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. Cry1518-35 is a crystal protein originally produced in Bacillus thuringiensis(Fig.1A). The Cry protein was first expressed in E.coli(Fig.1B) in large amounts for testing on nematodes which is under active progression. In the construction, we fused codon-optimised Cry coding sequence with 6x His tag and PET28a vector(Fig.1B). SDS-page was performed and the result shows that Cry protein was successfully expressed with induction(Fig.1C). The result was confirmed in Western Blot analysis(Fig.1D).

Fig.1 Cry1518-35 protein expression in E.Coli ; (A) Cry protein from B.Thuringiensis. (B) Construction of Cry protein with 6x His tag. (C) SDS-page analysis of Cry protein with His tag. (D) Western Blot analysis of Cry protein with His tag. M:marker; 1:PET28a-His-Cry; 2:PET28a-Cry-His; C:control, 1&2 without induction; W:whole cell; S:supernate; P:precipitate.

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(Fig.2A). After comparison, Cry was also connected with signal peptides(Fig.2A,Fig.2B).

Fig.2 Construction of different signal peptides with Cry and RFP proteins in S.Cerevisiae with 021 vector. (A) Genetic circuits for SP-Cry/RFP. (B) 3D Structure of Cry proteins with His tag and with different signal peptides. SP: different signal peptides, JF, JFm, ScMα, OPT.

SDS-page analysis showed that JFm was the most effective one in secretion RFP to supernate(Fig.3A). The fluorescence seen in JFm construction and absent in control CEN.Pk2(Fig.11D) and intensity quantitatively detected in supernates from different signal peptides construction further verified JFm was the most efficient one(Fig.3C). SDS-page analysis for Cry construction also showed that JFm was efficient which was consistent with the previous outcome in RFP construction(Fig.3B).

Fig.3 Verification of SP-RFP/Cry (A)SDS-page analysis of SP-Cry expression. 1:JF-His-Cry, 2:JFm-His-Cry; 3:ScMα-His-Cry; 4:OPT-His-Cry; 5:JF-Cry-His; 6:JFm-Cry-His; 7:ScMα-Cry-His; 8:OPT-Cry-His.(B) SDS-page analysis of SP-RFP expression. 1:JF-RFP; 2:JFm-RFP; 3:ScMα-RFP; 4:OPT-RFP. (C) Fluorescence intensity of RFP in supernates from samples with different signal peptides. (D) The fluorescence indication for JFm-RFP. M:marker; C:control, CENPK2-1C; S:supernate; P:precipitate.

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.(Fig.1A) 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

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 132
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]