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<p>BjaIT is an α-insect toxin derived from the Judean Black Scorpion (<i>Buthotus judaicus</i>). Although, α-toxins are known to affect both mammals and insects, BjaIT only shows lethality or envenomation in insects and not in mammals.<sup>1</sup></p>
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<h2>Usage and Biology</h2>
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<p>It is a neurotoxic polypeptide that acts on voltage-gated sodium channels. As all α-toxins do, it modifies the sodium conductance of sodium-gated sodium channels by slowing the inactivation kinetics.<sup>1</sup></p>
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<h2>Characterization</h2>
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<p>The construct pGGA-BBa_K206000-BjalT-OmpTSite-FLAG-HlyA was created. It contained the arabinose-inducible pBAD-promotor, the insect toxin and the protease site for the outer membrane protease OmpT. Furthermore, a FLAG-tag was added, to make the protein easily detectable with anti-FLAG-antibodies. Also it contained HylA, an excretion signal.
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The production of BjaIT by E. coli was verified by Western Blot analysis of cell lysates. Three different cultures were measured. Untransformed E. coli DH5α served as a negative control.
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<img src="https://static.igem.org/mediawiki/2018/2/22/T--Hamburg--Imager_2018-09-28_16hr_06min%2BImager_2018-09-28_16hr_04min.tiff"
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<p><b>Fig. 1:</b> Western Blot of cell lysates containing BjaIT and DH5α as a negative control. Different protein concentrations were added.</p>
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<p>Moreover, the toxicity of BjaIT for Anopheles mosquitoes was determined by experiments with mosquitoes. For information about these experiments, take a look at the <a href="http://2018.igem.org/Team:Hamburg/Demonstrate#main-content">mosquito experiments</a>.</p>
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<h2>Discussion</h2>
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<p>The Blot membrane of BjaIT showed bands at approximately 25 kDa for DH5α with pGGA-BBa_K206000-BjalT-OmpT-FLAG-HlyA both after induction with arabinose as well as uninduced. Furthermore, a very light band at the same height for DH5α without plasmids could be observed. We expected a band at 25 kDa in the induced cells only. The band in the uninduced DH5α cells carrying the plasmid can be explained by the used promotor pBAD, which is known to be very leaky. Moreover, the height of the bands might be lead back to posttranslational modifications or the secondary structure of the protein.</p>
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<h2>References</h2>
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<li>Arnon, T., Potikha, T., Sher, D., Elazar, M., Mao, W., Tal, T., Bosmans, F., Tygat, J., Ben-Arie, N., Zlotkin, E. (2005). BjαIT: a novel scorpion α-toxin selective for insects—unique pharmacological tool.</li>
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Revision as of 01:37, 18 October 2018


Black scorpion alpha insect toxin BjaIT

BjaIT is an α-insect toxin derived from the Judean Black Scorpion (Buthotus judaicus). Although, α-toxins are known to affect both mammals and insects, BjaIT only shows lethality or envenomation in insects and not in mammals.1

Usage and Biology

It is a neurotoxic polypeptide that acts on voltage-gated sodium channels. As all α-toxins do, it modifies the sodium conductance of sodium-gated sodium channels by slowing the inactivation kinetics.1

Characterization

The construct pGGA-BBa_K206000-BjalT-OmpTSite-FLAG-HlyA was created. It contained the arabinose-inducible pBAD-promotor, the insect toxin and the protease site for the outer membrane protease OmpT. Furthermore, a FLAG-tag was added, to make the protein easily detectable with anti-FLAG-antibodies. Also it contained HylA, an excretion signal. The production of BjaIT by E. coli was verified by Western Blot analysis of cell lysates. Three different cultures were measured. Untransformed E. coli DH5α served as a negative control.


Fig. 1: Western Blot of cell lysates containing BjaIT and DH5α as a negative control. Different protein concentrations were added.


Moreover, the toxicity of BjaIT for Anopheles mosquitoes was determined by experiments with mosquitoes. For information about these experiments, take a look at the mosquito experiments.

Discussion

The Blot membrane of BjaIT showed bands at approximately 25 kDa for DH5α with pGGA-BBa_K206000-BjalT-OmpT-FLAG-HlyA both after induction with arabinose as well as uninduced. Furthermore, a very light band at the same height for DH5α without plasmids could be observed. We expected a band at 25 kDa in the induced cells only. The band in the uninduced DH5α cells carrying the plasmid can be explained by the used promotor pBAD, which is known to be very leaky. Moreover, the height of the bands might be lead back to posttranslational modifications or the secondary structure of the protein.

References

  1. Arnon, T., Potikha, T., Sher, D., Elazar, M., Mao, W., Tal, T., Bosmans, F., Tygat, J., Ben-Arie, N., Zlotkin, E. (2005). BjαIT: a novel scorpion α-toxin selective for insects—unique pharmacological tool.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]