Difference between revisions of "Part:BBa K1660001"

Revision as of 12:34, 17 September 2015 (view source) BNU daisy (Talk | contribs) ← Older edit		Revision as of 12:37, 17 September 2015 (view source) BNU daisy (Talk | contribs) Newer edit →
Line 27:		Line 27:
	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.		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.
	<br/>		<br/>
−	[[File:BNU-RMpL_Fig_1A.jpg]]	+	<img src="https://static.igem.org/mediawiki/parts/0/0c/BNU-RMpL_Fig_1A.jpg">
	<h2>Function</h2>		<h2>Function</h2>
	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.		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.

---

Revision as of 12:37, 17 September 2015

rMpL

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

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.

Function

According to relevant research, Bace16 is the key reason for the high infection toxicity of Bacillus nematocida to Panagrellus redivivus and Bursaphelenchus xylophilus. 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^[4]. Researchers found that recombinant protease rm-Bace16(whose molecular weight is 34kDa) expressed in Escherichia coli 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.

Design

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 E. coli strain BW25113 to express and purify Bace16 protein. Then nematocidal activity test is conducted.

Reference

• [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.
• [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.
• [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.
• [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.