Difference between revisions of "Part:BBa K1668007"
 
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<h2>'''Characterization'''</h2>
 
<h2>'''Characterization'''</h2>
 
<h3> OVERVIEW </h3>
 
<h3> OVERVIEW </h3>
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Plu1537 is a small insecticidal toxin found in <i>Photorhabdus luminescens</i>, a native toxin storehouse. In our project, it is used for termite control.   
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Plu1537 is a small insecticidal toxin found in <i>Photorhabdus luminescens</i> TT01, a native toxin storehouse. In our project, it is used for termite control.   
 
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<h3> BACKGROUND </h3>
 
<h3> BACKGROUND </h3>
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[[File:Figure_1.png|200px|thumb|right|Figure 1, the 3D structure of cry34Ab1(<i>4</i>). Copyright 2014, Worldwide Protein Data.]]
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[[File:Figure_1.png|200px|thumb|right|Figure 1 The 3D structure of Cry34Ab1(<i>4</i>). Copyright 2014, Worldwide Protein Data.]]
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[[File:Figure_2.png|200px|thumb|left|Figure 2, comparison between cry34Ab1 and cry35Ab1(2). Copyright 2014, Public Library of Science.]]
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[[File:Figure_2.png|200px|thumb|left|Figure 2 Comparison between Cry34Ab1 and Cry35Ab1(2). Copyright 2014, Public Library of Science.]]
 
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In 2009 research<i> Expression and activity of a probable toxin from Photorhabdus Luminescens</i>, toxin protein pit, which is 94% homologous with plu1537, is expressed in <i>DH5α BL21(DE3)</i>. Engineered strain <i>BL21</i> was both orally fed and injected in hemocoel to two kind of moths(Galleria mellonella & Spodoptera litura)(<i>1</i>). As a result, hemocoel injection is more effective than oral feeding. However, our experiment showed that oral feeding is also effective.  
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In 2009 research<i> Expression and activity of a probable toxin from Photorhabdus Luminescens</i>, toxin protein pit, which is 94% homologous with plu1537, is expressed in <i>E.coli</i> BL21(DE3). Engineered strain BL21 was both orally fed and injected in hemocoel to two kind of moths(Galleria mellonella & Spodoptera litura)(<i>1</i>). As a result, hemocoel injection is more effective than oral feeding. However, our experiment showed that oral feeding is also effective.  
 
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Plu1537 shares 30% amino acid sequence similarity with a 13.6 kDa insecticidal crystal protein cry34Ab1 in <i>Bacillus thuringiensis</i> (figure 1), which belongs to Bt toxin family.  
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Plu1537 shares 30% amino acid sequence similarity with a 13.6 kDa insecticidal crystal protein Cry34Ab1 in <i>Bacillus thuringiensis</i> (Figure 1), which belongs to Bt toxin family.  
 
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Bt family may be the most famous insecticidal toxin up to now. It’s one of biological toxins first used for pest control. After years of study, Bt family is becoming bigger and bigger. Basically, most proteins in Bt family form pores in cell membrane to kill a cell, including cry34Ab1(<i>2</i>).  
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Bt family may be the most famous insecticidal toxin up to now. It’s one of biological toxins first used for pest control. After years of study, Bt family is becoming bigger and bigger. Basically, most proteins in Bt family form pores in cell membrane to kill a cell, including Cry34Ab1(<i>2</i>).  
 
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In 2014, the structure of cry34Ab1 was revealed and reported in PLOS ONE. As displayed in figure 1, the structure of cry34Ab1 is simpler, compared with other two toxins we used. However, cry34Ab1 (figure 2A) can only function with the assistant of cry35Ab1 (figure 2B)(<i>2</i>), which differentiate cry34 Ab1 from Plu1537, which is toxic without any other assistant.
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In 2014, the structure of Cry34Ab1 was revealed and reported in PLOS ONE. As displayed in Figure 1, the structure of Cry34Ab1 is simpler, compared with other two toxins we used. However, Cry34Ab1 (Figure 2A) can only function with the assistant of Cry35Ab1 (Figure 2B)(<i>2</i>), which differentiate Cry34 Ab1 from Plu1537, which is toxic without any other assistant.
 
