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Team:METU Turkey/project.html
From 2013.igem.org
Imidacloprid is a nicotine-derived systemic chemical which is commonly used in insecticides for agricultural purposes. It is also lethal for many beneficial insects, like honey bees. When imidacloprid is absorbed by bees, mainly from oral sources, it binds to bees nicotinic acetylcholine receptors and effects bees foraging abilities. After some time, imidacloprid gets degraded by bee’s own proteins, mainly into 5-hydroxyimidacloprid and olefin and these two metabolities are also lethal for insects due to their binding ability to nicotinic acetylcholine receptors.
However, one insect species is known for its resistance to imidacloprid, Drosophila melanogaster (fruit fly). One of the genes from the cytochrome P450 family, the gene CYP6G1, is responsible for this resistance. It mainly degrades imidacloprid into a different metabolite. This metabolite is 4-hydroxyimidacloprid and it neither affects insects nor is lethal to them.
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In our project, we create a circuit which contain modified CYP6G1 gene. In this modified form, gene is changed according to Barnes – MALLLAVF amino acid sequence, which enables gene to activate in prokaryotes and we give this circuit to the Bacillus subtilis.
Our construct is simple: it produces CYP6G1 protein continuously under the regulation of strong constitutive promoter Pveg and for the activation of this protein it produces cytochrome P450-reductase fCPR. After the production, protein is secreted from Bacillus subtilis which lives in Apis mellifera’s (western honey bee) midgut, it circulate through honey bees’ circulatory system and giving honey bee an imidacloprid resistance, which fruit fly has.
This is the first circuit. We used a constitutive promoter to produce the CYP6G1 protein and fCPR, which activates the main protein CYP6G1. Promoters and RBS's are chosen properly to work in B. subtilis. SacB tags are secretion tags. We added them to the proteins in order to get them out of the bacteria into the mid gut, where the proteins should work. And double terminator is there to stop the transcription process where it should stop.
In addition to fighting against the Colony Collapse Disorder by degrading a pesticide, our other aim is to protect the bees against living microorganisms that are dangerous for the bees. In order to accomplish that aim, we made a research and we realized that bees recently suffer from insufficient feed.
Since beekeepers prefer to feed the bees with high-fructose corn syrup or sucrose because of the low prices, bees started to suffer from insufficient nutrition. Bees lost their ability of fighting against the pathogens, parasites and pesticides and this situation creates a major problem, colony losses. P-coumaric acid is the most important substance as one of these nutritions.
P-coumaric acid, a monomer of sporopollenin, is the essential substance for the bees’ diet. As a main pollinator, bees get this phenolic substance from the pollens cell wall naturally. Presence of the p-coumaric acid in bees’ mid-gut induces the activity of all classes of detoxification genes that the bees have. With the help of coumaric acid, activity of P450 genes increases, especially the CYP6 and CYP9 gene families, and it gives immune boost to bees. In addition to up-regulation of these detoxification genes, p-coumaric acid also increases the metabolism of coumaphos in midgut significantly, and it also helps bee to fight against insects and mites.
In our project, we made system to produce p-coumaric acid in bees’ midgut. For this purpose, we used Sam8 gene from the bacterium Saccharothrix espanaensis with natural RBS and we supported this system with a constitutive promoter, Pveg, for Bacillus subtillis. Sam8 encodes a tyrosine ammonia lyase, which converts tyrosine to p-coumaric acid and tyrosine is one of the most abundant non-essential amino acid. Since tyrosine is already present in the bees’ diet, our system can always provide the production of p-coumaric and the bees become stronger against pesticides and pathogens. As a result, colony losses resulted from lack of nutrition can be prevented by this system; therefore, bees are protected from CCD in this manner, too.
In this year, in the kill switch part of our project, we use an mRNA interferase, MazF, which cleaves mRNA’s at specific sequences. In our kill switch, we used anti-sense RNA principle as a template. According to this principle, MazF, which is constantly produced via a constitutive promoter, is got inactivated by Anti-MazF construct. In order to trigger this mechanism, we used IPTG, a harmless molecule for the bee which at the same time does not appear in the honey too. When IPTG is present in the environment, LacI is inhibited by IPTG and therefore promoter gets activated. With the activated promoter of it, Anti-MazF is produced and inactive MazF. As long as IPTG exists, MazF gets inactivated continuously; therefore, the bacteria maintain their lives. On the other hand, if bacteria exists in a IPTG-free environment, Anti-MazF producing stops, which leads to MazF producing and bacteria get killed by MazF.
In this circuit, we used the parts K143053 as a constitutive promoter and SpoVG RBS due to their more efficient works in Bacillus subtilis. Besides these two, we used the part I732820 to produce LacI, but we had to change the RBS of this system since RBSelowitz is less effective in Bacillus then RBSspoVG. With this change in the activity of the RBS, we can compete with the production of MazF.
