Part:BBa_K5066001
Xpp81Aa1
Description
Xpp81Aa1 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; there are also the Xpp strains, which were renamed from Cry. 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 studies have demonstrated its role in assisting the mortality rate of mosquito larvae in its symbiotic relationship with Bt toxins (such as Cry2Aa and Cry4Aa). Chosen for its coactive properties, Xpp81Aa1 heightens the effectiveness of the other toxins.[2] The construct design can be further applied to other future iGEM teams and research related to vector control. Furthermore, it opens up possibilities for further exploration of this novel strain which has shown characteristics beneficial to the development of larvicidal toxin produced by Bt strains. This is pioneered by the implementation of this basic part in a composite part made up of basic parts Cyt2ba (BBa_K5066000), p20 (BBa_K5066008), and Xpp81Aa1. The sequence for this part can be found at BBa_K5066007 and serves as an important launch point for the further use of Xpp81Aa1's novel synergistic properties.
Plasmid Construct
Fig 1. Plasmid construct design in pET-28a
Design Considerations
Initially when designing, our sequence included a stop codon in the primer, which resulted in the His-tag after the fragment not being expressed, therefore we were unable to do western blot. To fix this issue, we reproduced the clone but removed the stop codon on the fragment, allowing the His-tag to be expressed.
Characterization
Western Blot
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.
Growth Curve Study
Production of a collection of biolarvicidal toxin systems in the future is important to improving the process of synthesizing any BioBricks of interest. It is important to rotate the use of insecticides to prevent build-up of resistance in Aedes populations[3]. Even though the risk of developing resistance from the treatment of biolarvicidal toxins from Bacillus thuringiensis and Buthus martensii Karsch is limited, we would like to implement integrated resistance management in our project. It is critical to have a thorough understanding of the recombinant bacteria that harbor plasmids that we designed and built to reduce to increase efficiency, hence combating possible resistance.
Therefore, we conducted a basic growth curve study to understand the growth trend of the recombinant bacteria so that we could determine the growth rate of each of the unique biolarvicidal toxins and the time to induce the bacterium to achieve maximum production of proteins from BL21(DE3). The results show that after the induction at the 6-hour mark, there was a significant increase in the OD600 value of E. coli in the broth, moreover, the curve flattened out after the 10-hour mark. From the results, it can be interpreted that the optimal time to obtain the highest growth rate is from 6 to 10 hours, with the IPTG induction beginning at 6 hours.
Larvicidal Assay
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 function, mechanism and structure of the protein.
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.
Immune responses of Aedes albopictus larvae by qPCR
Furthermore, to determine whether the mortality that we observed from the biolarvicidal assay is solely due to the mechanisms exerted by each of the toxins, such as the cytolytic effects of Xpp81Aa1, it is necessary to rule out the death of mosquito larvae due to E. coli.
The experiment was designed to observe the effect of Xpp81Aa1 toxins on Ae. albopictus larvae in comparison to control (negative control), BL21(DE3)-empty vector (control), and treatment groups. BL21(DE3)-empty vector is used to investigate whether the mortality observed was due to the cytotoxicity effect or bacterial infection of the E.coli or solely due to the potency of the Xpp81Aa1.
Hence, Real-time PCR, also known as quantitative PCR (qPCR), was used to confirm our hypothesis. This is a method used to determine the concentration of a target DNA [4]. PCR was performed to examine the expression levels of the target immune genes, including Gambicin, Rel1, Rel2, and Defensin of the larvae.
Gambicin
Figure 4 shows that after the 24-hour period for both 20°C and 37°C, the fold change of Gambicin in larvae left in Xpp81Aa1 solutions showed a significantly lower value than the controlled condition. However, only the results of the 37°C showed that the Xpp81Aa1 is effective after a 48-hour period. These results indicate that our Xpp81Aa1 is effective at targeting the larvae.
