Team:UAlberta/Demonstrate

DEMONSTRATE

The Beetector is composed of three main parts that assure the final product works as intended: the spore ligand, the reporter phage, and the paper strip. Team UAlberta worked arduously to design and evaluate each of these parts, and our progress is discussed below.

Spore Ligand

The spore wall of Nosema ceranae spores is uncharacterized and described in the literature as a general microsporidia fungal spore [1]. As detailed in our Design Page, we decided to use the NEB Ph.D.^TM-12 phage display library to find a specific ligand for these fungal spores. This is, to the best of our knowledge, the first time a phage display approach was used to search for a N. ceranae ligand.

Does our panning protocol work?

The first step was to design our biopanning protocol. Starting from the NEB Ph.D.^TM-12 phage library manual, we adapted some of the steps to our samples. Our detailed procedure can be seen on our Notebook: Protocols page.

To test if there was any unbound phage pelleting in our panning experiments, we performed the same procedure with a different phage, the lytic phage, T4, which should not bind to the spores. Our results showed that all the T4 added to the spores was in the supernatant rather than the pellet. Moreover, we also tested a phage that doesn’t belong to the NEB library (from one of our graduate advisors experiments) and observed a similar result.

Figure 1: Plaques of phage T4 (clear regions) on a lawn of the standard bacterial host E. coli 11303, titering the phage in the supernatant of the biopanning method. Virtually, all the T4 phage added to the spores was recovered in the supernatant.

Can we get good ligands through these experiments?

Through three rounds of biopanning with spores, we obtained multiple enriched plaques that are good candidates for displaying specific ligands. To obtain spores for the panning experiments, we used the cleanest samples we could find: Courtney McInnins kindly provided us with research bees that were sugar-fed, raised in a laboratory environment, and infected with N. ceranae since the larval stage.

Figure 2: Acid eluted phage plaques (blue dots) recovered from the third round of biopanning experiments.

Figure 3: Optical micrograph (1000x) of the sample used for the biopanning experiments.

Are our ligands specific enough?

To test if the peptides displayed by these phages are indeed specific towards N. ceranae spores, we proceeded with our panning protocol as usual, but instead of testing it against the spore-rich purified samples, we used whole bees crushed and diluted in sterile water. This procedure is detailed in our Notebook: Protocols page. The goal of this experiment was to see if our phages would pick up any part of uninfected bees, to confirm that the ligand is specific and will not bind to background information and aid in the prevention of false positives.

As seen in Figure 4, no plaques were observed in the pellet of the panning experiment. This strongly indicates no phages bound to the whole bee samples when they were not infected with N. ceranae. Further characterization, such as sequencing and characterization of individual phage plaques, can help us pick a suitable ligand for the Beetector in terms of specificity, fast binding, and ligand to spore ratio.

Figure 4: Acid elution plates from biopanning with whole bee samples, showing no phage plaques.

Reporter phage

The next component of the Beetector aims to make the phages displaying the spore ligand visible. We picked a chromoprotein, amajlime (BBa_K1033916), from the iGEM Registry. We were able to transform and express the standard part (BBa_K1033914), containing amajLime, a strong RBS, and a constitutive promoter, from the 2019 DNA distribution kit.

Figure 5: Plates with antibiotic showing the expressing colonies of cells transformed with amajLime.

Figure 6: Overnight cultures of E. coli DH10β transformed with BBa_K1033914 (right) and BBa_K1033916 (left).

We also purified amajLime by lysing these transformed E. coli DH10β cells and using a 10kDa size exclusion column, the buffer exchange was performed to prevent the protein from degradation from the B-PERII reagent. The protein was eluted into Tris-saline buffer with glycerol for storage and analysis. The amajLime visual signal resolution was determined by making multiple dilutions that were characterized by fluorescence in the microplate. The visual signal of amajLime informs us of the markers to be interpreted in the paper-based chromatography for the Beetector. The signal is also used as a scale to identify the appropriate phage ligand candidates from the model that binds to the spore in appropriate quantities, to show a range in resolution that is visually distinguishable.

Figure 7: Purification of amajLime by first lysing E. coli DH10β transformed and using a 10 kDa exclusion column.

