Saving the bees, one step at a time

Introduction

The UAlberta iGEM team decided to build on a similar problem that was tackled last year (Nosema ceranae), this time we aim to provide the beekeeping community with an effective, convenient, and accessible detection system. Our team achieved this by conducting exhaustive laboratory work along with consultations with experts in this field, i.e. bee researchers. We planned and consulted multiple beekeepers, both hobbyist and industrial, to acknowledge their main concerns and built our project around those concerns. Another reason we decided to continue working with bees and solving their issues was our relationship with the Alberta Beekeeping Commission. Their encouragement led us to realize that while it is beneficial to have a treatment for a disease, it may be of little use if we don’t exactly know which bee populations should be treated. After several interviews with beekeepers we realized that one of their main concerns was the accessibility to detection methods. The UAlberta iGEM team set their goal to use the principles of synthetic biology to create a portable detection system to replace the current time consuming detection method.

Abstract

Honey bees are an essential contributor to our food supply, in addition to producing honey, they pollinate one third of all plants. This makes them an integral part of our food supply system. Nosema ceranae is a debilitating fungal parasite that is the most widespread honey bee pathogen in Canada. In addition to the difficulties in treating N. ceranae infections - a problem the UAlberta team tackled last year - current detection methods are slow and costly, and infective. This means that hives are often diagnosed too late for effective treatment. Team UAlberta is working to change that by developing the Beetector, a field-ready paper-based test for the detection of N. ceranae in bee samples. The system is comprised of M13 phage labelled with a chromoprotein and displaying a ligand specific to N. ceranae spores. Based on the colour intensity of the diagnostic paper strip, the severity of the hive infection can be assessed, thus democratizing diagnosis and facilitating effective treatment of N. ceranae

Spore Ligand

The beginning of our Beetector system relies on phage to a receptor on the spore wall. Gaining access to the PhD. 12 library was key in determining a successful ligand that binds to the spore wall in optimal time without infecting. For this, we decided to use an M13 phage, a chronic infection phage, rather than an infection specific phage to prevent lysing of the spore (Paepe et al. 2010). This was completed by using a multitude of biopanning experiments narrowing down to selectable phages by: allowing the phage to bind to our spore, washing away unbound phages and then eluting the phages that are not bound to our spore of interest (Lim et al. 2019). We kept running biopanning experiments to optimize our conditions and to secure that our phages were securely matched to the spore wall and not to bring along phages by proximity (Lim et al. 2019).

Reporter Phage

The reporter phage will be a combination of 3 distinct parts: the chromoprotein, the spore ligand, and the sortase enzyme attaching the amajLime chromoprotein to the selected spore ligand. The sortase enzyme was found in Staphylococcus aureus gram positive bacteria and was found to be characterized as a transpeptidase on the exterior of the cell wall that covalently attaches to proteins (Melvin et al. 2011). The amajLime construct will be first grown in DH5alpha cells and then transformed into a T7 expression strain to allow for maximum expression of our protein.

Paper Strip

When creating our paper strip we needed to isolate N. ceranae spores from the honey bee midgut since that is where they are located (Kim et al. 2017). We decided to create our paper strip similar to a thin layer chromatography ideology; however, heavier masses like the spores will stay at the bottom whereas the phages will elute to the top creating different bands. In order to visualize the spore and try to test the paper strip we have created tests using various types of filter paper, sizes of paper and also different eluting speeds.

This was all partnered with modelling experiments helping us determine different concentrations of phages we should be adding, the dilution of amajLime and also how the paper stip should be used. The paper strip joins all three components of the Beetector system together, and thus showing if the beekeeper has N. ceranae in their hive.

QC + Safety

In order to ensure our product is not only pure but also safe to use in the environment, we will be using a quenching solution to neutralize the phages before discarding the solution in a separate waste basket. You will add the quenching solution to any tube that had our amajLime construct in it including the paper strip and then discard the quenching solution mixed with the tubes into the waste bin.

Also, ensuring the purity of our reporter phage is essential. Performing activities such as column based chromatography and selection and purification of phages is essential to maintain. Purification of our phages ensures that there will not be a mixture of phages but rather one select phage from the library that binds more efficiently. Also, verification of the sortase enzyme activity and amajLime is essential to then add to the phage.

End Product

The end product of the Beetector will be a solution of the amajLime chromoprotein attached through a sortase enzyme to our M13 phage. There will be a separate solution with our BPER to help create the bands and elute the phages up the paper strip. Protecting the phages from escaping to the environment is a priority and that is why they will be contained in a microfuge tube before being added to a falcon tube that contains the crushed bees. This will all get quenched with our solution to avoid contamination into the environment and then be disposed of into our supplied container.