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Application of heat shock protein gene in construction of high-heat-tolerance escherichia coli

A heat shock protein, Escherichia coli technology, applied in the field of genetic engineering, can solve the problems of increasing the cost of ethanol production, low microbial fermentation temperature and the like

Inactive Publication Date: 2015-11-25
SHANGHAI ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Many microorganisms can be used to produce ethanol, they are very important microbial cell factories in ethanol fermentation production, but the traditional microbial fermentation temperature is relatively low, (for example, the optimum fermentation temperature of Saccharomyces cerevisiae is 28~33°C, generally not exceeding 36°C ), which is a big bottleneck restricting ethanol fermentation production, which will greatly increase the cost of ethanol production

Method used

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  • Application of heat shock protein gene in construction of high-heat-tolerance escherichia coli
  • Application of heat shock protein gene in construction of high-heat-tolerance escherichia coli
  • Application of heat shock protein gene in construction of high-heat-tolerance escherichia coli

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0019] Example 1 Extreme high temperature resistance gene PhHSPI Synthetic

[0020] According to the PTDS (PCR-based two-step DNA synthesis, PTDS) method, the heat shock protein gene derived from the extreme thermophilic archaea (Pyrococcushorikoshii) was cloned PhHSP , for chemical synthesis, while maintaining the heat shock protein gene PhHSP On the basis of the unchanged amino acid sequence, design primers to synthesize the heat shock protein gene of the present invention PhHSPI , the designed primers are as follows:

[0021] 1. PHHSPI-1: Tm=54,60mer

[0022] ATG,CGT,GGT,GGT,GAA,CGT,ATG,GTT,CGT,CGT,CGT,CGT,TGG,GAC,ATC,TGG,GAC,CCA,TTC,GAC

[0023] 2. PHHSPI-2: Tm=54,60mer

[0024] CCC,ATT,CGA,CTT,GAT,TCG,TGA,AAT,CCA,AGA,AGA,GAT,CGA,TGC,CAT,GTT,CGA,CGA,GTT,CTT,C

[0025] 3. PHHSPI-3: Tm=54,60mer

[0026] GAC,GAG,TTC,TTC,AGT,CGT,CCA,CGT,CTC,TGG,ACC,TAT,CGT,CGT,TGG,AAG,GAA,CCA,GAA,CTC,TAC,GAA,G

[0027] 4.PHHSPI-4:Tm=54,60mer

[0028] GAA,CTC,TAC,GAA,GAG,GGA,ACA,GGT,...

Embodiment 2

[0043] Example 2 Synthesis PhHSPI genetic DNA-shuffling mutation

[0044] 1) Through the method of multi-site site-directed mutagenesis, eliminate PhHSPI in the gene Bam H I and Sac I and other restriction endonuclease sites.

[0045] 2) According to PhHSPI Determine the sequence and design primers at both ends, at both ends of the gene Bam HI and Sac I increase the enzyme cutting site to PhHSPI The gene is used as a template for PCR amplification, and the kit recovers the amplified fragment.

[0046] 3) The products degraded with DNaseI were separated by 12% polyacrylamide electrophoresis, and small fragments below 100 bp were recovered by dialysis bag method.

[0047] 4) Perform primerless PCR (Primerless PCR) using the recovered small fragments as templates. After 50 cycles, the amplified products are detected by 1.0% Agrose electrophoresis.

[0048] 5) According to the results of PrimelessPCR electrophoresis, take the amplified product of PrimelessPCR, add...

Embodiment 3

[0051] In vitro directed molecular evolution of embodiment 3 mutant genes

[0052] 1) Preparation of Escherichia coli E. coli EG50 chemically competent, the method refers to the molecular cloning experiment guide (Rainerie et al., 1990).

[0053] 2) Take 50 μL of Escherichia coli EG50 competent cells, add 1 μL of mutant DNA, mix thoroughly, and spread the mixture on a 2YT plate containing ampicillin after 20 minutes, restore the culture for 1 hour (37°C), and then place the plate at 55°C Grow in the incubator for 12 h.

[0054] 3) Pick high-temperature-resistant clones from the bacteria grown on the 2YT plate in step 2, and send them to Shanghai Sunny Company for sequencing after enzyme digestion and identification.

[0055] 4) Through sequencing, compare the high temperature resistant mutant gene with the original synthetic gene sequence, and obtain the gene of the high temperature resistant related mutation site PhHSPS .

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Abstract

The invention discloses a pyrococcus horikoshii heat shock protein gene (PhHSPS) with high heat tolerance. The nucleotide sequence of the mutant PhHSPS with high heat tolerance is shown as SEQ ID NO 1, and the coding amino acid sequence of the mutant PhHSPS with high heat tolerance is shown as SEQ ID NO2. In process of transforming mutant PhHSPS obtained by means of molecular evolution into escherichia coli, heat tolerance capacity of escherichia coli can be improved greatly by means of the mutant PhHSPS, and accordingly, the mutant PhHSPS can be applied to construction of bacterial strains of high heat tolerance.

Description

technical field [0001] The invention belongs to the field of genetic engineering, and specifically relates to an extreme thermophilic archaea (Pyrococcushorikoshii) heat shock protein gene PhHSPS and its application through directional molecular evolution in vitro, in particular to the application of the gene in the construction of high-temperature-resistant bacterial strains. Background technique [0002] Heat shock protein (Heat Shock Protein, HSP) is a protein synthesized by organisms in response to physical, chemical, biological, mental and other stimuli in the environment, also known as stress protein (Stress Protein, SP), is a highly conserved protein , are ubiquitous in prokaryotes and eukaryotes. The earliest report on HSP was found in 1962. Ritossa observed that the feeding temperature of Drosophila larvae was increased from 25°C to °C. After 30 minutes, special "bulges" appeared on the salivary gland chromosomes. Later studies found that the generation of this bul...

Claims

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Application Information

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IPC IPC(8): C12N15/31C07K14/195C12N15/70C12N1/21C12R1/19C12R1/01
Inventor 高建杰姚泉洪彭日荷
Owner SHANGHAI ACAD OF AGRI SCI