Rel1
Figure 7. Expression levels of Rel1 relative to the housekeeping gene S7 (A & B) Larvae exposed to biolarvicidal toxins with IPTG induction at 20°C overnight, and (C & D) IPTG induction at 37°C for 4 hours, respectively, compared to the control group treated with ddH2O and BL21(DE3)-empty vector at 24 hours and 48 hours
Figure 7 shows that after both the 24 and 48-hour periods, the fold change of Rel1 in larvae left in Xpp81Aa1 solutions showed a significantly lower value than the controlled condition in the two temperature conditions. These results indicate that our Xpp81Aa1 is effective at targeting the larvae. This result is different to the results of Gambincin where only the 24 hours condition at 37°C was effective.
Rel2
Figure 8. Expression levels of Rel2 relative to the housekeeping gene S7 (A & B) Larvae exposed to biolarvicidal toxins with IPTG induction at 20°C overnight, and (C & D) IPTG induction at 37°C for 4 hours, respectively, compared to the control group treated with ddH2O and BL21(DE3)-empty vector at 24 hours and 48 hours
Figure 8 shows that after both the 24 and 48-hour periods, only the protein inducted at 37°C showed fold changes lower than the control. These results indicate that only when incubated at 37°C is Xpp81Aa1 effective at inducing changes in the expression of Rel2.
Defensin
Figure 9. Expression levels of Defensin relative to the housekeeping gene S7 (A & B) Larvae exposed to biolarvicidal toxins with IPTG induction at 20°C overnight, and (C & D) IPTG induction at 37°C for 4 hours, respectively, compared to the control group treated with ddH2O and BL21(DE3)-empty vector at 24 hours and 48 hours
Figure 9 shows that after both the 24 and 48-hour periods, the protein inducted at 20°C showed fold changes lower than the control, moreover, the results from the 37°C condition also showed positive results after the 24 period. These results indicate that when incubated at 20°C and 37°C is Xpp81Aa1 effective at inducing changes in the expression of Defensin, but only at 24 and 48 hours or 24 hours periods, respectively.
Morphology of Larvae
These larvicide assays were all carried out within the same time frame, but the larvae were mounted at different times. The control larvae were in the late stage three of their development during mounting, while most of the larvae from the treatment group were mounted at various time frames when they died. Therefore, the length of the larvae reflected the true length of their size in real life. We washed and preserved the larvae in absolute alcohol and mounted them with the mounting solution. The difference observed in length, therefore suggests the relative efficacy of the toxins.
This graph shows that the treated group was shorter in length compared to the control group. We deduced that the shorter length was due to the early mortality experienced by the treatment groups compared to the control groups.
We started the biolarvicidal assay with the larvae of the same stage, stage 3, and mounted the control group 2 days after the test. Due to the larvae’s early mortality, they were unable to grow past the stage at which they were upon death. This allows us to see that the effects of Xpp81Aa1 manifested in the form of early death, showing that the larvicides indeed impact larvae mortality.
Conclusion from results
In conclusion, we gained insight from the qPCR study that the potency observed from the biolarvicidal toxins was probably due to larvicidal activities of Xpp81Aa1 to the epithelium of midgut by Xpp81Aa1. In this study, we observed that BL21(DE3)-empty vector does not trigger immune responses in Aedes albopictus larvae. Therefore, the mortality recorded was postulated to be due to the toxicity of the Xpp81Aa1. Previous studies showed that Xpp81Aa1 shared about 37.9% homology with Cry37Aa, and hence, we postulated that Xpp81Aa1 could share a similar mechanism as Cry37Aa. Cry proteins are endotoxin and have the ability to penetrate the membrane of the cell. We observed some abnormalities in the morphology of the larvae that were treated with Xpp81Aa1 as compared to the control group. The larvae treated with Xpp81Aa1 have darker exterior integument than control group. Further investigation to be conducted to further explore the mechanism, structure and functionality of Xpp81Aa1.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE 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
[3] Dusfour, I., Vontas, J., David, J. P., Weetman, D., Fonseca, D. M., Corbel, V., ... & Chandre, F. (2019). Management of insecticide resistance in the major Aedes vectors of arboviruses: Advances and challenges. PLOS Neglected Tropical Diseases, 13(10), e0007615. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6786541/
[4] Dymond, J. S. (2013, January 01). Chapter twenty three - Explanatory chapter: Quantitative PCR (J. Lorsch, Ed.). Retrieved from ScienceDirect website: https://www.sciencedirect.com/science/article/abs/pii/B9780124186873000239?via%3Dihub
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