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Method of breaking wall of bacteriophage expressing lytic gene under control of magnesium ion

A technology for cracking genes and phages, which is applied in the field of genetic engineering, can solve the problems of destroying the target product, time-consuming and labor-intensive, etc., and achieve the effect of effective self-cleavage

Active Publication Date: 2007-11-07
YANGZHOU UNIV
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Problems solved by technology

Prokaryotic expression is usually intracellular expression, which requires mechanical breaking or the use of strong surfactants to release the expression product, which is time-consuming and laborious and may destroy the target product

Method used

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  • Method of breaking wall of bacteriophage expressing lytic gene under control of magnesium ion
  • Method of breaking wall of bacteriophage expressing lytic gene under control of magnesium ion
  • Method of breaking wall of bacteriophage expressing lytic gene under control of magnesium ion

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Embodiment 1

[0023] Embodiment 1: Escherichia coli induced lysis test

[0024] Cloning of cleavage genes and construction of plasmids

[0025] The construction of pYS can refer to the previous Chinese invention patent application "Expression Vector Regulated by Magnesium Ion" with the application number of 200710020573.0: using primers P1 and P2 to amplify a fragment of about 600 bp from the genome of Salmonella typhimurium X4550, and link it into pGEM- T vector, confirmed by sequencing to be the same as the P of Salmonella typhimurium in GenBank mgt [Serial Number: M57715] Exactly the same. P was then excised with restriction enzymes Bst1107 I and Xba I mgt Load the pET-30a(+) vector with the same digestion, and the new plasmid is named pYS (Figure 1), which removes the lacI gene and T7 promoter sequence in the original vector.

[0026] (P1: 5'- GTATAC TCCGGAGCAAACGCCTGAACTCCC-3'[Bst1107I],

[0027] P2: 5'- AGATCT GGAAGAATAAGTACGTGCTATATTTAG-3'[Xba I])

[0028] Using primers P3 an...

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Abstract

The present invention relates to gene engineering, and is especially method of expressing bacteriophage lytic gene under the control of magnesium ion. The method features that bacteriophage lytic gene is first cloned to the expression vector pYS controlled by magnesium ion and then transformed into the host bacteria, and the magnesium ion concentration is then controlled for the lytic gene to be expressed in the host bacteria. The present invention makes it possible to express lytic gene in colibacills for the host bacteria to self crack effectively under mild condition.

Description

technical field [0001] The invention relates to the field of genetic engineering, in particular to a method for breaking walls of expressing phage lysing genes under the regulation of magnesium ions. Background technique [0002] The prokaryotic expression system of Escherichia coli is currently the most widely used protein expression method in genetic engineering research. It has the characteristics of simple gene manipulation, wide selection of host strains and plasmids, and easy growth and control of bacteria. Difficult purification of expression products is a major difficulty in prokaryotic expression systems. Prokaryotic expression is usually intracellular expression, which requires mechanical breaking or the use of strong surfactants to release the expression product, which is time-consuming and laborious and may destroy the target product. Some reports use the method of expressing the lytic gene of bacteriophage to lyse the host bacteria. Resch et al. used thermal c...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N15/33
Inventor 焦新安张晓明潘志明黄金林孙林殷月兰唐丽华刘秀梵
Owner YANGZHOU UNIV
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