Designed by: JiXiao Wu   Group: iGEM20_SZ-SHD   (2020-10-25)

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].

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, 28(3), 609-619.

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.


Codon-optimized for expression in Escherichia coli.

Optimization details:

Cry7ca GC.jpeg

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:

Cry7ca express in BL21.jpeg
Cry7ca express in DH10B.jpeg

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.

Figure 1. Plasmid construction of pCAMBIA-Cry7Ca1-eGFP.


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)


Fluorescence microscope to observe the GFP in leave

Figure 2(a). Positive results showed successful expression of pCAMBIA-Cry7Ca1-eGFP in Nicotiana
Figure 2(b). Negative results without Cry7Ca1-eGFP in Nicotiana benthamiana.
T--SZ SHD--lyj2.jpg

Figure 3. Western Blot result of Cry7Ca1-eGFP protein express in wheat leaf.

T--SZ SHD--lyj1.jpg

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

Assembly Compatibility:
  • 10
  • 12
  • 21
  • 23
  • 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
  • 1000