Part:BBa_K3686010
Insecticidal protein from Bacillus thuringiensis (BTH-3), complete CDs, codon-optimized
Bt toxins refers to the toxic proteins produced by insect pathogenic bacteria Bacillus thuringiensis. Each Bt toxin display unique conformations of peptide chain thus have a specificity to certain invertebrates. Where Cry7Ca1 is a differentiate of Bt toxins with a molecular mass of 129kDa, recently isolated from Bt strain BHT-13. According to the crystalline analysis, it could be inferred that this toxin displays a similar mode of action, the pre-pore forming model, like its relatives [1][2][3]. Which the lethality of this toxin against locusts have been confirmed at an LC50 (50% lethal concentration) value of 8.98 μg/ml [1].
Once ingested by insects, Cry7Ca1 undergoes a series of digestion by protease in insect gut juice. Which cleaves the protective shell and release the core toxin inside. Furthermore, the core toxin binds to the membrane-bonded protease on epithelial cells. The proteolytic degradation removes the N-terminal alpha chain, unreal the core into smaller oligomers that embed onto the cell membrane and result in cell disruption. [4]
[1]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.
[2]Jimenez-Juarez, N., Munoz-Garay, C., Gómez, I., Gill, S. S., Soberón, M., & Bravo, A. (2008). The pre-pore from Bacillus thuringiensis Cry1Ab toxin is necessary to induce insect death in Manduca sexta. Peptides, 29(2), <318-323..
[3]Vachon, V., Laprade, R., & Schwartz, J. L. (2012). Current models of the mode of action of Bacillus thuringiensis insecticidal crystal proteins: a critical review. Journal of invertebrate pathology, 111(1), 1-12.
[4]Melo, A. L. D. A., Soccol, V. T., & Soccol, C. R. (2016). Bacillus thuringiensis: mechanism of action, resistance, and new applications: a review. Critical reviews in biotechnology, 36(2), 317-326
Source of sequence
Genome of Bacillus thuringiensis (BTH-3), full amino acid sequence submitted under GenBank accession no. EF486523.
Considerations:
Codon-optimized for expression in Escherichia coli.
Optimization details:
Expression in pTac system:
Gene fore Cry7Ca1 has been his-tagged and sealed onto vector pET28a and transformed into BL21 and DH10B(E. coli) for pTac regulated expression.
When induced by 1mM IPTG under 37℃ for 6 hours, the cell were harvested and disrupted by ultrasonic homogenizer. Which the Cry7Ca1 in homogenate then purified using Ni-NTA system where samples were isolated from raw lysate and elusion buffer flowthrough.
We have successfully identified the desired band after SDS-PAGE which indicates the presence of Cry7Ca1:
SZ-SHD 2021's Improvement
The part BBa_K3895012 was improved from SZ-SHD 2020's bt toxin Cry7Ca1 (BBa_K3686010) for tobacco wheat instant expression, where Cry7Ca1 is a differentiate of Bt toxins with a molecular mass of 129kDa, recently isolated from Bt strain BHT-13. Cry7Ca1 is connected with GFP (BBa_E0040),and constructed into pCAMBIA1301 vector.
Protocol
1. Transfection of pCAMBIA-Cry7Ca1-eGFP vector through Carbon dots nanocomposite (CDP) to Nicotiana benthamiana
Coupling CDP with plant expressing vector Mix the following ingredients:
| Ingredients | Volumn |
|---|---|
| MES buffer(50X) | 10ul |
| CDP(50X) | 10ul |
| DNA(237ng/ul >10ng/ul final con) | 22ul |
| 10% glycerol | 25ul |
| ddH2O | 433ul |
| total | 500ul |
Gently mix and incubate at 37℃ for 30min.
2. Brush the mixture gently on the leaf of Nicotiana benthamiana(leaf length>10cm,Growing well) ，mark the area of brushing
3. Put the plant back in the light incubator(28℃，12h light 12h dark), repeat the process for four days at 3:00pm each day(2/4)
Results
Fluorescence microscope to observe the GFP in leave
Figure 3. Western Blot result of Cry7Ca1-eGFP protein express in wheat leaf.
Figure 4. Fluorescence microscopy images of the CDP transfected on plant leaf. 1/3. Blank control group with CDP only; 2/4. CDP with 10ng/ul plant expressing vector.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 205
Illegal NgoMIV site found at 220
Illegal NgoMIV site found at 1567
Illegal NgoMIV site found at 1828
Illegal NgoMIV site found at 1990 - 1000COMPATIBLE WITH RFC[1000]
| None |