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Magnesium ion-regulating new expression vector

A technology of expressing vectors and magnesium ions, applied in the field of genetic engineering

Active Publication Date: 2007-10-03
YANGZHOU UNIV
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  • Abstract
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Problems solved by technology

The pET series is a commonly used prokaryotic expression system. It controls the activity of the T7 promoter by regulating the expression of T7 RNA polymerase. This system has a strong transcription function, but at the same time, it needs to add an exogenous inducer, and it is easy to form high-efficiency expression in a short time. Disadvantages of inclusion bodies

Method used

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Examples

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Embodiment 1

[0019] Example 1: Salmonella typhimurium P mgt Cloning of and its induction activity in Escherichia coli

[0020] Plasmid construction

[0021] Using primers P1 and P2, a fragment of about 600 bp was amplified from the genome of Salmonella typhimurium X4550, connected into the pGEM-T vector, and sequenced confirmed that it was identical to the P gene 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, which removes the lac I gene and T7 promoter sequence in the original vector. Use Nco I and EcoR I to cut out the DsRed gene on the plasmid pDsRed-Express and load it into the corresponding site of pYS to construct pYS-DsRed (Figure 1). (P1: 5'- GTATAC TCCGGAGCAAACGCCTGAACTCCC-3'[Bst1107I], P2:5'- AGATCT GGAAGAATAAGTACGTGCTATATTTAG-3'[Xba I])

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Abstract

The present invention relates to gene engineering, and is especially magnesium ion regulated new expression vector. The magnesium ion regulated new expression vector pYS contains the promoter sequence Pmgt of magnesium ion transport protein gene mgtC / B. The expression vector of the present invention is controlled by Mg2+ concentration in the culture medium and has no need of adding exogenous inducer. The present invention provides new way for the improvement and development of colibacillus pronucleus expression system.

Description

technical field [0001] The invention relates to the field of genetic engineering, in particular to an expression carrier regulated by 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. The pET series is a commonly used prokaryotic expression system. It controls the activity of the T7 promoter by regulating the expression of T7 RNA polymerase. This system has a strong transcription function, but at the same time, it needs to add an exogenous inducer, and it is easy to form high-efficiency expression in a short time. Disadvantages of inclusion bodies. This problem also exists in other prokaryotic expression systems. Therefore, a prokaryotic vector capable of self-induced expression of the target ...

Claims

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Application Information

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IPC IPC(8): C12N15/63C12N15/70
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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