Difference between revisions of "Part:BBa K5066001"

Revision as of 18:41, 1 October 2024

Xpp81Aa1

Description

Xpp81Aa is one of the Bacillus thuringiensis toxins, or Bt toxins, that derive from Bt bacteria and are commonly used as insecticides as they can target specific insects without causing harm to other species. There are a wide variety of strains derived from a selection of Bt bacteria and each has similar effects but targets different species of insects. There are three main categories of the Bt toxin, Cry, Cyt, and Vip, and the Xpp strains we renamed from Cry strains. It has also shown synergetic effects with Bt toxins, heightening the toxic effects.[1]

Usage and Biology

Currently, there is a lack of information regarding Xpp81Aa1. Previous study has demonstrated its role in assisting the mortality rate of mosquito larvae is its symbiotic relationship with Bt toxins (such as Cry2Aa and Cry4Aa). Chosen for its coactive properties, Xpp81Aa heightens the effectiveness of the other toxins.[2]

Fig 1. Western blot analysis on His-tagged protein induced with IPTG and control (no IPTG)

Lane 1: pET-28a-Xpp81Aa1 incubated without IPTG; Lane 2: pET-28a-Xpp81Aa1 with 1 mM IPTG; Lane 3: pET-28a-Cyt2Ba without IPTG; Lane 4: pET-28a-Cyt2Ba incubated with 1mM IPTG

The Xpp81Aa1 was expressed using E. coli BL21 (DE3) cells. Results on lanes 1 and 2 indicate the success of the induction of Xpp81Aa1 via IPTG. The band on Lane 1 is significantly lighter compared to the band on Lane 2, hence we can deduce that IPTG has an impact on the expression of our protein and we have successfully produced a protein. The Protein was further confirmed to be Xpp81Aa1 as the band size is correct, showing 45KDa.

Fig 2. Larvicidal results indicating the mortality of larvae at different concentrations

Larvicidal mortality of Aedes albopictus larvae exposed to various concentrations of the biolarvicidal toxins collected under different conditions including Xpp81Aa1 37°C overnight, Xpp81Aa1 37°C for 4 hours and Xpp81Aa1 20°C overnight. The LC₅₀ was calculated to be 3.16x10⁷ CFU/mL, 9.24x10⁷ CFU/mL and 1.98x10⁷ CFU/mL, respectively.

The larvae from our larvicidal assay were inspected under a light microscope. In the larvae treated by Xpp81Aa1, we observed a total of 6 larvae with visibly darkened areas, specifically from the anus through to the rectum, making the surrounding area darker. Darkening can also be observed through the rest of the larvae, generally in the head, thorax, and lower half of the abdomen. Some larvae were found to retain food in the midgut until death. To date, the structure and mechanism of how Xpp81Aa1 is not known. Further research studies could be done on Xpp81Aa1 to elucidate the functional, mechanism and structure of the protein.

Fig 3. Microscope amplification of darkening in larvae exposed to Xpp81Aa1.

The larvae from our larvicidal assay were inspected under a light microscope. In the larvae treated by Xpp81Aa1, we observed a total of 6 larvae with visibly darkened areas, specifically from the anus through to the rectum, making the surrounding area darker. Darkening can also be observed through the rest of the larvae, generally in the head, thorax, and lower half of the abdomen. Some larvae were found to retain food in the midgut until death. To date, the structure and mechanism of how Xpp81Aa1 is not known. Further research studies could be done on Xpp81Aa1 to elucidate the function, mechanism and structure of the protein.

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]

Reference

[1] Shilling, P. J., Mirzadeh, K., Cumming, A. J., Widesheim, M., Köck, Z., & Daley, D. O. (2020). Improved designs for pET expression plasmids increase protein production yield in Escherichia coli. Communications Biology, 3(1). https://doi.org/10.1038/s42003-020-0939-8

[2]Wu, J., Wei, L., He, J., Fu, K., Li, X., Jia, L., Wang, R., & Zhang, W. (2021). Characterization of a novel Bacillus thuringiensis toxin active against Aedes aegypti larvae. Acta tropica, 223, 106088. https://doi.org/10.1016/j.actatropica.2021.106088

@@ Line 4: / Line 4: @@
 ==Description==
-Xpp81Aa is one of the <i>Bacillus thuringiensis</i> toxins, or Bt toxins, that derive from Bt bacteria and are commonly used as insecticides as they can target specific insects without causing harm to other species. There are a wide variety of strains derived from a selection of Bt bacteria and each has similar effects but targets different species of insects. There are three main categories of the Bt toxin, Cry, Cyt, and Vip, and the Xpp strains were renamed from Cry strains. It has also shown synergetic effects with Bt toxins, heightening the toxic effects.[1]
+Xpp81Aa is one of the <i>Bacillus thuringiensis</i> toxins, or Bt toxins, that derive from Bt bacteria and are commonly used as insecticides as they can target specific insects without causing harm to other species. There are a wide variety of strains derived from a selection of Bt bacteria and each has similar effects but targets different species of insects. There are three main categories of the Bt toxin, Cry, Cyt, and Vip, and the Xpp strains we renamed from Cry strains. It has also shown synergetic effects with Bt toxins, heightening the toxic effects.[1]
@@ Line 36: / Line 36: @@
 Larvicidal mortality of <i>Aedes albopictus</i> larvae exposed to various concentrations of the biolarvicidal toxins collected under different conditions including Xpp81Aa1 37°C overnight, Xpp81Aa1 37°C for 4 hours and Xpp81Aa1 20°C overnight. The LC<sub>50</sub> was calculated to be 3.16x10<sup>7</sup> CFU/mL, 9.24x10<sup>7</sup> CFU/mL and 1.98x10<sup>7</sup> CFU/mL, respectively.
+The larvae from our larvicidal assay were inspected under a light microscope. In the larvae treated by Xpp81Aa1, we observed a total of 6 larvae with visibly darkened areas, specifically from the anus through to the rectum, making the surrounding area darker. Darkening can also be observed through the rest of the larvae, generally in the head, thorax, and lower half of the abdomen. Some larvae were found to retain food in the midgut until death. To date, the structure and mechanism of how Xpp81Aa1 is not known. Further research studies could be done on Xpp81Aa1 to elucidate the functional, mechanism and structure of the protein.
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