PROBEEOTIC
HOW IT WORKS
1. CRISPR CPF1
To obtain the cpm gene from Rhodopseudomonas capsulata, CRISPR-Cpf1 will be performed. R. capsulata will be isolated from wastewater using high-pressure liquid chromatography (HPLC), which separates compound mixtures to purify individual components.
2. EXTRACTING GUT MICROBIOTA
The Lactobacillus spp bacteria will be extracted from the bee’s gut microbiome by sampling a bee’s gut microbiome using a swab, inoculating it in a liquid culture that will distinguish other gut microbiota, and growing a larger culture of it.
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3. DNA CLONING
DNA cloning introduces the cpm gene to the Lactobacillus spp gut microbiome bacteria by reconstructing plasmid vectors to create recombinant DNA.
To ensure that the cpm gene remains in the bacteria after it reproduces, ampicillin will act as an anti-resistance marker placed at the end of the gene. Transformation of the Lactobacillus spp bacteria culture via heat shock will allow the reconstructed vector to enter short-lived pores within the bacterial cell membrane.
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4. FREEZE-DRYING TECHNIQUE
The bacteria will be prepared by cultivating them in water with oxygen and testing to ensure that they have duplicated with the detoxifying gene. The bacteria will then be distributed into airtight cryotubes, which will be transferred to the freeze-dryer. From there, the machine will pump air by raising pressure, dehydrate bacteria by modifying the temperature, causing it to dry, and freeze bacteria formulating a powder form.
5. EXPERIMENTATION
We will have three groups: a control group where we will refrain from exposing honey bees with the probiotic and neonicotinoid , an experimental group where we will expose the honey bee with the probiotic and 1.10 +0.77 ppb concentration of neonicotinoid, and another experimental group where the honey bee is exposed to neonicotinoid without the probiotic.