Figure 8: AmajLime serial dilutions under white light (top) and UV light (bottom) to determine the visual signal resolution.

Figure 9: Pellets of 50 mL of overnight cultures of E. coli DH10β transformed with amajLime, a strong RBS, and a constitutive promoter (BBa_K1033914).

We used these purified amajLime samples to further characterize this chromoprotein and to determine parameters for our model.

Can we engineer coloured phages?

We learned early on that we couldn’t directly express amajLime in the M13 phage structural protein pVIII. Inspired by the approach adopted by Hess and colleagues [2] to tag M13 pVIII with GFP using sortase, we designed parts containing a strong inducible promoter, a strong RBS, the amajLime coding region, the sortase tag, a His-Tag for purification, and a double terminator.

Figure 10: Schematic of one of our designed part composites, containing a T7 promoter, amajLime coding region, the Sortase-A tag, a His-Tag, and a double terminator.

The other tag was designed on the distal region of the pVIII encoding gene in M13. Both parts were synthesized as gBlocks but unfortunately could not be transformed despite multiple attempts

The Sortase-A pentamutant (eSrtA) in pET29 was a gift from David Liu (Addgene plasmid # 75144) [3]. We transformed the Addgene plasmid into E. coli T7 Express lysY/Iq, tested different expression protocols, and purified the enzyme from the cellular lysate of B-PERII reagent using a His GraviTrap column. The buffer exchange to Tris-saline buffer and glycerol solution was performed on the elution of the column for storage. The enzyme can be seen in the SDS-PAGE gel in Figure 11.

Figure 11: SDS-PAGE gel of the purified Sortase-A pentamutant that was transformed.

Paper Strip

Can a paper strip separate the components of our mixture?

Figure 12: Elution of Whatman P5 filter paper in a sample of crushed bees with pollen present.

We tested different types of paper filter against crushed bee samples, our chromoprotein, and a mixture of the two to check which one would allow for better separation of the components and allow for better visualization of our Beetector signal. Ideally, the paper strip would separate all the unbound phages (that would elute farther to the upper end of the strip) from the spore-bound ones (that would form a concentrated band at the bottom region of the strip). Out of the ones tested, Whatman P5 filter paper allowed for the best elution of the larger compounds present in the bee, namely pollen, as seen in Figure 12.

Upon testing the untagged amajLime purified from cell lysates, we noticed the elution of the unbound protein varied with the viscosity of the solvent used - samples diluted in buffer would elute farther in the paper, forming a dim green-yellow region, whereas the chromoprotein diluted in more viscous liquids (B-PER or glycerol) wouldn’t elute far but rather formed a visible band at the bottom of the paper.

Figure 13: Whatman P5 filter paper eluted with 2 mL of purified amajLime in B-PERII.

Once we obtain a phage displaying a N. ceranae spore ligand and amajLime, we can test different solvent mixtures to evaluate which one would improve the signal visualization of the Beetector.

Product prototype

The sessions above focused on each main part of our project in the lab, but it is very important that our Beetector setting works in its intended real world application.

Can a beekeeper use the Beetector easily?

A central part of our project design was the focus on the end-user - commercial and hobbyist beekeepers - as our diagnostic tool has to be convenient, simple, and usable to them. To test this, we used lab supplies and mock Beetector solutions to come up with a project prototype. We also wrote simplified instructions to guarantee a safe and reliable use of our product.

Figure 14: The instruction diagnostic procedure and disposal for beekeepers when using our prototype.

We then asked a hobbyist beekeeper to follow these instructions and use our prototype in front of us. This was done at their house - an environment this beekeeper is comfortable in, and most likely similar to where our final product would be used in real life.

Figure 15: Image of our prototype package with the instruction.

By observing this beekeeper use our prototype, we realized we are on the right path, and were able to use his reviews and input to improve the instruction for the utilization of our final product. Please see our Integrated Human Practices page to learn more about the reviews we received regarding our prototype.

This made us confident the Beetector can be used to aid beekeepers to diagnose and fight N. ceranae infections, saving the bees one hive at a time.