Difference between revisions of "Part:BBa K1660001"

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=='''Introduction'''==
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<h2>Introduction</h2>
  
 
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<p>rMpL has a good toxicity toward the nematodes. It is a novel lectin isolated from a kind of parasol mushroom (<em>Macrolepiota procera</em>). It is a kind of intracellular expression protein. MpL could bind with glycan of the nematodes specifically. So it can stop the growth of the nematodes from L1 phase to adults.</p>
 
<p>rMpL has a good toxicity toward the nematodes. It is a novel lectin isolated from a kind of parasol mushroom (<em>Macrolepiota procera</em>). It is a kind of intracellular expression protein. MpL could bind with glycan of the nematodes specifically. So it can stop the growth of the nematodes from L1 phase to adults.</p>
 
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<partinfo>BBa_K1660001 parameters</partinfo>
 
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<h2>Structure</h2>
 
<h2>Structure</h2>
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rMpL has a typical β-trefoil fold, consisting of α-,β- and γ- repeats(Fig. 1A). The β-trefoil fold seems like a tree, which has a short trunk(in red) and an expanded crown(in blue)(Fig. 1B). The trunk is a six stranded β-barrel composed of β-strands(β1, β4, β5, β8, β9, β12).And the crown is constituted by the other three pairs of β-strands(β2, β3, β6, β7, β10 and β11) and its connective loops.
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rMpL has a typical β-trefoil fold, consisting of α-, β- and γ- repeats (Fig. 1A). The β-trefoil fold seems like a tree, which has a short trunk (in red) and an expanded crown (in blue) (Fig. 1B). The trunk is a six stranded β-barrel composed of β-strands (β1, β4, β5, β8, β9, β12). And the crown is constituted by the other three pairs of β-strands (β2, β3, β6, β7, β10 and β11) and its connective loops.
 
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<figcaption>Fig.1A</figcaption>
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<figcaption>Fig.1B</figcaption>
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<figcaption>Fig.1 Three-dimensional structures of rMpL in complex with carbohydrates.</figcaption>
 
<figcaption>Fig.1 Three-dimensional structures of rMpL in complex with carbohydrates.</figcaption>
 
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<p class="fig">Fig.1 Three-dimensional structures of rMpL in complex with carbohydrates. 1A: The structure of rMpL with a-, b- and c-repeats shown in green, cyan and yellow; 1B:The structure of rMpL in a tree-like orientation. The trunk is shown in red and the crown is shown in blue. Galactose is represented as sticks.</p>
 
<p class="fig">Fig.1 Three-dimensional structures of rMpL in complex with carbohydrates. 1A: The structure of rMpL with a-, b- and c-repeats shown in green, cyan and yellow; 1B:The structure of rMpL in a tree-like orientation. The trunk is shown in red and the crown is shown in blue. Galactose is represented as sticks.</p>
 
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<h2>Function</h2>
 
<h2>Function</h2>
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According to relevant research, Bace16 is the key reason for the high infection toxicity of <em>Bacillus nematocida</em> to <em>Panagrellus redivivus</em> and <em>Bursaphelenchus xylophilus</em>. Bioassay with purified Bace16 has showed that 90% of the nematodes could be killed within 24 h at the concentration of 1.79 μg/ml; after 48 h, all of the tested nematodes were almost killed and degraded<sup>[4]</sup>. Researchers found that recombinant protease rm-Bace16(whose molecular weight is 34kDa) expressed in <em>Escherichia coli</em> also presented a nematotoxic activity. And both Bace16 and rm-Bace16 could degrade a broad range of substrates including casein, denatured collagen, and nematode cuticle.  
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According to the related literature，rMpL is toxic to <em>C.elegans</em> larvae. Only 20% of rMpL-expressing <em>E. coli</em> is sufficient to inhibit the development of most N2 larvae into adulthood(Fig. 2). The potential glycan targets in the nematode is ‘GalFuc’, for 30% of the worms developed to adulthood when nematodes lacks additional modifications in the antennae of N-glycans, and 20% of worms reach adulthood when nematodes lack the ‘GalFuc’ epitope in the N-glycan core, compared with almost all the nematodes which cannot reach to L4-adults with normal N-Glycans.
 
