Part:BBa_K4825004
Cry1518-35
"Cry1518-35 is a crystal protein gene isolated from the strain of Bacillus thuringiensis YBT-1518, performing insecticide bioactivity to nematodes. In our project, we will modify both E.coli and S.cerevisiae to express this part as an effective killing agent, in order to eliminate the parasites inside Giant African Snails, A.cantonensis.
This part has specific toxicity to more than 500 species of insects such as Lepidoptera, Diptera, Coleoptera, Hymenoptera, and Homoptera, as well as protozoa, linear animals, and flat animals. However, it is environmentally friendly and has negligible impacts on other aquatic animals, birds and mammals.
While considering the future parasitic control, Cry1518-35 can provide future iGEM teams that dedicate in achieving ecological balance with more choices of nematode pesticides. "
characterisation
After successful attraction of snails, the elimination of parasitic nematode A. cantonensis in the snails are completed by Cry1518-35 proteins.In the construction, we fused codon-optimised Cry coding sequence with 6x His tag and PET28a vector. SDS-page was performed and the result shows that Cry protein was successfully expressed with induction. The result was confirmed in Western Blot analysis. For continuous secretion expression in modified Saccharomyces cerevisiae, an effective signal peptide is necessary. The different signal peptides, JFm, JF, ScMα were primarily constructed with RFP to compared their functionality. After comparison, Cry was also connected with signal peptides. SDS-page analysis showed that JFm was the most effective one in secretion RFP to supernate. The fluorescence seen in JFm construction and absent in control CEN.Pk2 and intensity quantitatively detected in supernates from different signal peptides construction further verified JFm was the most efficient one. SDS-page analysis for Cry construction also showed that JFm was efficient which was consistent with the previous outcome in RFP construction.
Usage and Biology
Cry1518-35 is a crystal protein that is able to eliminate certain nematodes originally produced in Bacillus thuringiensis. Its toxicity functions by binding with the cadherin receptor in the nematode's midgut and activating the magnesium ion dependent signal transduction pathway that possibly results in the death of living cells of the nematodes.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 132
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
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