Difference between revisions of "Part:BBa K1668006"

 
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<partinfo>BBa_K1668006 short</partinfo>
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<partinfo>BBa_K166806 short</partinfo>
  
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metK is the S-adenosylmethionine synthetase gene from Streptomyces avermitilis, which was found to stimulate the production of avermectins. When wild-type strain ATCC31267 was transformed with pYJ02 and pYJ03, two metK expression plasmids, avermectin production was increased about 2.0-fold and 5.5-fold compared with that in the control strains, respectively. The avermectin productivity of each culture was quantitatively measured by HPLC analysis.  
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The part CDS<i>plu0840</i> is coding sequence of toxin protein Plu0840, which is used for termite control in our project.
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As the kinetic study revealed, avermectin overproduction in recombinant strain was not caused by the change of cell growth rate or copy number effect. Instead, metK stimulates the avermectin production by increasing the intracellular concentration of S-adenosylmethionine (SAM), an important intermediate product in avermectin production. However, there may be a maximum concentration of SAM for the production of avermectin in S. avermitilis, over which it has no any effect on the antibiotic production.
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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.
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The results of experiments showed that there were different effects of metK expression levels on avermectin production in various S. avermitilis strains. The gene expression levels of metK in two engineered strain, GB-165 and 76-05, were much higher then those in wild-type strain, whereas the avermectin productivity in these two strains were not significantly improved. It probably because the high expression level of metK limited the increasement of avermectin by overexpression of metK.  
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===Usage and Biology===
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===Functional Parameters===
 
===Functional Parameters===
 
<partinfo>BBa_K1668006 parameters</partinfo>
 
<partinfo>BBa_K1668006 parameters</partinfo>
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<h2>'''Characterization'''</h2>
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<h3> OVERVIEW </h3>
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Plu0840 is a 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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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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h3> BACKGROUND </h3>
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[[File:ZJU-CHINA_0840_3D_structure.jpg|200px|thumb|right|Figure 1, the 3D structure of Plu0840. 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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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. 
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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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<h3> RESULTS </h3>
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<h4> PLASMID CONSTRUCTION </h4>
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[[File:Digestion_A1%2BpSB1A2.png|200px|thumb|left|Figure 4 digestion confirmation of tcdA1-device in pSB1A2 backbone.]]
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5μl samples of the double enzyme digestion products for tcdA1-device were loaded onto a 1% BioRad Ready Agarose Mini Gel, then subjected to AGE. See (protocol) for AGE parameters. Sizes of the XbaI and PstI–cleaved assemblies were determined by AGE analysis. The DNA size standards were the DL5,000 DNA Marker (M2; TaKaRa, Cat#3428A) and 1kb DNA Ladder (Dye Plus)(M2; TaKaRa, Cat#3426A). Bands were visualized with a Shanghai Peiqing JS-380A Fluorescence Imager.
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First we construct the tcdA1 device in pSB1A2. Our target fragments can be clearly seen in the right position (figure 4). As the fragment is a little big(7.2k), the efficiency is low when we change the backbone to pSB1C3 and the unwanted fragment is hard to explain(figure 5).
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<h4> PLASMID 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.
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Revision as of 12:40, 16 September 2015

No part name specified with partinfo tag.

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.


h3> BACKGROUND </h3>

Figure 1, the 3D structure of Plu0840. 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 DH5α 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 4 digestion confirmation of tcdA1-device in pSB1A2 backbone.



5μl samples of the double enzyme digestion products for tcdA1-device were loaded onto a 1% BioRad Ready Agarose Mini Gel, then subjected to AGE. See (protocol) for AGE parameters. Sizes of the XbaI and PstI–cleaved assemblies were determined by AGE analysis. The DNA size standards were the DL5,000 DNA Marker (M2; TaKaRa, Cat#3428A) and 1kb DNA Ladder (Dye Plus)(M2; TaKaRa, Cat#3426A). Bands were visualized with a Shanghai Peiqing JS-380A Fluorescence Imager.

First we construct the tcdA1 device in pSB1A2. Our target fragments can be clearly seen in the right position (figure 4). As the fragment is a little big(7.2k), the efficiency is low when we change the backbone to pSB1C3 and the unwanted fragment is hard to explain(figure 5).






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.