Difference between revisions of "Part:BBa K3895012"

(→‎Protocol)
(→‎Protocol)
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===Protocol===
 
===Protocol===
 
1. Transfection of pCAMBIA-Cry7Ca1-eGFP vector through Carbon dots nanocomposite (CDP) to Nicotiana benthamiana
 
1. Transfection of pCAMBIA-Cry7Ca1-eGFP vector through Carbon dots nanocomposite (CDP) to Nicotiana benthamiana
 +
 
Coupling CDP  with plant expressing vector
 
Coupling CDP  with plant expressing vector
 
Mix the following ingredients:
 
Mix the following ingredients:
 +
 +
{|border=1 width="90%" align="center"
 +
|-
 +
!width="20%" style="background:#CCCCFF"|Ingredients
 +
!width="20%"|Volumn
 +
 +
|-align="center"
 +
|style="background:#EEEEFF"|MES buffer(50X)
 +
|10ul
 +
|-align="center"
 +
|style="background:#EEEEFF"|CDP(50X)
 +
|10ul
 +
|-align="center"
 +
|style="background:#EEEEFF"|DNA(237ng/ul >10ng/ul final con) 
 +
|22ul
 +
|-align="center"
 +
|style="background:#EEEEFF"|10% glycerol
 +
|25ul
 +
|-align="center"
 +
|style="background:#EEEEFF"|ddH2O 
 +
|433ul
 +
|-align="center"
 +
|style="background:#EEEEFF"|total                                         
 +
|500ul
 +
 +
 +
 
MES buffer(50X)                                10ul
 
MES buffer(50X)                                10ul
 
CDP(50X)                                      10ul
 
CDP(50X)                                      10ul
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Gently mix and incubate at 37℃ for 30min.
 
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
 
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)
 
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 17:25, 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:

Ingredients Volumn
MES buffer(50X) 10ul
CDP(50X) 10ul
DNA(237ng/ul >10ng/ul final con) 22ul
10% glycerol 25ul
ddH2O 433ul
total 500ul


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.