Team:DTU-Denmark/ExperimentalDesign
Experimental Design
Venom composition
Snake venom is a complex mixture of proteins, enzymes and other substances with toxic and lethal properties. Its function it to defend against threats, immobilize the prey, and help the digestion. Some of the proteins in the snake venom have very specific effects on biological functions, such as blood coagulation, blood pressure regulation and transmission of nervous or muscular impulses.
Proteins constitute 90-95% of venom’s dry weight and are responsible for most of its biological effects. Enzymes make up 80-90% of the Viperidae family venom and 25-70% of the Elapidae family venom [1]. The most common enzymes are oxidases, phospholipases, metalloproteinases, hydrolases and serine proteases. Polypeptide toxins include cytotoxins (effects mainly on local tissue), cardiotoxins (effects on heart tissue) and neurotoxins (effects on nerve tissue).
Venomic studies have enabled the characterization of venom composition, from snakes species of medical importance [2]. From these, it can be shown that the venom between snake species, even within families, is more similar than expected [3]. Major protein families like metalloproteinases (SVMPs), Phospholipases A2 (PLA2), cysteine rich secretory proteins (CRISPs) are present in all snake families, with the relative abundance in the venom composition being the only difference [4].
However, these studies have also shown that there are certain enzymes that are probably innate to snake families, for example the prominent serine proteases in the Viperidae venom, or the much more abundant three finger toxins (3FTx) in Elapidae venom.
Choice of enzymatic assay
In Australia, immunoassays based on venom-specific antibodies have been developed to distinguish between the most lethal snakes in that region [5]. Immunoassays have been used or suggested to be used as a detection mechanism for snake venom for many years [5,6].
We apply an alternative approach to detect snake venom circumventing immunoassay based methods. We want to create a proteolytic enzyme assay based on a linker sequence which contains a cleavage site for proteases that are characteristic for the venom of different snakes.
Proteins that give off signals when a linker sequence is cleaved, are not a new invention. FRET (Föster resonance energy transfer) has been used for many years in research, to gain a better understanding of protein-protein interactions.
Our approach to snake venom detection, utilising the proteolytic activity of different venoms, has several potential advantages compared to current methods used for snake venom detection:
- - The recombinant proteins can be produced at a lower price compared to the current immunological methods, which involves inoculating snake venom in horses.
- - With this approach, we are able to find new potential target peptides for snake venom cleavage in a relatively fast and cheap way. Screening of these peptides is much less time consuming and easier than trying to identify suitable antibodies for the venom enzymes and toxins.
- - If reliable measurements can be made, we might be able to not only detect which snake people have been bitten by, but also to determine the level of venom injected into the blood.
Choice of snakes
The focus of our project was sub-Saharan Africa, where snakebites are more common than other regions of the continent. Approximately 1 million snakebites occur in sub-Saharan Africa annually, resulting in up to 500.000 envenomations, 25.000 deaths and another 20.000 permanent disabilities. Due to no reliable reporting system in place, victims often never report their injury to clinical facilities, which makes these numbers uncertain [7].
In sub-Saharan Africa, over 50% of snakebite incidents are not appropriately treated. Most victims, who receive treatment by healthcare professionals, have nevertheless delayed seeking medical attention sometimes up to 1 to 2 weeks. In many sub-Saharan countries, poor availability of expensive antivenom contributes to increased morbidity and mortality, whilst snakebites continue to remain a neglected health problem.
In that region, approximately 60% of all bites are caused by vipers, with the puff adder (Bitis arietans) being responsible for the most fatalities overall. For that reason, we chose these species to be of primary focus in our experiments.
Sub-Saharan Africa also hosts cobra species of the Elapidae family, which in forested areas, cause 30% of all venomous bites. One of the most prominent of them is black-necked spitting cobra (Naja nigricollis), which was also chosen as a representative of the Elapidae family for our project.
Finally, another important snake species of that area is a close relative of the puff adder, the gaboon viper (Bitis gabonica). This snake is not responsible for a major percentage of deaths, but is also very dangerous due to the enormous amount of venom that it injects with its bite, which is the highest among all snakes. This snake was chosen in order to explore the possibility of distinguishing snake venom at the species level.
AMC experiment
7-Amino-4-MethylCoumarin is a fluorescent compound that can be used to create fluorogenic oligopeptides for detection of proteolytic activity. AMC has been used for protease activity assays since 1976 [8], and other amine species, such as nitroanilides, have been used for even longer [9].
AMC emits light at 460 nm, with a maximum excitation at 366 nm.
Due to the primary amine in the aromatic ring (see figure 4), AMC is able to bind to the C-terminal end of peptides. Thus, the AMC molecule will emit light only if the bond between the C-terminal peptide and AMC is cleaved.
