Results

The AMC experiment showed that we are able to detect a significant difference from the Bitis species and the Naja nigricollis. The initial AMC substrate experiment led to a more comprehensive substrate screening experiment that resulted in multiple substrate candidates. We managed to assemble the peptide sequences into the plasmid backbone (pSB1C3). The venom degradation test of the reporter molecules amilCP and β-galactosidase showed no reduction in the colorimetric or enzymatic properties. We improved part BBa_K592009 by adding a His-tag, the expression of color was not reduced and the color protein could easily be retained by a His-tag purification. To further improve our diagnostic device a reduction on the response time for the result was undertaken. Instead of the amilCP the reporter enzyme β-galactosidase were to be attached to the substrate linker. However, there were no successful assembly of ScAvidin with the linker to the β-galactosidase.

Visit experimental design page for the theory and design behind our experiments, and protocols for the protocols of our experiments.

AMC Experiment

In this experiment, we used the AMC fluorescent molecule, coupled with a peptide sequence (A-L-K) known to be cleaved by serine proteases from literature. The molecule emits fluorescence when it is released from the peptide it is coupled with. For that reason, we expected to see fluorescence when the peptide was cleaved by proteases in the snake venom. We first generated a standard curve of the AMC molecule without the peptide sequence as seen in figure 1. The raw data for the standard curve can be found here.

We tested the AMC-substrate peptide against the venom from our three snakes of interest, Bitis arietans, Bitis gabonica and Naja nigricolis. We made measurements in five different timepoints, with five different venom concentrations. The background noise was deducted from all measurements. The experiment showed that this particular substrate is cleaved significantly by the two venoms from Bitis arietans and Bitis gabonica, but not by the venom of Naja nigricolis as seen in figure 2-4. Different concentrations of venom had great effect in the fluorescence intensity. You can find the raw data here.

We repeated the experiment with two different substrate concentrations, along with three different venom concentrations, similar results were obtained. The bigger substrate concentration produced much higher fluorescence intensity as seen in figures 5-7. You can find the raw data here.

Peptide substrate screening

In order to find more suitable substrates, we conducted screening experiments using JPT Peptide Technologies’ Protease Substrate Sets. They are consisted of 360 oligopeptides with cleavage sites described in scientific literature, flanked by a fluorescent molecule (EDANS) and a quencher (DABCYL). Fluorescence is obtained when the fluorescent molecule is released from the complex due to cleavage by proteases.

The plates with the peptides were incubated with the three different venoms. Background fluorescence was deducted from the measurements. A great number of wells exhibited different fluorescence patterns, showing that some peptides had different specificities depending on the venom. The highest cleavage activity was observed when incubating with the venom of Bitis arietans. As expected, no peptide showed unique specificity for the venom of Naja nigricolis. From these peptides, the most significant ones that can be used for distinguishing between the three venoms were selected and submitted as parts. The plots with fluorescence intensity when incubating the three different venoms of the submitted peptides are shown here.

These two peptides, J12 and D20, are cleaved by all three venoms. They can be used as a positive control, or as the start of a decision tree classification.

This peptide is not cleaved by any of the three venoms, and can be consequently used as a negative control.

These peptides are cleaved by Bitis arietans and Bitis gabonica. They can be used to distinguish between these two snakes and Naja nigricolis.

These three peptides are cleaved only by Bitis arietans.

This peptide is cleaved only by Bitis gabonica.

You can find the raw data from this experiment here (link for excel file, measurements files).