Part:BBa_K3895012

Insecticidal protein

Bt toxins refer to the toxic proteins produced by insect pathogenic bacteria Bacillus thuringiensis[1]. This part was adjusted 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.

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 2(a). Positive results showed successful expression of pCAMBIA-Cry7Ca1-eGFP in Nicotiana

Figure 2(b). Negative results without Cry7Ca1-eGFP in Nicotiana benthamiana.

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

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE 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
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 4079
  

References

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.