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<img width="50%" src="https://static.igem.org/mediawiki/parts/c/ca/BNU-RMpL_Fig2.png">
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<figcaption>Fig.2 Toxicity of rMpL against wild-type (N2) <em>C. elegans</em>. Percentages in brackets represent the proportion of rMpL-expressing <em>E. coli</em> mixed with bacteria transformed with empty vector control. The dose dependence of MpL-mediated toxicity on development of L1 to L4 larvae is shown.</figcaption>
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<h2>Design</h2>
 
<h2>Design</h2>
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The DNA sequence of bace16 precursor is from NCBI(GenBank: AY708655.1), taking the fold and secretion of Bace16 into consideration, the presequence signal peptide of 30 amino acids and propeptide of 77 amino acids are retained before the mature peptide. And in order to produce this toxin, a pBAD promoter(BBa_K206000), which is said to be suitable for toxic protein expression is add upstream the functional gene.In order to change the promoter and RBS to control the expression rate of Bace16, a XhoI restriction site is added between RBS and the start codon. bace16-pSB1C3 plasmid is transformed into <em>E. coli</em> strain BW25113 to express and purify Bace16 protein. Then nematocidal activity test is conducted.  
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In <em>Macrolepiota procera</em>, the MpL gene is 791 bp long (including start and stop codons) which is composed of four exons and three introns. By knocking out the introns, we will optimize this gene which comes from eukaryotic cells so that it can express efficiently in <em>E.coli</em>. Furthermore, we will add the pBAD promotor ([https://parts.igem.org/Part:BBa_K206000 BBa_K206000]) induced by L-arabinose as well as the RBS ([https://parts.igem.org/Part:BBa_B0034 BBa_B0034]) in the upstream of rMpL gene sequence, for pBAD promoter is suitable for the expression of the toxin. At the same time, the Xho I restriction site will also be added between the RBS and start codon, which will give us a lot of convenience to replace different promoter with different intensity.
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After acquiring the recombinant vector successfully, we will transfer the recombinant vector into the <em>E.coli</em> BW25113. Then we will design a series of concentrations of the L-arabinose to induce the expression of rMpL.
 
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<h2>Results</h2>
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After we successfully built the vectors, we transferred the vectors into different <em>E.coli</em> strains depending on the kinds of vectors. Vectors with pBAD promoter were transferred into <em>E.coli</em> BW25113. After rMpL gene was expressed in the bacteria, we did SDS-PAGE to testify the expression of rMpL protein. And according to the SDS-PAGE figure, we expressed rMpL in <em>E.coli</em> strains.
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<img width="50%" src="https://static.igem.org/mediawiki/parts/c/c6/Bnu-Fig_3_SDS-PAGE%28BW25113%29.jpg">
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<figcaption>Fig.3 SDS-PAGE of rMpL</figcaption>
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<figcaption>Lane 1, molecular weight standards (kDa); Lane 2-5, supernatant after 1.2, 1.2, 1.0, 1.0 uM L-Ara induction respectively; lane 7, supernatant after 0 uM L-Ara induction; Lane 8: supernatant of the pSB1C3; Lane 9, 10, 12, 13, 14: homogenate after 1.2, 1.2, 1.0, 1.0uM L-Ara induction respectively; Lane 15: homogenate of pSB1C3.</figcaption>
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<h2>Reference</h2>
 
<h2>Reference</h2>
 
<ul>
 
<ul>
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             <li>[1] Huang X W, Niu Q H, Zhou W, et al. Bacillus nematocida sp. nov., a novel bacterial strain with nematotoxic activity isolated from soil in Yunnan, China[J]. Systematic and applied microbiology, 2005, 28(4): 323-327.</li>
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             <li>[1] Sacchettini JC, Baum LG & Brewer CF (2001) Multivalent protein–carbohydrate interactions. A new paradigm for supermolecular sssembly and signal transduction. Biochemistry (US) 40, 3009–3015.</li>
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             <li>[2] Niu Q, Huang X, Zhang L, et al. Functional identification of the gene bace16 from nematophagous bacterium Bacillus nematocida[J]. Applied microbiology and biotechnology, 2007, 75(1): 141-148.</li>
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             <li>[2] Jerica Saboti, Simon Zurga&Jure Pohleven(2014) A novel b-trefoil lectin from the parasol mushroom(Macrolepiota procera) is nematotoxic. FEBS Journal(UK)281,3489-3505.</li>
−
            <li>[3] Day R M, Thalhauser C J, Sudmeier J L, et al. Tautomerism, acid‐base equilibria, and H‐bonding of the six histidines in subtilisin BPN′ by NMR[J]. Protein Science, 2003, 12(4): 794-810.
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            </li>
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            <li>[4] Qiuhong N, Xiaowei H, Baoyu T, et al. Bacillus sp. B16 kills nematodes with a serine protease identified as a pathogenic factor[J]. Applied microbiology and biotechnology, 2006, 69(6): 722-730.</li>
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         </ul>
 
         </ul>

Latest revision as of 17:26, 17 September 2015

rMpL


K1660001

Introduction

rMpL has a good toxicity toward the nematodes. It is a novel lectin isolated from a kind of parasol mushroom (Macrolepiota procera). It is a kind of intracellular expression protein. MpL could bind with glycan of the nematodes specifically. So it can stop the growth of the nematodes from L1 phase to adults.


