Substrate Set

Our substrate screening experiment produced a large number of interesting substrates for our detection assay. We looked at the raw fluorescence measurements, but we also applied statistical analysis to identify the peptides that had significant differences between the three venoms. In addition, we modelled the enzymatic activity of the venoms based on our measurements.

You can find the plots of logarithmic fluorescence signal and time of all the substrates here

The relative signal of each substrate between the three snakes was also analyzed. You can find that part here

Lastly, we looked at the fluorescence output in terms of enzymatic activity. We fit a non-linear model in the reactions and calculated their rates. You can find the models for all the substrates here

LacZ Device

Purpose

If β-galactosidase is used as a reporter in our biosensor, we would have an output of our device that depends on two subsequent enzymatic reactions. We want to model how the enzymatic activity of the first reaction affects the kinetics of the secondary reaction, and find some optimal time for the first reaction to run, if we want the combined time of both enzymatic reactions to be as short as possible.

Background

In investigating if our prototype would be a viable option for in-field detection we decided to meet with Andreas Lausten, Co-founder of VenomAB, a producer of breakthrough solutions to modern antivenom monovalent production . As per his recommendation our system would ideally have to give a rapid response. As of yet, our assays had shown response time above 45 minutes for certain venoms, and we should seek to reduce the response time. In doing this we would have to amplify the output of our device compared to that of just using chromoproteins such as AmilCP.

Central assumptions

Ideally we would like to do this with different enzymes, especially enzymes with a high turnover rate and a robustness towards snake venom. However, due to time constraint we decided to establish a proof of concept for using β-galactosidase as an amplifier of the snake venom cleavage reaction, using the following assumptions

- Incubation times are static (no flow or dilution through the system)

- Volume is kept constant

- The concentration of ONPG in the secondary reaction is in great excess. This means that our released reporter enzyme from reaction 1 will be saturated with substrate throughout the secondary reaction, and will catalyse reactions at Vmax throughout the incubation period

Design of the Model

We imagine a device with two chambers (CH): CH1 and CH2 (See figure 1). When testing for snake envenoming, a blood sample will be loaded into CH1 with a set volume. CH1 will contain some sort of biotinylated surface e.g. biotinylated beads. Our biobrick testing device will attach to the biotinylated surface via the ScAvidin domain, and if venom is present, the linker peptide of the biobrick device will be cleaved, thereby releasing β-galactosidase. After a certain incubation time in venom, the sample is transferred from CH1 to CH2.

Primary reaction

As we needed a lot of kinetic parameters for our lacZ composite part, which would require a lot of kinetic experiments on our expressed part, we did a first iteration of our model, using a mixture of arbitrary values as well as values from literature to create a proof-of-concept that it would, in theory, be possible to determine an optimal timepoint for the incubation device to let the sample flow from CH1 to CH2.

In the first reaction, our expressed composite lacZ part (BBa_K2355313) is converted to ScAvidin and free enzyme by the reaction described in figure 2:

Besides the central parameter k1, which is the reaction rate constant for the reaction, both snake venom concentration and the concentration of the expressed composite part seems to have an influence on the rate of the reaction. As the snake venom proteases are not depleted during the reaction, no differential equation for concentration of venom is made. That leaves us with a very manageable set of ODE’s (Figure 3):

When simulating these ODE’s, the initial venom concentration is set to be 100 ug/mL, as this was the highest concentration of venom that we used in our experiments. The initial fusion protein concentration is set as being 2.47*10^-6 mol/L (2,47 uM), as this is the maximum binding capacity of our biotinylated beads [1]. The reaction rate constant has been determined from the data of our protease substrate assay, where we calculated a rate constant of k1 = 0,014 on the basis of the data from Bitis gabonica venom on well O22 of the substrate set.