Difference between revisions of "Part:BBa K3895012"
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This part was adjusted from BBa_K3686010 (<partinfo>K3686010</partinfo>), 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 (<partinfo>E0040</partinfo>),and constructed into pCAMBIA-Cry7Ca1-eGFP vector.
 
This part was adjusted from BBa_K3686010 (<partinfo>K3686010</partinfo>), 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 (<partinfo>E0040</partinfo>),and constructed into pCAMBIA-Cry7Ca1-eGFP vector.
  
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[[file:T--SZ SHD--cryjpg.png]]
 
  
 
[[File:T--SZ SHD--cryjpg.png|center|350px|thumb|'''Figure 1.''' Plasmid construction of pCAMBIA-Cry7Ca1-eGFP vector.]]
 
[[File:T--SZ SHD--cryjpg.png|center|350px|thumb|'''Figure 1.''' Plasmid construction of pCAMBIA-Cry7Ca1-eGFP vector.]]
  
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===Protocol===
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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:
 +
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)
  
  

Revision as of 17:20, 10 October 2021


Insecticidal protein

Bt toxins refer to the toxic proteins produced by insect pathogenic bacteria Bacillus thuringiensis[1]. This part was adjusted from BBa_K3686010 (BBa_K3686010), 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 pCAMBIA-Cry7Ca1-eGFP vector.


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

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)


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.