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 125
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 65
    Illegal XhoI site found at 151
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 208
    Illegal AgeI site found at 304
    Illegal AgeI site found at 519
  • 1000
    COMPATIBLE WITH RFC[1000]



Structure

rMpL has a typical β-trefoil fold, consisting of α-, β- and γ- repeats (Fig. 1A). The β-trefoil fold seems like a tree, which has a short trunk (in red) and an expanded crown (in blue) (Fig. 1B). The trunk is a six stranded β-barrel composed of β-strands (β1, β4, β5, β8, β9, β12). And the crown is constituted by the other three pairs of β-strands (β2, β3, β6, β7, β10 and β11) and its connective loops.
Fig.1 Three-dimensional structures of rMpL in complex with carbohydrates.

Fig.1 Three-dimensional structures of rMpL in complex with carbohydrates. 1A: The structure of rMpL with a-, b- and c-repeats shown in green, cyan and yellow; 1B:The structure of rMpL in a tree-like orientation. The trunk is shown in red and the crown is shown in blue. Galactose is represented as sticks.



Function

According to the related literature，rMpL is toxic to C.elegans larvae. Only 20% of rMpL-expressing E. coli is sufficient to inhibit the development of most N2 larvae into adulthood(Fig. 2). The potential glycan targets in the nematode is ‘GalFuc’, for 30% of the worms developed to adulthood when nematodes lacks additional modifications in the antennae of N-glycans, and 20% of worms reach adulthood when nematodes lack the ‘GalFuc’ epitope in the N-glycan core, compared with almost all the nematodes which cannot reach to L4-adults with normal N-Glycans.

Fig.2 Toxicity of rMpL against wild-type (N2) C. elegans. Percentages in brackets represent the proportion of rMpL-expressing E. coli mixed with bacteria transformed with empty vector control. The dose dependence of MpL-mediated toxicity on development of L1 to L4 larvae is shown.


Design

In Macrolepiota procera, the MpL gene is 791 bp long (including start and stop codons) which is composed of four exons and three introns. By knocking out the introns, we will optimize this gene which comes from eukaryotic cells so that it can express efficiently in E.coli. Furthermore, we will add the pBAD promotor (BBa_K206000) induced by L-arabinose as well as the RBS (BBa_B0034) in the upstream of rMpL gene sequence, for pBAD promoter is suitable for the expression of the toxin. At the same time, the Xho I restriction site will also be added between the RBS and start codon, which will give us a lot of convenience to replace different promoter with different intensity. After acquiring the recombinant vector successfully, we will transfer the recombinant vector into the E.coli BW25113. Then we will design a series of concentrations of the L-arabinose to induce the expression of rMpL.


Results

After we successfully built the vectors, we transferred the vectors into different E.coli strains depending on the kinds of vectors. Vectors with pBAD promoter were transferred into E.coli BW25113. After rMpL gene was expressed in the bacteria, we did SDS-PAGE to testify the expression of rMpL protein. And according to the SDS-PAGE figure, we expressed rMpL in E.coli strains.

Fig.3 SDS-PAGE of rMpL
Lane 1, molecular weight standards (kDa); Lane 2-5, supernatant after 1.2, 1.2, 1.0, 1.0 uM L-Ara induction respectively; lane 7, supernatant after 0 uM L-Ara induction; Lane 8: supernatant of the pSB1C3; Lane 9, 10, 12, 13, 14: homogenate after 1.2, 1.2, 1.0, 1.0uM L-Ara induction respectively; Lane 15: homogenate of pSB1C3.


Reference

  • [1] Sacchettini JC, Baum LG & Brewer CF (2001) Multivalent protein–carbohydrate interactions. A new paradigm for supermolecular sssembly and signal transduction. Biochemistry (US) 40, 3009–3015.
  • [2] Jerica Saboti, Simon Zurga&Jure Pohleven(2014) A novel b-trefoil lectin from the parasol mushroom(Macrolepiota procera) is nematotoxic. FEBS Journal(UK)281,3489-3505.