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Recombinant vector, expression vector, genetically engineered bacterium and application thereof

A technology of genetically engineered bacteria and recombinant vectors, applied in the field of recombinant vectors, can solve the problems of difficult large-scale screening of regulatory proteins and high technical requirements, and achieve the effects of short test period, simple equipment and experimental conditions, and low cost

Active Publication Date: 2021-11-09
SHANXI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At the same time, since channel proteins and regulatory proteins can only be expressed one-to-one in the same Xenopus egg, it is difficult to use for large-scale screening of regulatory proteins that potentially affect channel protein activity due to high experimental equipment and technical requirements.

Method used

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  • Recombinant vector, expression vector, genetically engineered bacterium and application thereof
  • Recombinant vector, expression vector, genetically engineered bacterium and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0028] Example 1: Preparation of pKY2in1-Dest recombinant vector

[0029] The pPrey-PartnerSUS-2in1-Dest vector is used to determine the three-protein interaction in yeast, and has nothing to do with the function of the present invention. This vector only provides the background backbone for vector screening with Amp resistance and Leu synthesis ability of the pKY2in1-Dest vector of the present invention. Artificially synthesized DNA sequence SEQ ID NO.1 (Shanghai Sangong), including the pmet25-attR1-CmR-ccdb-attR4-Myc-ptdh3-HA-attR3-LacZ-attR2 component, retaining NruI and NdeI restriction sites at both ends of the sequence . Promoter is an important element of yeast expression regulation, and its activity can significantly affect the initiation, duration and expression level of heterologous gene transcription. The pTDH3 selected in the present invention is the yeast glyceraldehyde-3-phosphate dehydrogenase 3 promoter, which is a constitutive promoter; pmet25 is an ATP sulfu...

Embodiment 2

[0030] Example 2: Using the pKY2in1-Dest vector to construct VAMP721-KAT1, SYP121-KAT1 and VAMP727-KAT1 clones and transform yeast

[0031] 1. Construction of expression vector

[0032]SYP121, VAMP721 and VAMP727 are Arabidopsis SNARE family membrane transporters, and KAT1 is a potassium ion channel in Arabidopsis. pDONR221-L3L2-KAT1, pDONR221-L1L4-SYP121, pDONR221-L1L4-VAMP721, and pDONR221- L1L4-VAMP727 Gateway entry clone. The pKY2in1-Dest recombinant vector and the above entry clone DNA were purified by PureLinkTM PCR Purification Kit (Thermo) and diluted to a concentration of 150ng / ul. Take 1ul pKY2in1-Dest recombinant vector, according to the number of DNA molecules as the carrier: KAT1 entry clone: ​​SNARE entry clone = 1:3:3 to configure a 2in1 LR reaction system, add 1ul of LR enzyme, mix well and overnight at room temperature, transform Trans10 (full format Gold) Competent cells, spread on LB plates containing Amp at 37°C overnight, pick single clones and culture t...

Embodiment 3

[0039] Example 3: Exploring the effects of SYP121, VAMP721 and VAMP727 on KAT1 potassium uptake function using the genetically engineered bacteria of Example 2

[0040] 1. pick the yeast colony that example 2 obtains, in the CSM containing potassium ion 100mM -L Amino acid deficient medium (CSM -L , 100mM K), culture overnight at 28°C, 150rpm.

[0041] 2. Reserve 750ul of the overnight yeast culture product, mix it with 250ul 60% glycerol, freeze it in liquid nitrogen, and store it at -80°C.

[0042] 3. Centrifuge at 3000rpm for 1min at room temperature to harvest yeast cells and resuspend in CSM -L 100mM K,ddH 2 O diluted to OD600 1.0 and 0.1.

[0043] 4. In CSM containing different concentrations of potassium ions (1, 5, 10, 100mM) and different concentrations of methionine (0.5 and 500mM) -L Add 4ul of the bacterial solution obtained in the previous step to the solid plate dropwise, and incubate at 28°C for 24-72 hours. Yeast growth is checked and documented by takin...

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Abstract

The invention relates to the technical field of potassium ion channel regulatory proteins, in particular to a recombinant vector, an expression vector, a genetically engineered bacterium and application thereof. The recombinant vector is obtained by inserting a DNA (Deoxyribose Nucleic Acid) sequence as shown in SEQ ID NO.1 between NruI and NdeI sites of a pPrey-PartnerSUS-2in1-Dest vector, and is named as pKY2in1-Dest, and the expression vector is obtained by transferring a potassium channel gene and other regulatory protein genes into the pKY2in1-Dest in one step through Gateway cloning reaction. The genetically engineered bacterium is obtained by transferring the expression vector into a recipient bacterium. The genetically engineered bacterium can be applied to large-scale screening of regulatory proteins affecting the potassium absorption function of the potassium ion channel, influences on the potassium channel function are directly shown by observing yeast growth under a low-potassium condition, and the genetically engineered bacterium has the advantages of being simple in condition, low in cost and short in experimental period.

Description

technical field [0001] The invention relates to the technical field of potassium ion channel regulation proteins, in particular to a recombinant vector, an expression vector, genetically engineered bacteria and applications thereof. Background technique [0002] Potassium ion channels play a key role in plant potassium uptake and osmotic pressure balance. The regulation of potassium ion channels is the basis for plants to absorb potassium to maintain growth and respond to external stress. Potassium ion channels function, mainly relying on changes in the structure of channel proteins under changes in membrane potential, allowing potassium ions to pass through. This channel protein is influenced by changes in membrane potential to allow potassium to pass through a property known as channel gating. [0003] Potassium ion channel regulation, including the regulation starting from the number of effective proteins (such as the regulation of protein transcription and expression l...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/19C12N15/12C12N15/81C12Q1/04C12R1/865
CPCC12N15/81C07K14/463C12Q1/04
Inventor 张犇郭悦王慧
Owner SHANXI UNIV
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