at temperature T₁ and T₂ and Boltzmann constant R.

where R is the gas constant and k₁ and k₂ are the rate constant at temperature T₁ and T₂ The activation energy difference E_A was fitted to a barrier that follows the common rule of thumb that lysis should increase twice in efficiency for every temperature increase of 10 °C. The model for lysis is shown derived in the following:

The full model can then be described by the coupled ordinary differential equations:

(9)

(10)

with k_lysis being the rate constant of bacterial lysis, k_RNase the rate constant of RNA degradation, count of target RNA [targetRNA] and count of bacteria Baks(t). The solution to equation 3 is of course simply:

(11)

Plugging equation 5 into equation 4 gives

(12)

where ratio determines the copy number of a target RNA in a single cell. This differential equation has the form and thus the analytical solution:

Equation 7 + 8

(13)

(14)

with initial condition of

(15)

we get the final solution to the lysis equation:

(16)

The full model at different temperatures looks as follows:

Figure 3: Effect of lysis temperature on the lysis efficiency of bacterial cells and determination of the released concentration of target RNA from lysis assuming a ratio of 30 RNA molecules per cell.

References

1. Moody, C., et al. (2016). “A mathematical model of recombinase polymerase amplification under continuously stirred conditions.” Biochemical Engineering Journal 112: 193-201.
2. Moody, C., et al. (2016). “A mathematical model of recombinase polymerase amplification under continuously stirred conditions.” Biochemical Engineering Journal 112(Supplement C): 193-201.
3. Li, L., et al. (2011). “Kinetics of hydrothermal inactivation of endotoxins.” Appl Environ Microbiol 77(8): 2640-2647.
4. Valente, W., et al. (2009). “A Kinetic Study of In Vitro Lysis of Mycobacterium smegmatis.” Chem Eng Sci 64(9): 1944-1952.
5. Fabritz, H. (2007). “Autoclaves Qualification & Validation.” Experts Meeting in Baden
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8. Chiruta, J. (2000). “Thermat Sterilisation Kinetics of Bacteria as Influenced by Combined Temperature and pH in Continuous Processing of Liquid.” Thesis, The Universit of Adelaide Department of Chemical Engineering Faculty of Engineering.
9. Rauhut, R. and G. Klug (1999). “mRNA degradation in bacteria.” FEMS Microbiol Rev 23(3): 353-370.
10. Licciardello, J. J., & Nickerson, J. T. R. (1963). “Some Observations on Bacterial Thermal Death Time Curves.” Applied Microbiology, 11(6), 476–480.
11. Deindoerfer, F. H. (1957). “Calculation of Heat Sterilization Times for Fermentation Media.” Applied Microbiology, 5(4), 221–228.
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13. V. Mekler1, L. Minakhin, E. Semenova, K. Kuznedelov and K. Severinov "Kinetics of the CRISPR-Cas9 effector complex assembly and the role of 3′-terminal segment of guide RNA" Nucleic Acids Research, Vol. 44(6): 2837–2845