Composite

Part:BBa_K3407017

Designed by: Alicia Rodrguez Molina   Group: iGEM20_TUDelft   (2020-10-23)


Cry7Ca1 toxin with inducible T7 promoter, RBS and T7 terminator


Assembly Compatibility:
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    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 688
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 1388
    Illegal XhoI site found at 367
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    COMPATIBLE WITH RFC[23]
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    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]

Usage and Biology

Microbial-based insecticides represent an alternative to chemical pesticides used for insect control. These types of insecticides are mainly based on the bacteria Bacillus thuringiensis (Bt) and its insecticidal toxins such as crystal (Cry) and cytolytic (Cyt) toxins, known as delta endotoxins [1]. These toxins are being used for biological control of pests worldwide, either through spray formulations based on spore-crystal preparations, or by introducing the cry toxin genes (Cry protein or some active fragment) into transgenic crops. Some Cry proteins are also being used against mosquitoes to control vector transmission [2][3]. The Cry toxins are highly specific to their target insects, and therefore kill a limited number of species [1]. Cry proteins have shown to have toxicity against insects belonging to the orders such as Hymenoptera, Coleoptera, Homoptera, Orthoptera, and Mallophaga, as well as nematodes, mites, and protozoa [4][5]. These Cry proteins remain inactive (as protoxin) until consumed by the insect and being activated by its gut proteases. Once processed, the Cry toxin must recognize the insect midgut receptors and bind to them, thus piercing holes in the insect’s gut that cause leakage and insect death [6] [2] (Figure 1).

  • Figure 1: Overview of Cry toxins pore formation mechanism in the insect gut epithelium

The Cry7Ca1 toxin was identified in the B. thuringiensis strain BTH-13 for its toxic activity against Locusta migratoria manilensis, from the order Orthoptera[7][8]. The same research group had previously largely tested a large number of Bt strains isolated from soil samples and performed toxicity tests against different pests such as: Plutella xylostella, Heliothis armigera, Laphygma exigua, Leptinotarsa decemlineata, Tenebrio molito, Culex fatigans, and L. migratoria manilensis. The strain BTH-13 was the most potent locust-active isolate identified, and they showed that its crystal proteins BTH-13 had low or no toxicity towards Lepidoptera, Coleoptera and Diptera. They also showed by immunofluorescent analysis that the locust-active Bt toxin was located in the midgut only [7]. Later studies performed by the same research group further characterized the Cry7Ca1 toxin, both the protoxin and activated toxin[8], and obtained the crystal structure of the toxin.[9]. The specificity of Cry proteins is intrinsically determined by its structure. 3D-Cry toxins such as Cry7Ca1 are characterized by three domains. Domain I is implicated in membrane insertion, toxin oligomerization and pore formation, whereas Domain II and III interact with different midgut insect proteins and therefore are implicated in insect specificity [3]. The Cry7Ca1 receptors in L. migratoria manilensis have yet not been identified, so further research including adequate genomic information should be done to identify them to later characterize the toxin-receptor interaction that will determine the specificity of this insecticidal protein [9].

The Cry protein, Cry7Ca1 (GenBank accession no. EF486523) from the Bt strain BTH-13 is a three-domain Cry protein with a molecular weight of 129,196.50 Da, whereas the activated toxin has a molecular weight of 68 kDa (Figure 2). The Cry7Ca1 toxin (activated Cry7Ca1 protein) crystal structure has been published and can be found in the Protein Data Bank (PDB) under the accession number 5ZI1 [9].

  • Figure 2: Schematic diagram of the Cry7Ca1 protoxin and toxin. The yellow boxes represent protein sequences of the different domains, the arrows and numbers indicate the amino acids of the Cry7Ca1 sequence.

Experimental results

Optimal conditions for Cry7Ca1 toxin overexpression

In order to check the overexpression of the Cry7Ca1 toxin, we incubated the E. coli BL21 (DE3) (Negative control) and E. coli BL21 (DE3) transformed with the plasmid pTWIST_Cry7Ca1 containing the Biobrick BBa_K3407017 at 37ºC until the OD600 reached approximately 0.6. In order to find the best induction conditions, we added different IPTG concentrations (0.5 and 1 mM) and incubated the cultures for 4h and overnight. The total protein content of the cells and the soluble proteins obtained after cell lysis were analysed by SDS-PAGE electrophoresis (Figure 3) .

  • Figure 3: SDS-PAGE to verifyCry7Ca1 overexpression at different IPTG conditions.A) Total protein content. B) Soluble proteins after lysis using FastBreak™ Cell Lysis Reagent (Promega). E. coli BL21 (DE3) is the negative control, induced with the higher IPTG concentration used in this experiment (1 mM). E. coli BL21 (DE3) Cry7Ca1 contains the plasmid pTWIS_Cry7Ca1 (BBa_K3407017), and it is induced with 0.5 mM IPTG and 1 mM IPTG. The non-induced sample is a negative control to check leaky expression. MW (Molecular weight marker, #1610363 Bio-Rad), PI (pre-induction), 4h (4 hours after induction), ON (overnight). All the samples used corresponded to the same OD600.

