View source for Part:BBa K3895012


__NOTOC__
<partinfo>BBa_K3895012 short</partinfo>

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 (<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.]]

===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:

{|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
|}
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
{|border=0 width="90%" align="center"
|-align="center"
|[[File:T--SZ SHD--cry1.jpg|400px|thumb|center|'''Positive''' ]]
|[[File:T--SZ SHD--cry2.jpg|400px|thumb|center|'''Negative''' ]]
|}

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===Usage and Biology===

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<span class='h3bb'>Sequence and Features</span>
<partinfo>BBa_K3895012 SequenceAndFeatures</partinfo>


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===Functional Parameters===
<partinfo>BBa_K3895012 parameters</partinfo>
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===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.