For our project, we chose to design a detection system for various factors, in order to determine beehive health state. Those factors include the bee pathogen Paenibacillus larvae, heavy metals levels, and sugar levels, which will all be detected in bees' excrements.
We chose to use bees’ excrements for testing as it is the less invasive solution we found. The first options we had was to use crushed bees but we highly disliked the idea of killing more bees. We considered our project was to save bees and that option was not feasible with our convictions. Another option was to put the modified bacteria directly in larvae which are naturally part of their microbiota. In this case the color change could be observable in the living bees but we encountered two issues. The obvious is the GMO aspect of the project which 90% of researchers we met with raised and insisted on an alternative. The other being that the pigment may not be as visible as we would expect in the living organism plus larvae’s cells are closed at some point and even if they do change color we won’t be able to observe it. We finally chose bee excrements where P. larvae is still present, which will not be a big concern regarding public opinion and finally which is not invasive.
Paenibacillus larvae detection system
We based our detection system on the iron uptake system of P. larvae and we chose Bacillus subtilis to detect it since they both have a similar iron regulation system. Soluble Iron, which is crucial for bacteria survival, is scare in nature. Thus, bacteria develop ways to use the available source. For instance, during low iron concentrations conditions in the extracellular environment, P. larvae and B. subtilis produce iron chelating agents, known as siderophores, and in our case it is the bacillibactin (BB).
BB is a catechol-based siderophore-secreted by members of the genus Bacillus (antracis, subtilis). It is involved in the chelation of ferric iron (Fe3+) from the surrounding environment and is subsequently transferred into the bacterial cytoplasm via the use of ABC transporters and siderophore binding proteins (SBP).
Once in the intracellular environment, first the Fe3+ is converted into Fe2+ then the FUR (Ferric Iron Regulator) takes over. In the presence of Fe, FUR, a negative transcription factor, dimerizes upon iron II binding and inhibits its target genes expression.
The concept of our project was to first delete B. subtilis’s BB, and replace its SBP and ABC transporter by the ones of P. larvae. On one hand, the idea being that by putting our modified B. subtilis in the same environment, B. subtilis will be forced to use the P. larvae siderophore which is possible now with the help of the ABC transporter and SBP specific to P. larvae. On the other hand, we want to put a pigment under a LacI promotor (for example), itself being regulated by FUR. The amount of intracellular iron in B. subtilis will increase with the use of P. larvae’s BB, which will activates FUR and therefore repress LacI. This chain of events will lead to the expression of our pigment, silent in normal conditions, indicating the presence of P. larvae.
Proof of concept
In a lab settings, the ideal would be to test our modified bacteria simply with P. larvae’s BB instead of working with the pathogen itself. Due to multiple reasons, we also had to adapt our project for this competition. Most importantly, most P. larvae sequences including the SBP and ABC transporter sequences are discontinued. For the sake of a proof of concept, we modified our system where we will instead remove all other iron uptake system, and its own BB from B. subtilis. We will then transfect it with a plasmid containing our repressed pigment, with the repressor under FUR regulation. In this case, we will put our modified bacteria next to one that still produces BB. If our bacteria changes color, it would indicate that they were able to take up the BB from the unmodified B. subtilis, and also that our pigment expression system is functional.
Other factors’ detection
Other factors than pathogens can indicate beehives health. We decided to look into sugar levels (sucrose and fructose), and heavy metals levels, still in the same samples. Our idea is to create a system that could be adapted to study any type of factors we are interested in by simply using the same system but each time modifying promotors to make it specific to the factor of interest. We went from the basic idea of P. larvae detection system with a promotor repressed by a transcription factor in normal condition. With the presence of the component to detect, that transcription factor will no longer block the expression of the pigment, thus the staining of B. subtilis.