Figure 3 shows successful overexpression of Cry7Ca1 toxin with all IPTG concentrations used, as a band corresponding to the molecular weight of the Cry7Ca1 toxin can be observed in samples from E. coli BL21 (DE3) Cry7Ca1 (Figure 3A). After lysis with FastBreak™ Cell Lysis Reagent (Promega), bands corresponding to the size of the Cry7Ca1 toxin are also observed (Figure 3B), although at lower intensity. These results indicated that a final IPTG concentration of 0.5 mM is enough for the overexpression of our BioBrick. They also suggest that our protein may not be completely soluble after lysis, as the fraction of soluble protein recovered seems lower than that of the total overexpressed amount. Moreover, we noticed a leaky expression of our protein, as a band was observed after an overnight culture of E. coli BL21 (DE3) pTWIST_Cry7Ca1 without inducer. This may be due to the absence of a repressor in our constructed plasmid. In order to avoid leaky expression, another host could be used such as E. coli BL21 (DE3) pLysS. The BBa_K3407017 could also be modified to include a repressor such as the lacI gene.

Purification of Cry7Ca1 toxin

We purified the Cry7Ca1 using the identified IPTG concentration for overexpression. As the recombinantly expressed Cry7Ca1 toxin contains a His-tag at the N-terminal part, we purified it using affinity chromatography with the HisLink protein purification system (Promega). All samples obtained from the purification process were analysed by SDS-PAGE electrophoresis (Figure 4).

  • Figure 4: SDS-PAGE of Cry7Ca1 toxin purification by affinity chromatography. E. coli BL21 (DE3) is the negative control, and E. coli BL21 (DE3) Cry7Ca1 contains the pTWIS_Cry7Ca1 (BBa_K3407017). MW (Molecular weight marker, #1610363 Bio-Rad), PI (pre-induction), ON (overnight), FT (flow-through), W (Washing), E (Elution). All the samples used corresponded to the same OD600.

A clear band of the expected size was obtained in the elution sample (Figure 4), indicating that the Cry7Ca1 toxin has successfully been purified. The fact that the toxin protein is also present in the flow through sample suggests that further optimization is possible.

Conclusions

From these results, we conclude that the Cry7Ca1 toxin is overexpressed in E. coli cells after induction with 0.5 mM IPTG (although there is some leaky expression). In this expression conditions, the Cry7Ca1 toxin can be further successfully purified by affinity chromatography thanks to its N-terminal Histag. This part is functional and can be expressed in E. coli.

References

Ordered List

  1. Pardo-López, L., Soberón, M., & Bravo, A. (2013). Bacillus thuringiensis insecticidal three-domain Cry toxins: mode of action, insect resistance and consequences for crop protection. FEMS microbiology reviews, 37(1), 3–22.
  2. Bravo, A., Likitvivatanavong, S., Gill, S. S., & Soberón, M. (2011). Bacillus thuringiensis: A story of a successful bioinsecticide. Insect biochemistry and molecular biology, 41(7), 423–431.
  3. Rubio-Infante, N., & Moreno-Fierros, L. (2016). An overview of the safety and biological effects of Bacillus thuringiensis Cry toxins in mammals. Journal of applied toxicology : JAT, 36(5), 630–648.
  4. Schnepf, E., Crickmore, N., Van Rie, J., Lereclus, D., Baum, J., Feitelson, J., Zeigler, D. R., & Dean, D. H. (1998). Bacillus thuringiensis and its pesticidal crystal proteins. Microbiology and molecular biology reviews : MMBR, 62(3), 775–806.
  5. Palma, L., Muñoz, D., Berry, C., Murillo, J., & Caballero, P. (2014). Bacillus thuringiensis toxins: an overview of their biocidal activity. Toxins, 6(12), 3296–3325.
  6. Mendoza-Almanza, G., Esparza-Ibarra, E. L., Ayala-Luján, J. L., Mercado-Reyes, M., Godina-González, S., Hernández-Barrales, M., & Olmos-Soto, J. (2020). The Cytocidal Spectrum of Bacillus thuringiensis Toxins: From Insects to Human Cancer Cells. Toxins, 12(5), 301.
  7. Song, L., Gao, M., Dai, S., Wu, Y., Yi, D., & Li, R. (2008). Specific activity of a Bacillus thuringiensis strain against Locusta migratoria manilensis. Journal of invertebrate pathology, 98(2), 169–176.
  8. Wu, Y., Lei, C. F., Yi, D., Liu, P. M., & Gao, M. Y. (2011). Novel Bacillus thuringiensis δ-endotoxin active against Locusta migratoria manilensis. Applied and environmental microbiology, 77(10), 3227–3233.
  9. Jing, X., Yuan, Y., Wu, Y., Wu, D., Gong, P., & Gao, M. (2019). Crystal structure of Bacillus thuringiensis Cry7Ca1 toxin active against Locusta migratoria manilensis. Protein science : a publication of the Protein Society, 28(3), 609–619.

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