Team:UCSC/Detection

Detection

Detecting Shiga Toxin

In addition to a way to eliminate STEC, professionals in the agricultural industry stressed a need for a better method of detecting STEC before products are sent out to be sold. The presence of Shiga toxin contamination in manure or water is typically detected by PCR [1], a lengthy protocol that requires access to a laboratory. Thus, we explored ways to detect Shiga toxin in the field using DNA aptamers.

What are Aptamers?

Aptamers are short, single stranded DNA or RNA molecules whose specific three-dimensional folds lead to high binding affinity for specific ligands. Unlike antibodies which take months to culture and extract from animals, aptamers are generated in vitro and take weeks to produce [2]. Aptamers also have the advantage of greater stability than antibodies which is important for conditions in which there is a continuous need for detection of pathogens such as Shiga toxin. Once tested and amplified, the best aptamers can be made available to farmers and ranchers, allowing them to detect STEC and use Progenie to eliminate it where needed on their own farms.

Designing Shiga toxin-specific aptamers

We plan to generate DNA aptamers that bind specifically to Shiga toxin using the Systematic Evolution of Ligands by Exponential enrichment (SELEX) protocol. In the SELEX protocol, a library of oligonucleotides is prepared with random DNA sequences in the middle, and fixed sequences on either end. The unique DNA sequence of each aptamer dictates the specific three-dimensional fold, which in turn dictates binding affinity to certain ligands [3]. The library of potential aptamers is then tested against the Shiga toxin protein to identify candidates with a high affinity for the target. Target-specific aptamers get PCR amplified, and the procedure is repeated until the library of aptamers is reduced to a manageable size. The remaining molecules in the aptamer library at the end of SELEX selectively bind to Shiga toxin [3].

Working with a recombinant form of Shiga toxin

To produce aptamers that bind to Shiga toxin, we aimed to produce a non-toxic, recombinant form of the Shiga toxin protein, referred to here as Dead Shiga (structure shown below). Producing the actual toxin would require permission from EHS and the iGEM committee, our design only uses the non-toxic subunits. The Shiga toxin A and B genes encode for an A subunit consisting of the active site (A1 subunit) and an anchor (A2 subunit) to five identical B chains that form a pentamer. The B subunits bind to Gb3 receptors on the surface of intestinal epithelial cells in humans, then cleave the A1 subunit so it can enter the cell where it disrupts protein translation by cleaving ribosomal RNA [4]. The A2 and B subunits confer no toxicity, thus our Dead Shiga protein consists only of these subunits.

Dead Shiga, the recombinant Shiga toxin protein consisting of the A2 subunit (blue) and the surrounding B subunits (green).

Expression of Dead Shiga

To express Dead Shiga, we obtained the sequences for subunit B from UCSC microbial genome browser [5] and for A2 subunit from Tu et al. [6]. Additionally, we based our construct on Tu et al.’s design consisting of: BfuAI restriction sites, A2 sequence, 11bp linker, the full B subunit, and a 6x HisTag, all under one promoter [6]. The A2 and B subunit sequences feature respective signal sequences to signal the cell to secrete the proteins into their surroundings. The 11bp linker contains an ribosomal binding site (RBS) that increases production the B subunit in order to form the pentamer [6]. The 6x HisTag was added for protein purification as well as a protein immobilization method for the SELEX protocol. The destination vector pET52-Express will be used to clone via Golden Gate in this gene block using the BfuAI sites. Once expressed, the Dead Shiga protein will be used as the antigen for SELEX.

Construction of expression vector

To construct the destination vector pET52-Express, we used a pET-52b(+) backbone containing a ccdB toxin gene insert flanked with type IIS restriction enzyme sites for Golden Gate assembly (as seen on the plasmid map below). The insert was constructed by amplifying the BBa_1016 BioBrick part from pOSIP-KC (Addgene), which contains the ccdB gene and a promoter, using flagged primers to add the type IIS sites as well as NcoI and BlpI sites for standard restriction cloning. Procedure for the construction of the destination vector can be found here.

Further Directions

We submitted a check-in form to iGEM but have not yet gotten approval, thus we were unable to produce Dead Shiga. However once Dead Shiga is produced and isolated, we can use it to produce Shiga toxin-targeting aptamers using SELEX. The aptamers can then be combined with a lateral flow device to provide a fast and effective way for farmers to detect Shiga toxin in their products before they are sold to consumers (more details).