Part:BBa_K3407001

Microbial-based insecticides represent an alternative to chemical pesticides used for insect control. These types of insecticides are mainly based on the bacteria Bacillus thuringiensis (Bt) and its insecticidal toxins such as crystal (Cry) and cytolytic (Cyt) toxins, known as delta endotoxins.[1] These toxins are being used for biological control of pests worldwide, either through spray formulations based on spore-crystal preparations, or by introducing the cry toxin genes (Cry protein or some active fragment) into transgenic crops. Some Cry proteins are also being used against mosquitoes to control vector transmission.[2][3] The Cry toxins are highly specific to their target insects, and therefore kill a limited number of species. [1] Cry proteins have shown to have toxicity against insects belonging to the orders such as Hymenoptera, Coleoptera, Homoptera, Orthoptera, and Mallophaga, as well as nematodes, mites, and protozoa. [4][5] These Cry proteins remain inactive (as protoxin) until consumed by the insect and being activated by its gut proteases. Once processed, the Cry toxin must recognize the insect midgut receptors and bind to them, thus piercing holes in the insect’s gut that cause leakage and insect death. [6][2] (Figure 1)

The Cry7Ca1 toxin was identified in the B. thuringiensis strain BTH-13 for its toxic activity against L. migratoria manilensis, from the order Orthoptera[7][8]. The same research group had previously largely tested a large number of Bt strains isolated from soil samples and performed toxicity tests against different pests such as: Plutella xylostella, Heliothis armigera, Laphygma exigua, Leptinotarsa decemlineata, Tenebrio molito, Culex fatigans, and L. migratoria manilensis. The strain BTH-13 was the most potent locust-active isolate identified, and they showed that its crystal proteins BTH-13 had low or no toxicity towards Lepidoptera, Coleoptera and Diptera. They also showed by immunofluorescent analysis that we locust-active Bt toxin was located in the midgut only.[7] Later studies performed by the same research group further characterized the Cry7Ca1 toxin, both the protoxin and activated toxin[8], and obtained the crystal structure of the toxin.[9] Specificity of Cry proteins is intrinsically determined by its structure. 3D-Cry toxins such as Cry7Ca1 are characterized by three domains. Domain I is implicated in membrane insertion, toxin oligomerization and pore formation, whereas Domain II and III interact with different midgut insect proteins and therefore are implicated in insect specificity.[3] The Cry7Ca1 receptors in L. migratoria manilensis have yet not been identified, so further research including adequate genomic information should be done to identify them to later characterize the toxin-receptor interaction that will determine the specificity of this insecticidal protein. [9] In order to determine the allergenicity of the Cry7Ca1 in concrete, we followed the protocol described by Baltimore Biocrew 2017 team (http://2017.igem.org/Team:Baltimore_Bio-Crew/Experiments).