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Transglutam kinase coding gene interrupted streptomyces hygroscopicus and application thereof

A technology for encoding transglutaminase and Streptomyces hygroscopicus is applied in the field of genetic engineering to achieve the effect of simple, easy to implement and short-required gene blocking method.

Inactive Publication Date: 2012-07-25
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] There is no report on the function of transglutaminase in Streptomyces and the blockage of transglutaminase gene in Streptomyces hygroscopicus

Method used

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  • Transglutam kinase coding gene interrupted streptomyces hygroscopicus and application thereof
  • Transglutam kinase coding gene interrupted streptomyces hygroscopicus and application thereof
  • Transglutam kinase coding gene interrupted streptomyces hygroscopicus and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Interruption of Transglutaminase Encoding Gene in Streptomyces hygroscopicus in Example 1

[0029] 1. Acquisition of the target fragment gene TG1

[0030] Using primers:

[0031] Y1: 5′-TTTAAGCTT CCACTGTGAGTGCGGCGATA-3′ (underlined, boldface is the Hind III site); Y2: 5′-AAA GAATTC TTCCGTCTGAGCCCCAAACC-3' (underlined, boldface is the Eco RI site) was amplified from the genome of Streptomyces hygroscopicus to obtain the knockout fragment TG1.

[0032] The total volume of the PCR reaction was 25 μL, and 1 μL of Streptomyces hygroscopicus total DNA was used as a template for the reaction. The reaction conditions are: 95°C for 5 min; 95°C for 1 min, 65°C for 1 min, 72°C for 10 min, 30 cycles. The PCR product was a fragment of the MTG gene, named TG1, and verified by sequencing.

[0033] 2. Blocking vector construction

[0034] Digest bifunctional thermosensitive shuttle plasmid pKC1139 (6.5kb) with Eco RI and Hind III (Hopwood D, Bibb M, Chater KF e a. Genetic manipul...

Embodiment 2

[0057] Example 2. Verification of Streptomyces hygroscopicus transglutaminase gene function

[0058] 1. Inoculate recombinant strains for liquid medium and solid culture of Streptomyces hygroscopicus, and detect enzyme activity at different time periods. Wild strains can detect MTG enzyme activity, but recombinants cannot detect MTG enzyme activity during solid and liquid culture. see results image 3 .

[0059] 2. S.h-ΔTG was cultured on a solid plate, and the wild strain was used as a control, and the wild strain and the recombinant were inoculated to different areas of the same plate ( Figure 4 ), cultured at 32°C. During the early growth process of the bacteria, the hyphae in the substrate could grow normally. At 48 hours, the wild strain had completely differentiated to form aerial hyphae, while the recombinant S.h-ΔTG was still in the state of vegetative mycelium growth, and no aerial mycelium was formed. After continuing to cultivate for 120 hours, the wild strain S...

Embodiment 3

[0060] Example 3. Transglutaminase gene blocking Streptomyces hygroscopicus is used to express and produce transglutaminase

[0061] 1. The TGase gene fragment and the signal peptide fragment on the vector pIB13 vector and the expression vector pIJ8600 (Hopwood D, Bibb M, Chater KF e a. Genetic manipulation of Streptomyces[M]. A Laboratory Manual. In: Norwich: John Innes Foundation Press; 2000) respectively carry out Nde I and Xba I double enzyme digestion, after recovery, use T4 ligase to connect Figure 5 . The ligation reaction system is (10 μL): target gene fragment 2 μL, carrier DNA 2 μL, 10×T4 ligase Buffer 1 μL, T4 DNA ligase 1 μL, ddH 2 O 4 μL.

[0062] The ligation product was transformed into competent Escherichia coli JM109 for transformation. The conversion method is as follows:

[0063] (1) Under sterile conditions, take 200 μL of competent cells and place them in a sterile microcentrifuge tube;

[0064] (2) Add 1-2 μL of recombinant plasmid to each tube, rot...

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Abstract

The invention discloses a transglutam kinase coding gene interrupted streptomyces hygroscopicus and an application thereof. The invention mainly adopts a homologous recombination technique, and makes transglutam kinase coding genes subjected to insertional inactivation through single exchange so as to obtain recon not producing transglutam kinase. The differentiation of the streptomyces hygroscopicus obtained by the invention is interrupted without influencing growth of substrate myceliums. A genetically engineered microorganism constructed by using the interrupted streptomyces hygroscopicus as a host is used for producing the transglutam kinase. The interrupted streptomyces hygroscopicus can be used as a good streptomyces hygroscopicus expressing host. Metabolin changes caused by the differentiation process in the ferment process of streptomyces hygroscopicus can be solved, the ferment production quantity and stability can be improved, subsequent streptomyces hygroscopicus gene change and microorganism physiology study can be greatly promoted, and a new type strain is provided for production and ferment of streptomyces hygroscopicus.

Description

technical field [0001] The invention relates to a transglutaminase encoding gene blocking Streptomyces hygroscopicus and application thereof, belonging to the field of genetic engineering. Background technique [0002] Transglutaminase (protein-glutamic acid-transglutaminase, transglutaminase, EC2.3.2.13, TGase for short) is a transferase that catalyzes acyl transfer reaction. It can catalyze the intramolecular and intermolecular crosslinking of proteins, the connection between proteins and amino acids, and the hydrolysis of glutamine groups in protein molecules, thereby directly changing the structure and function of the protein itself and the cells and tissues to which the protein is attached. properties of nature. Therefore, MTG has broad application prospects in food, textile, biopharmaceutical and other fields. Transglutaminase exists in animals, plants, and microorganisms. At present, the strain for industrial production of transglutaminase is Streptomyces. [0003]...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/20C12N15/76C12P19/00C12P1/06C12R1/55
Inventor 陈坚陈康康张东旭马腾博堵国成
Owner JIANGNAN UNIV
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