Difference between revisions of "Part:BBa K3895012"
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Bt toxins refer to the toxic proteins produced by insect pathogenic bacteria ''Bacillus thuringiensis''[1].  
 
Bt toxins refer to the toxic proteins produced by insect pathogenic bacteria ''Bacillus thuringiensis''[1].  
−
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 SZ-SHD 2020's bt toxin Cry7Ca1 (<partinfo>K3686010</partinfo>) 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 (<partinfo>E0040</partinfo>),and constructed into pCAMBIA1301 vector.
  
  
−
[[File:T--SZ SHD--cryjpg.png|center|350px|thumb|'''Figure 1.''' Plasmid construction of pCAMBIA-Cry7Ca1-eGFP vector.]]
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[[File:T--SZ SHD--cryjpg.png|center|350px|thumb|'''Figure 1.''' Plasmid construction of pCAMBIA-Cry7Ca1-eGFP.]]
  
 
===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:
−
MES buffer(50X)                               10ul
+
 
−
CDP(50X)                                       10ul
+
{|border=1 width="90%" align="center"
−
DNA(237ng/ul >10ng/ul final con)               22ul
+
|-
−
10% glycerol                                   25ul
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!width="20%" style="background:#CCCCFF"|Ingredients
−
ddH2O                                         433ul
+
!width="20%"|Volumn
−
total                                          500ul
+
 
−
      - Gently mix and incubate at 37℃ for 30min.
+
|-align="center"
−
2. Brush the mixture gently on the leaf of Nicotiana benthamiana(leaf length>10cm,   Growing well) ，mark the area of brushing
+
|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
 +
|}
 +
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)
 
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
 +
{|border=0 width="90%" align="center"
 +
|-align="center"
 +
|[[File:T--SZ SHD--cry1.jpg|400px|thumb|center|'''Figure 2(a). Positive results showed successful expression of pCAMBIA-Cry7Ca1-eGFP in Nicotiana''' ]]
 +
|[[File:T--SZ SHD--cry2.jpg|400px|thumb|center|'''Figure 2(b). Negative results without Cry7Ca1-eGFP in Nicotiana benthamiana.''' ]]
 +
|}
 +
 +
[[File:T--SZ SHD--lyj2.jpg|300px|center]]
 +
'''Figure 3.''' Western Blot result of Cry7Ca1-eGFP protein express in wheat leaf.
 +
[[File:T--SZ_SHD--lyj1.jpg|300px|center]]
 +
'''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.
  
 
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<!-- Add more about the biology of this part here
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<partinfo>BBa_K3895012 parameters</partinfo>
 
<partinfo>BBa_K3895012 parameters</partinfo>
 
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===References===
 
===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.
 
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.

Latest revision as of 17:16, 21 October 2021


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.
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
    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.