Part:BBa_K4825053

JFm-Cry

This composite part contains part JFm and part Cry1518-35. It codes for protein Cry1518-35 that can be secreted in extracellular space of S.cerevisiae.

"JFm is a mutated version of the signal peptide originated in K.smarxianu, JF, and utilized in our project's chassis, S.cerevisiae, to target heterologous proteins to the extracellular environment of yeast cells.

This part as a signal peptide can function in S.cerevisiae to enhance the secretion level of heterologous proteins or recombinant enzymes by determining and trafficing the secretion pathway of target genes,and the modifications in the genetic sequence remarkably increase the secretion level in yeasts, compared to the original JF signal peptide

In our project, this part is used to secrete heterologous protein Cry1518-35 out of S.cerevisiae, of which the efficiency is examined by secreting red fluorescence protein (RFP). This part can be applied as for a more efficient expression of heterologous proteins in S.cerevisiae than the yeast's native signal peptide, especially for the expression of large molecule proteins. "

"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. "

Usage and Biology

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. The Cry protein was first expressed in E.coli 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. SDS-page was performed and the result shows that Cry protein was successfully expressed with induction. The result was confirmed in Western Blot analysis.

Cry1518-35 is a crystal protein that is able to eliminate certain nematodes. 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. JFm, optimized from the inulinase signal peptide, JF, in K.smarxianu, is a 74bp signal peptide located in the N-terminal of targeted proteins. Its role in translocating proteins to the periplasmic space or out of the cell starts with the N-region orienting the secretion by interactions with negatively charged phosphate group of the lipid bilayer of the cells; and because of the presence of Lys-Arg at the C-terminal of JF that can be recognized by S.cerevisiae's endoprotease, the translocation ends with the C-region being cleaved by signal peptidase. As a result, the targeted heterologous proteins will be secreted to extracellular space with a much higher level of bioactivity than the bioactivity inside the cells, whereas the signal peptide itself remains in the cytosol.

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.

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]