Difference between revisions of "Part:BBa K1668006"
 
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We clone and standardize the gene into standard plasmid pSB1C3, and confirmed the part by PCR and sequencing. Then we combine the CDS <i>plu0840</i> with arabinose inducible promoter pBad in front reporter mCherry and double terminator behind into the device <i>plu0840</i> to strongly express the toxin.
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We clone and standardize the gene into standard plasmid pSB1C3, and confirmed the part by PCR and sequencing. Then we combine the CDS <i>plu0840</i> with arabinose inducible promoter <i>pBad</i> in front reporter mCherry and double terminator behind into the device <i>plu0840</i> to strongly express the toxin.
 
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[[File:ZJU-CHINA_0840_3D_structure.jpg|200px|thumb|right|Figure 1 The 3D structure of Plu0840(<i>4</i>). Copyright 2014, Worldwide Protein Data.]]
 
[[File:ZJU-CHINA_0840_3D_structure.jpg|200px|thumb|right|Figure 1 The 3D structure of Plu0840(<i>4</i>). Copyright 2014, Worldwide Protein Data.]]
 
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In 2009 research<i> Cloning and expression analysis of a predicted toxin gene from Photorhabdus sp. HB78</i>, <i>plu0840</i> fused with GST 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 moth (<i>S. litura </i>and <i>S.exigua</i>)(<i>1</i>).  
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In 2009 research<i> Cloning and expression analysis of a predicted toxin gene from Photorhabdus sp. HB78</i>, <i>plu0840</i> fused with GST is expressed in <i>E.coli</i> BL21(DE3). Engineered strain BL21 was both orally fed and injected in hemocoel to two kind of moth (<i>S. litura </i>and <i>S.exigua</i>)(<i>1</i>).  
 
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The research mentions that Plu0840 (figure 1) shares 55% sequence identity with an enterotoxin Ast from <i>aeromonas hydrophila</i>. <i>Aaeromonas hydrophila</i>, which is connected with gastroenteritis, may lead to altered fluid secretion in mouse. According to a 2002 research, enterotoxin Ast makes weak contributions to fluid secretion compared with two other genes (<i>2</i>) However, judging that <I>TT01</I> is nontoxic to animals at all, we think Plu0840 may share little similarity with Ast.  
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The research mentions that Plu0840 (Figure 1) shares 55% sequence identity with an enterotoxin Ast from <i>aeromonas hydrophila</i>. <i>Aaeromonas hydrophila</i>, which is connected with gastroenteritis, may lead to altered fluid secretion in mouse. According to a 2002 research, enterotoxin Ast makes weak contributions to fluid secretion compared with two other genes (<i>2</i>) However, judging that <I>TT01</I> is nontoxic to animals at all, we think Plu0840 may share little similarity with Ast.  
 
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It can be clearly seen the recombinants shown in figure 2 is positive cloning.
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It can be clearly seen the recombinants shown in Figure 2 is positive cloning.
 
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Latest revision as of 09:43, 18 September 2015

CDS plu0840

The part CDSplu0840 is coding sequence of toxin protein Plu0840, which is used for termite control in our project.

Plu0840 is a 72kDa insecticidal toxic protein, which had weak oral toxicity against two kinds of moth according to a 2007 research and showed weak toxicity to termites by oral feeding.

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

Plu0840 is a insecticidal toxin found in Photorhabdus luminescens, 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 plu0840 with arabinose inducible promoter pBad in front reporter mCherry and double terminator behind into the device plu0840 to strongly express the toxin.


BACKGROUND

Figure 1 The 3D structure of Plu0840(4). Copyright 2014, Worldwide Protein Data.


In 2009 research Cloning and expression analysis of a predicted toxin gene from Photorhabdus sp. HB78, plu0840 fused with GST is expressed in E.coli BL21(DE3). Engineered strain BL21 was both orally fed and injected in hemocoel to two kind of moth (S. litura and S.exigua)(1).

According to the result, on the one hand, oral feeding effectively inhibits the growth of larva while has only weak oral toxic effect. On the other hand, hemocoel injection showed negative results.

The research mentions that Plu0840 (Figure 1) shares 55% sequence identity with an enterotoxin Ast from aeromonas hydrophila. Aaeromonas hydrophila, which is connected with gastroenteritis, may lead to altered fluid secretion in mouse. According to a 2002 research, enterotoxin Ast makes weak contributions to fluid secretion compared with two other genes (2) However, judging that TT01 is nontoxic to animals at all, we think Plu0840 may share little similarity with Ast.


RESULTS

PLASMID CONSTRUCTION

Figure 2 PCR confirmation results of CDS plu0840 with standard primers VF2 and VR.



The template we use is recombinant plasmids.


It can be clearly seen the recombinants shown in Figure 2 is positive cloning.









PLASMID 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. J. Sha, E. V. Kozlova, A. K. Chopra, INFECT IMMUN 70, 1924 (2002).
3. Plu0840 (uniprot): http://www.uniprot.org/uniprot/Q7N895.
4. 3D structure of Plu0840: http://www.proteinmodelportal.org/?pid=modelDetail&provider=MODBASE&template=1uasA&pmpuid=1000725544546&range_from=1&range_to=639&ref_ac=Q7N895&mapped_ac=Q7N895&zid=async.