Genetically engineered bacteria for efficiently secreting, expressing and reconstructing cutinase and method for constructing same

A genetically engineered bacteria, secretion and expression technology, applied in the field of bioengineering, can solve the problems of increased host metabolic burden, low expression level of cutinase, etc., and achieve low cost, high recovery rate and obvious effect

Active Publication Date: 2010-08-04
JIANGNAN UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the type I secretion pathway also has two unavoidable disadvantages: first, the signal peptide is not excised during the secretion process, and the recombinant protein is still connected to the signal peptide after being secreted out of the cell, which needs to be excised to obtain the natural protein; second, usually Co-expression of relevant system elements is required to improve secretion efficiency, but this will increase the metabolic burden of the host, often resulting in low expression levels (F.J.M. )
Although there is a large demand for cutinase in the domestic and foreign markets, there is no commercial cutinase product on the market. The main reason is that the expression level of cutinase in the original strain is low, and there is an urgent need for a strain of cutinase high-producing bacteria in the market.

Method used

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  • Genetically engineered bacteria for efficiently secreting, expressing and reconstructing cutinase and method for constructing same
  • Genetically engineered bacteria for efficiently secreting, expressing and reconstructing cutinase and method for constructing same

Examples

Experimental program
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Effect test

Embodiment 1

[0035] Example 1: Extraction of E.coli CFT073 Total DNA

[0036] Cultivate the E.coli CFT073 strain in LB liquid medium (peptone 10g / L, yeast extract 5g / L, NaCl 10g / L) overnight, take 3mL of the bacterial liquid, and centrifuge at 12000rpm for 2min to collect the bacterial cells. Technology Service Co., Ltd. Bacterial Genomic DNA Extraction Kit Method (EZ SpinColumn Bacterial Genomic DNA Isolation Kit UNIQ-10 Column Bacterial Genomic DNA Extraction Kit) to extract the total DNA of E.coli CFT073;

Embodiment 2

[0037] Embodiment 2: Construction of hlyBD / pSTV28 recombinant plasmid

[0038] Primers P1 and P2 were designed according to the gene sequence of hlyBD.

[0039] P1: 5'-CggCgAgCTCggATTCTTgTCATAAAATTg-3'

[0040] P2: 5'-CCACggATCCTTAACgCTCATgTAAAC-3'

[0041] The hlyBD gene was amplified by PCR using the total DNA of E.coli CFT073 as a template and P1 and P2 as primers. The PCR reaction was carried out in a 50 μL system, and the reaction conditions were 30 cycles of denaturation at 94°C for 1 min, followed by denaturation at 94°C for 30 s, annealing at 60°C for 30 s, and extension at 72°C for 4 min. A PCR fragment of 3579bp was amplified and recovered by tapping the gel. The recovered fragment was ligated with the pMD18-Tsimple vector, the ligated product was transformed into Escherichia coli JM109, and the transformed product was coated on an LB plate containing 30 mg / L chloramphenicol. After culturing overnight at 37°C, single clones were selected, inserted into LB liquid m...

Embodiment 3

[0105] Embodiment 3: Construction of Tfu_0883-hlyAs / pET20b (+) recombinant plasmid

[0106] Two pairs of primers P3, P4 and P5, P6 were designed according to the sequences of cutinase and hlyAs.

[0107] P3: 5'-gTAATCCATATggCCAACCCCTACgAgCgC-3'

[0108] P4: 5'-gACTTCCATAggCTAAgAACgggCAggTggAg-3'

[0109] P5: 5'-CTCCACCTgCCCgTTCTTAgCCTATggAAgTC-3'

[0110] P6: 5'-CCgCTCgAgTTATgCTgATgCTgTCAAAg-3'

[0111]Using the Tfu_0883-pET20b(+) plasmid DNA constructed earlier in our laboratory as a template and using P3 and P4 as primers, the cutinase gene was amplified by PCR. The hlyAs gene was amplified by PCR using the total DNA of E.coli CFT073 as a template and P5 and P6 as primers. The PCR reaction was carried out in a 50 μL system, and the reaction conditions were denaturation at 94°C for 1 min, followed by a cycle of denaturation at 94°C for 30 s, annealing at 60°C for 30 s, and extension at 72°C for 1 min and 20 s, respectively. . PCR fragments of 783bp and 180bp were respec...

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Abstract

The invention relates to genetically engineered bacteria for efficiently secreting, expressing and reconstructing cutinase and a method for constructing the same, belonging to the field of the bioengineering technology. The genetically engineered bacteria are escherichia coli BL21 (DE3) which carry two recombinant plasmids, and the recombinant plasmids are respectively plasmid pSTV28 carrying the specific genes in the Alpha-hemolysin A (hly A) pathway and plasmid pET20b(+) containing the cutinase-hly As genes. The method for constructing the genetically engineered bacteria for efficiently secreting, expressing and reconstructing cutinase comprises the following steps: constructing two key recombinant plasmids and transforming the constructed recombinant plasmids in the escherichia coli BL21 (DE3) to obtain the genetically engineered bacteria for efficiently secreting, expressing and reconstructing cutinase. The cutinase is produced by using the genetically engineered bacteria through culturing liquid and inducing and expressing the cutinase. The cutinase Tfu_0883 for thermophilic monospore bacteria of Thermobifida Fusca WSH03-11 is used as the report protein. The shaking flask fermentation shows that the extracellular output of the cutinase is 306U / mL which is 1.7 times of the output of the cutinase which adopts the II-type secreting pathway in the preliminary working process in the research laboratory. The cutinase is secreted and expressed efficiently.

Description

technical field [0001] The invention relates to a genetic engineering bacterium for efficiently secreting and expressing recombinant cutinase and a construction method thereof, belonging to the technical field of bioengineering. Background technique [0002] Cutinase is a multifunctional enzyme that can hydrolyze various soluble esters, emulsified triglycerides, insoluble fatty acid esters, insoluble polymer cutin, etc., and is widely used in detergents, food, agriculture, and chemical industries, especially In the textile industry, it can be used for the biorefining of cotton fiber and the modification of synthetic fiber ethylene terephthalate (PET). At present, the above two processes adopt the traditional alkali treatment process, which has high water consumption and energy consumption. Fiber damage, environmental pollution and other defects, therefore, enzymatic treatment process as an environmentally friendly technology for energy saving and consumption reduction, will ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12N15/70C12N9/16C12R1/19
Inventor 吴敬陈晟宿玲恰陈坚吴丹
Owner JIANGNAN UNIV
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