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Construction method and application of engineering bacteria by surface display of black nightshade herb metallothionein

A metallothionein, surface display technology, applied in the field of engineering strain construction, can solve the problems of high cost, weak, incomplete removal, etc., and achieve the effect of improving cadmium tolerance and strong adsorption capacity

Inactive Publication Date: 2015-12-23
QUFU NORMAL UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0004] In order to solve the disadvantages of the physical and chemical methods commonly used in the prior art to remove heavy metals in the environment, such as high cost, incomplete removal, secondary pollution, and low specificity, the present invention provides a dragon that uses microorganisms to adsorb cadmium. Construction method of sunflower metallothionein surface display engineering bacteria

Method used

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  • Construction method and application of engineering bacteria by surface display of black nightshade herb metallothionein
  • Construction method and application of engineering bacteria by surface display of black nightshade herb metallothionein
  • Construction method and application of engineering bacteria by surface display of black nightshade herb metallothionein

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Embodiment 1

[0047] 1. Acquisition of Solanum vulgare metallothionein gene

[0048] 1. Extraction of Solanum RNA

[0049] Take 50-100 mg of plant tissues, grind them into powder in liquid nitrogen, and extract the total RNA of nightshade according to the operating instructions of RNAisoPlus, an RNA extraction kit purchased from Takara.

[0050] 2. cDNA synthesis

[0051] Using the total RNA of nightshade as a template, cDNA was synthesized according to the following reaction system

[0052] 5×ReverseTranscriptaseBuffer: 4μL

[0053] dNTPmix(10mM): 2μL

[0054] RNaseinhibitor (40U / μL): 0.5μL

[0055] Oligo(dT)primer: 0.5μL

[0056] AMVReverseTranscriptase: 2μL

[0057] RNAtemplet: 2μL

[0058] RNase-freeddH 2 O: dilute to 20μL

[0059] The reaction mixture was heated in a 37°C water bath for 60 minutes, heated at 70°C for 10 minutes to complete the reaction, and placed on ice for subsequent experiments or frozen.

[0060] 3. Acquisition of metallothionein gene

[0061] Using cDNA as a template, the metallothi...

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Abstract

The invention relates to the technical field of construction of engineering strains, and in particular relates to a construction method of engineering bacteria by surface display of black nightshade herb metallothionein. The construction method comprises the following steps: extracting a black nightshade herb metallothionein gene; taking pYES2 as a starting vector; replacing an inducible promoter on the vector with a constitutive expression promoter; separately connecting a secretion signal peptide gene and an anchoring protein agglutinin gene of saccharomyces cerevisiae; connecting the black nightshade herb metallothionein gene between the secretion signal peptide gene and the agglutinin gene; transferring an expression vector into escherichia coli; extracting vector plasmids; and transferring into the saccharomyces cerevisiae. The engineering bacteria by surface display of black nightshade herb metallothionein, constructed by the construction method provided by the invention, has stronger adsorptivity to Cd<2+>, the cadmium tolerance is also promoted significantly, and the capability of adsorbing ultra-trace heavy metals is significantly higher than that of wild yeast strains and control strains; the influence caused by pH values is small; and the engineering bacteria can adsorb a heavy metal namely cadmium in the coexistence of Cd<2+> and Cu<2+>, or Cd<2+> and Hg<2+>.

Description

Technical field [0001] The present invention relates to the technical field of engineering strain construction, in particular to a construction method for the surface display engineering bacteria of Solanum vulgare metallothionein, and also relates to the use of the surface display engineering bacteria of Solanum vulgare metallothionein. Background technique [0002] With the rapid development of industry, various heavy metal ions are released into the environment through various industrial channels. Due to the strong toxicity, non-degradability and enrichment of heavy metals in the food chain, the environmental pollution of heavy metals has received more and more attention (NriaguandPacyna1988; DhankharandHooda2011). Moreover, excessive intake of heavy metals can cause various hazards to human health (Wuetal. 2004; Batayneh 2012). Many methods of processing heavy metals, such as ion conversion, physical infiltration, chemical precipitation, activated carbon adsorption, electroc...

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Application Information

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IPC IPC(8): C12N15/81C12N1/19C02F3/34C12R1/865C02F101/20
Inventor 张洪海韦钦国郭东革马士胜田李
Owner QUFU NORMAL UNIV
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