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.

In conclusion we were able to detect a significant difference from the Bitis species and the Naja nigricollis.

Peptide substrate screening

In order to find more suitable substrates, we conducted screening experiments using JPT Peptide Technologies’ Protease Substrate Sets. They 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 nigricollis as seen in figure 8. 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 is shown below.

As seen in figure 9, this peptide is not cleaved by any of the three venoms, and can be consequently used as a negative control.

The peptides below, are cleaved by Bitis arietans and Bitis gabonica, as seen in figure 10. They can be used to distinguish between these two Bitis snakes and Naja nigricollis.

Three peptides were specifically cleaved only by Bitis arietans see figure 11.

The peptide in figure 12 is cleaved only by Bitis gabonica and can be used as a single positive response for that snake.

You can find the raw data from this experiment here. For more on the analysis of this experiment, click here.

Assembly of amilCP with and without His-tag

As part of our collaboration with the Biosensor project one of the response genes used in the kit was amilCP (BBa_K592009), which we decided to improve by adding a His-tag. The results of the assembly of amilCP (BBa_K592009) with promoter and RBS (BBa_K608003) is shown in figure 13 by a digestion. The expected bands for a double digestion with EcoRI and PstI are 2029 bp for the backbone and 774 bp for the amilCP. For the single digests the bands should be 2803 bp which corresponds to the bands on the picture.

The His-tagged amilCP sequence was ordered as a gBlock and assembled into the pSB1C3 backbone. The results of the modified amilCP with the His-tag is shown in figure 14. The expected bands for a double digestion with EcoRI and PstI are 2029 bp for the backbone and 792 bp for the amilCP with His-tag. For the single digests the bands should be 2821 bp which corresponds to the bands on the picture.

A PCR was run on the amilCP with His-tag the results is shown in figure 15. Well number 4 and 5 was amilCP with His-tag (BBa_K2355002) and it showed clear bands at 1065 bp.

Below two pictures of amilCP with His-tag (BBa_K2355002) is displayed see figure 16. The left picture is amilCP with His-tag spun down in LB media. The picture on the right is a picture of amilCP with His-tag after the lysis procedure by sonication. It is clear that the expression of the blue/purple color is very strong. In conclusion the amilCP has been successfully modified by adding a His-tag without compromising the protein.

AmilCP with His-tag purification test

To test whether the amilCP with His-tag worked as expected, we purified two solutions of amilCP; one with His-tagged amilCP, and one with just amilCP. Cells producing the His-tagged amilCP were lysed in accordance with the cell lysis protocol before being used in this experiment. The non His-tagged amilCP producing cells were not lysed because they secreted the amilCP out of the cells and into the media.

Both solutions of amilCP were diluted to an equal absorbance before the solutions were loaded onto Ni-NTA columns to be purified. The purification is based on loading the columns, washing away residual proteins, and eluting the target protein.

As shown in table 1 below, the vast majority of amilCP without His-tag seems to run right through the column, as high concentrations can be detected in the flow through after adding the samples to the column. In addition, the absorbance of the eluant is not far from that of the blank, indicating that no amilCP was retained in the column and eluted. It seems like it makes no difference to run the sample through the column multiple times before washing and eluting, which makes sense if the amilCP is not bound to the column.

Table 2 shows a different pattern for the His-tagged amilCP. For the His-tagged amilCP, the flow-through after sample loading yields concentrations of amilCP that are lower or equal to that of the eluates. The number of times the sample is run through the column seems to make a big difference, especially in the 2nd elution, where it seems there are a linear correlation between the number of times cell lysate were loaded onto the column, and the concentration of eluted amilCP with His-tag.

The difference between the purification of amilCP with and without His-tag is illustrated by figure 16. It is clear that amilCP without His-tag just runs directly through the column, whereas the His-tagged amilCP actually binds to the column and elutes as expected.

From these results we can conclude that our his-tag purification of amilCP has worked.