Among the major classes of proteases, serine proteases are one that stands out when analysing snake venoms. As explained in the background page, serine proteases are found predominantly in the venoms of snakes belonging in the viperidae family, with only a few exceptions in other families, such as the elapids. By targeting serine proteases with synthetically designed oligopeptides, our team hypothesised that it would be possible to distinguish snake venom from viperidae from other snake families.
To test our hypothesis, we identified some oligopeptides that have been used in the past to characterise snake venom from different Bitis species. Although studies have been able to document the ability of Bitis venom to cleave blood factors such as fibrin and bradykinin/kallidin [10, 11], no comparative study has ever been carried out, researching the ability to cleave these substrates by snake venoms from across taxonomical families.
We found a suitable peptide substrate in literature [11], and we conducted our initial experiment to test our hypothesis with that specific peptide. The results can be found here.
Substrate screening
In order to distinguish different snake species, we needed to be able to detect enzymatic activity that was unique to the given species. Our study of literature indicated that some proteases found in the snake venom, are unique to the venom of the Viperidae family [2]. For this reason, we decided to focus specifically on these proteases.
One of the most efficient ways to study protease activities and substrate specificity is the incubation of a collection of potential substrate peptides with the proteases, or in our case, the venom of interest.
As a collection of these substrates, JPT Peptide Technologies’ Protease Substrate Set was found to be the one more suited to our needs. This set is comprised of 360 peptides derived from cleavage sites described in the scientific literature. These peptide derivatives contain the cleavage site sequences, flanked by a quencher molecule (DABCYL) and a fluorophore Glu(EDANS)-amide at the N-terminus and the C-terminus, respectively. Subsequent to incubation with the snake venom, these two moieties are separated. The emitted fluorescence can be detected using standard microtiter plate readers. An illustration of how the substrate screening works can be seen in figure 5.
The results of this experiment can be found here.
Biosensor design
Our team aims to develop a novel biosensor to detect and distinguish between snake venoms from different families of snakes. Our biosensor is based on the different proteolytic activity of snake venom from different species.
The biosensor consists of a fusion protein. Inspired by the design of different FRET reporters, our fusion protein contains two larger protein domains, separated by a flexible linker containing one or more substrate sequences. However, unlike FRET reporters, where fluorescent proteins make up the two larger domains, the two large protein domains in our biosensor are a chromoprotein domain and a binding domain. The linker between the two domains is designed to contain the specific cleavage sites found through the previously described AMC- and substrate screening experiments.
In this project, our team has used the RFC[25] standard (Freiburg standard) to assemble our protein domains in-frame. The standard RFC[10] standard of the iGEM competition does not give the option to assemble parts in-frame, as the ligation between XbaI and SpeI will leave an asymmetric scar.
Complying with the iGEM paradigm of protein domains, we fused the DNA sequences of our different parts in silico, and ordered most of our fusion-proteins as single DNA fragments.
Ideally, when using our BioBrick device to detect snake venom in a clinical setting, the output signal should be sufficiently strong to give a definite result within a short amount of time. In order to make the output signal of our test easy to analyse without unnecessary equipment, we chose to incorporate a reporter molecule that could be analysed without the use of expensive equipment. Using chromoproteins, it would in theory be possible to visually analyse whether or not a person has been bitten by a specific type of snake.
AmilCP is a chromoprotein isolated from Acropora millepora, a coral native to the indo-pacific ocean. The chromoprotein absorbs light at 588 nm, giving it a purple-blue colour [12].
The Uppsala iGEM team has been working with a collection of different chromoproteins since 2011. From their continuous collection of chromoprotein BioBricks, amilCP was chosen from Uppsalas collection, as it has a high molar extinction coefficient and can absorb light at wavelengths that are far away from the red blood cells from the blood we are testing on. Furthermore, our team used amilCP as a reporter during the BioBrick Tutorial, and had thereby gained experience with expressing this particular BioBrick from a very early starting point.
We aimed to improve the amilCP part from the distribution kit by adding a 6x his-tag to the C-terminal end of the chromoprotein. By doing this, it would theoretically be possible to purify both the amilCP part, and composite parts based on the his-tagged amilCP, using affinity chromatography. The designed part of amilCP with his-tag was submitted to the parts registry as BBa_K2355002.
Avidin can form non-covalent bonds to biotin with an unusually low association constant ( Ka=1015 M-1 ) [13], which is a much higher affinity compared to normal protein-ligand interactions that have association constant of ~ 1011 M−1. This high affinity has been utilised for years for different chromatography purposes.
