| NDSU - iGEM 2023

Our Project

Each year, almost half of all beehives suffer from colony collapse disorder (CCD) (1). CCD is a complex problem caused by various factors like disease, temperature, and food availability. However, one significant contributor to CCD is Varroa mites. These mites infiltrate beehives by entering as adult females and laying eggs inside brood cells (8). Once the eggs hatch and the bees emerge, the female mites attach themselves to the bees, reproducing and infesting the colony. If left untreated, these mites eventually lead to mass population loss, culminating in CCD.

Currently, there are only two main diagnostic methods for detecting Varroa mites: the powdered sugar test and the alcohol test. Both methods involve taking around one cup of bees out of the hive and placing them in a container. The bees are then covered in confectioners’ sugar or ethanol, depending on the method used. The container is vigorously shaken to make the mites fall off, and a mesh cover is used to separate the mites from the bees. The mites are then counted to determine the infestation rate (number of mites per 100 bees). Unfortunately, these methods are highly disruptive and prone to inaccuracy.

Our project aims to address this diagnostic challenge by developing a biosensor-based testing method to detect Varroa mites. We exploit the fact that Varroa mite excrement consists of 95% guanine, which provides us with a suitable target (3). Our biosensor functions by triggering a genetic pathway in the presence of guanine, resulting in the production of mScarlet-I, a protein that can be visually analyzed or measured using a spectrophotometer. This allows us to determine the number of Varroa mites present. The data obtained can then assist beekeepers in making informed treatment decisions.

We chose this project for this year's iGEM because our home state, North Dakota, is the top honey-producing state in the United States (9). North Dakota also has the second highest number of bee colonies and the highest number of bee colonies per capita. Moreover, North Dakota's economy is heavily focused on agriculture and has recently embraced autonomous systems and agricultural technology (Agtech). Therefore, we believe our project can provide significant value to the local beekeeping and agricultural ecosystems.

Guanine-II Riboswitch

design-riboswitch2

The first genetic approach was based on the Guanine-II Riboswitch (2). Riboswitches are regulatory agents located within mRNA that modify the production of the protein produced by its mRNA based on the concentration of an effector molecule. In this case, the effector molecule of Guanine-II Riboswitch is guanine. If the concentration of guanine is high enough, the riboswitch upregulates production of the protein it is attached to. In our case, it increases protection of our bioreporter mScarlet-I, a red fluorescent protein that is also easily visible to the naked eye.


purR Purine Sensor

design

The second genetic approach was based on the purR Purine Sensor (4,5,6,7). This system depends on the purR purine sensor and a tetR regulation switch to produce the same mScarlet-I reporter. If the guanine concentration is high enough, then the purR sensor upregulates production of the PurR repressor protein. The PurR repressor protein represses production of the TetR protein. With a lack of TetR repressor protein, expression of mScarlet-I is upregulated.


References

  1. Aurell, D., Bruckner, S., Wilson, M., Steinhauer, N., Williams, G., for the Bee Informed Partnership (2022). United States Honey Bee Colony Losses 2021-2022: Preliminary Results. https://beeinformed.org/2022/07/27/united-states-honey-bee-colony-losses-2021-2022-preliminary-results-from-the-bee-informed-partnership/ (Accessed 16 June 2023).
  2. Dhakal S.H., Panchapakesan S.S.S., Slattery P., Roth A., Breaker R.R. Variants of the guanine riboswitch class exhibit altered ligand specificities for xanthine, guanine, or 2′-deoxyguanosine. Proc. Natl. Acad. Sci. U.S.A. 2022; 119:e2120246119.
  3. Erickson, E., A. Cohen, and B. Cameron. "Mite excreta: a new diagnostic for varroasis." Bee Science 3 (1994): 76-78.
  4. Houlberg U, Jensen KF. Role of hypoxanthine and guanine in regulation of Salmonella typhimurium pur gene expression. J Bacteriol. 1983 Feb;153(2):837-45. doi: 10.1128/jb.153.2.837-845.1983. PMID: 6401706; PMCID: PMC221703.
  5. Rolfes RJ, Zalkin H. Escherichia coli gene purR encoding a repressor protein for purine nucleotide synthesis. Cloning, nucleotide sequence, and interaction with the purF operator. J Biol Chem. 1988 Dec 25;263(36):19653-61. PMID: 3058704.
  6. R J Rolfes, H Zalkin,Regulation of Escherichia coli purF. Mutations that define the promoter, operator, and purine repressor gene., Journal of Biological Chemistry, Volume 263, Issue 36,1988,Pages 19649-19652, ISSN 0021-9258, https://doi.org/10.1016/S0021-9258(19)77685-6.
  7. Smith, J & Daum, Henry. (1987). Identification and nucleotide sequence of a gene encoding 5'-phosphoribosylglycinamide transformylase in Escherichia coli K12. The Journal of biological chemistry. 262. 10565-9. 10.1016/S0021-9258(18)60999-8.
  8. Vilarem C, Piou V, Vogelweith F, Vétillard A. Varroa destructor from the Laboratory to the Field: Control, Biocontrol and IPM Perspectives-A Review. Insects. 2021 Sep 7;12(9):800. doi: 10.3390/insects12090800. PMID: 34564240; PMCID: PMC8465918.
  9. Apiary (Honey Bees) | North Dakota Department of Agriculture." North Dakota Department of Agriculture, https://www.ndda.nd.gov/divisions/plant-industries/apiary-honey-bees#:~:text=North%20Dakota%20is%20the%20No,and%20registers%20hive%20locations%20annually.
  10. Dhakal S.H., Panchapakesan S.S.S., Slattery P., Roth A., Breaker R.R. Variants of the guanine riboswitch class exhibit altered ligand specificities for xanthine, guanine, or 2′-deoxyguanosine. Proc. Natl. Acad. Sci. U.S.A. 2022; 119:e2120246119.