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It can be clearly seen the recombinants shown in figure 3 is positive cloning.
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It can be clearly seen the recombinants shown in Figure 3 is positive cloning.
 
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<h4> PLASMID SEQUNCING </h4>
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<h4> DNA SEQUNCING </h4>
 
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We have sequenced the parts with standard primers VF2 and VR. The sequence of the 1.8k part shows 100% agreement with the desired sequence.
 
We have sequenced the parts with standard primers VF2 and VR. The sequence of the 1.8k part shows 100% agreement with the desired sequence.

Latest revision as of 09:47, 18 September 2015

CDS plu1537

The part CDSplu1537 is coding sequence of toxin protein Plu1537, which is used for termite control in our project.

Plu1537 is a 14kDa insecticidal toxic protein, which has strong toxicity against termites by oral feeding according to our termites experiment. It has 30% homology with a kind of Bt toxic protein, therefore it may play a similar role.

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
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


Characterization

OVERVIEW

Plu1537 is a small insecticidal toxin found in Photorhabdus luminescens TT01, a native toxin storehouse. In our project, it is used for termite control.

We clone and standardize the gene into standard plasmid pSB1C3, and confirmed the part by PCR and sequencing. Then we combine the CDS plu1537 with arabinose inducible promoter pBad in front reporter mCherry and double terminator behind into the device plu1537 to strongly express the toxin. Termites in vivo experiments results show that Plu1537 is strongly toxic to termites.


BACKGROUND

Figure 1 The 3D structure of Cry34Ab1(4). Copyright 2014, Worldwide Protein Data.
Figure 2 Comparison between Cry34Ab1 and Cry35Ab1(2). Copyright 2014, Public Library of Science.


In 2009 research Expression and activity of a probable toxin from Photorhabdus Luminescens, toxin protein pit, which is 94% homologous with plu1537, is expressed in E.coli BL21(DE3). Engineered strain BL21 was both orally fed and injected in hemocoel to two kind of moths(Galleria mellonella & Spodoptera litura)(1). As a result, hemocoel injection is more effective than oral feeding. However, our experiment showed that oral feeding is also effective.

Plu1537 shares 30% amino acid sequence similarity with a 13.6 kDa insecticidal crystal protein Cry34Ab1 in Bacillus thuringiensis (Figure 1), which belongs to Bt toxin family.

Bt family may be the most famous insecticidal toxin up to now. It’s one of biological toxins first used for pest control. After years of study, Bt family is becoming bigger and bigger. Basically, most proteins in Bt family form pores in cell membrane to kill a cell, including Cry34Ab1(2).

In 2014, the structure of Cry34Ab1 was revealed and reported in PLOS ONE. As displayed in Figure 1, the structure of Cry34Ab1 is simpler, compared with other two toxins we used. However, Cry34Ab1 (Figure 2A) can only function with the assistant of Cry35Ab1 (Figure 2B)(2), which differentiate Cry34 Ab1 from Plu1537, which is toxic without any other assistant.






RESULTS

PLASMID CONSTRUCTION

Figure 3 PCR confirmation results of CDS plu1537 with standard primers VF2 and VR.



The template we use is recombinant plasmids.


It can be clearly seen the recombinants shown in Figure 3 is positive cloning.












DNA SEQUNCING


We have sequenced the parts with standard primers VF2 and VR. The sequence of the 1.8k part shows 100% agreement with the desired sequence.


REFERENCE

1. M. Li et al., MOL BIOL REP 36, 785 (2009).
2. M. S. Kelker et al., PLOS ONE 9, (2014).
3. cry34Ab1(uniprot): http://www.uniprot.org/uniprot/Q939T0
4. 3D structure of cry34Ab1: http://www.ebi.ac.uk/pdbe/entry/pdb/4JOX