We intend to use this strong interaction between biotin and avidin to immobilise our fusion protein to a column containing some sort of biotinylated surface e.g. biotinylated beads. By expressing an Avidin protein as part of our fusion protein, we can attach our expressed fusion protein to any biotinylated surface.
The avidin used in this project is derived from the Single chain avidin (Scavidin) part from the 2016 TUMunich team (BBa_K2170205). A ribosome binding site was added to the Scavidin part to ensure a high expression of the desired fusion protein.
The oligopeptides found in both our AMC experiment and our High Throughput screening experiment were used to design protein linkers for our fusion protein. Beside the linkers we found through testing and screening, we have tested an empirical linker molecule. The FRET linker with the peptide sequence RPPGFSPFRQ has previously been used on the venom of different Bitis sp., to test the neutralizing ability of newly developed antivenoms [14].
We aimed to design our own flexible linker that would allow for our two major protein domains to move independently from each other. Empirical flexible linkers found in fusion proteins usually contain either Gly or Ser/Thr residues [15]. Ser/Thr residues have the advantage of being hydrophilic residues that allow linkers to be in contact with water, enabling them to come into contact with proteases. The linkers designed by our team generally had a lot of Gly residues, in order to ensure the aforementioned flexibility. To increase the chance of cleavage, repeats of the same cleavage sequence were introduced in some of our linkers. The resulting rationally designed linker, with the ALK/ sequence identified by our AMC experiment, was submitted to the registry as BBa_K2355001.
Amplification step: Introducing β-galactosidase
In order to lower the incubation time needed in our prototype, we wanted to add an amplification step to the device. This was done by using an enzyme as a reporter instead of using chromoproteins.
By cleavage of the linker molecules by snake venom, the enzymes would be released from the Avidin/biotin surface into the eluent, just as the chromoproteins would. However, when using enzymes as reporters, the eluent would subsequently flow into a secondary reaction chamber, where the concentration of eluted reporter enzyme would be determined by a colorimetric assay. As each enzyme would catalyse multiple substrate molecules, we would expect a decrease in the necessary incubation time between the venom and our fusion protein.
To demonstrate the concept, we used β-galactosidase as a reporter, and ortho-Nitrophenyl-β-D-Galactopyranoside (ONPG) as a substrate for the secondary colorimetric assay.
The Single chain Avidin (ScAvidin) is the same as the one used for the amilCP based composite parts, with the only difference being in the ribosome binding site, where a stronger RBS (B0030) was selected.
As this composite part was a proof-of-concept for the effect of using an enzyme as a reporter, the linker used for this composite part was the empirical FRET linker mentioned earlier, as we were fairly sure our venom would exhibit some activity against this linker.
The composite β-galactosidase part was used in several proof-of-concept experiments. It was used without the addition of snake venom to prove that the ScAvidin part binds the expressed fusion protein to the biotinylated beads. It was also used to evaluate the effect of snake venom on the empirical FRET linker.
Alternative approach
In our project, we wanted to express the whole biosensor construct as a single, composite part. However, that approach has some disadvantages. Namely, the complexity of the part creates difficulties in transforming and expressing it in competent hosts, and avidin containing recombinant proteins had low rate of success in our experiments.
An alternative approach to our design of the biosensor, would be to express a protein that contains a linker and the chromophore protein amilCP that has the His tag on its C-terminus. The expressed protein would then be His-tag purified, and biotinylated at the N-terminus with a chemical reaction. The biotinylated construct can be bound to streptavidin beads, which are widely used in molecular biology applications.
The final biosensor would share the same principles with our original design, but would result in our opinion in a better success rate of production and functionality. However, this semi-synthetic design would require additional modifications after being expressed, which would increase the amount of time and cost needed to be manufactured.
References
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[10] Nikai T, Momose M, Okumura Y, Ohara A, Komori Y and Sugihara H. (1993). TKallidin-Releasing Enzyme from Bitis arietans (Puff Adder) Venom Archives of Biochemistry and Biophysics, 307(2), pp 304-310
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[13] Weber PC, Wendoloski JJ, Pantoliano MW, Salemme FR (1992). Crystallographic and thermodynamic comparison of natural and synthetic ligands bound to streptavidin J. Am. Chem. Soc., 114(9), pp 3197–3200
[14] Paixão-Cavalcante D, Kuniyoshi AK, Portaro FCV, Dias da Silvan W and Tambourgi DV (2015). African Adders: Partial Characterization of Snake Venoms from Three Bitis Species of Medical Importance and Their Neutralization by Experimental Equine Antivenoms PLoS Negl Trop Dis 9(2):e0003419
[15] Chen X, Zaro J and Shen W (2013). Fusion Protein Linkers: Property, Design and Functionality Adv Drug Deliv Rev., 65(10): 1357–1369
