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Fluorescent clone screening vector and preparation and application thereof

A cloning vector and fluorescent screening technology, applied in the fields of molecular biology and microbiology, which can solve the problem of poor chromophore efficiency

Active Publication Date: 2011-09-14
生工生物工程(上海)股份有限公司
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Problems solved by technology

GFPS65T is a mutant in which Thr replaces Ser65. Under the same conditions, its fluorescence intensity is 4-6 times stronger than that of wtGFP, and its only Redshifted excitation peak is located at 490nm, but the efficiency of chromophore formation at 37°C is not as good as that of EGFP

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  • Fluorescent clone screening vector and preparation and application thereof
  • Fluorescent clone screening vector and preparation and application thereof
  • Fluorescent clone screening vector and preparation and application thereof

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Embodiment 1

[0042] Construction and verification of embodiment 1 fluorescence screening cloning vector

[0043] The design idea and technical route of constructing the fluorescent screening cloning vector are as follows:

[0044] 1. Sequence design of enhanced green fluorescent protein gene. The green fluorescent protein gene in the vector pEGFP-N1 is EGFP, its detailed sequence is SEQ ID NO.2, and the corresponding amino acid sequence is SEQ ID NO.3, see the structure diagram image 3 . The crystal structure of GFP is a columnar structure composed of 11 β-sheets, and the GFP chromophore tyg is tightly packed in the β-box surrounded by β-sheets, which is protected from the surrounding environment. The present invention appropriately modifies the nucleic acid sequence without changing its protein structure, introduces a commonly used enzyme cutting site during the modification process, and modifies 4 amino acids at the same time, namely G20V, V29G, A37L, V55G ( Figure 4 ), the modifie...

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Abstract

The invention relates to the field of molecular biology and microbiology, and discloses a fluorescent screening vector, the construction and application thereof. Polynucleotide and / or a complementary strand of the polynucleotide which encodes an enhanced green fluorescent protein are taken as a positive clone selection marker of the fluorescent screening vector; and an amino acid sequence of the enhanced green fluorescent protein is SEQ ID NO.5. The vector can be widely applied to the screening of positive clone in gene clone.

Description

technical field [0001] The present invention relates to the fields of molecular biology and microbiology. Specifically, the present invention relates to a fluorescent screening carrier, a construction method of the carrier, and an application of the carrier in precisely screening bacterial positive colonies. Background technique [0002] After the 1970s, the emergence of genetic engineering technology as a new milestone marked the beginning of a new era in which human beings have a deep understanding of the essence of life and can transform life. Major achievements during this period include the establishment and development of recombinant DNA technology. Recombinant DNA (Recombinant DNA) technology is the core technology of genetic engineering, and it is also a technology for human beings to operate at the level of genes and DNA molecules. It includes the following steps: 1) Construction of recombinant DNA molecules; 2) Transformation of recombinant DNA molecules into host...

Claims

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Application Information

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IPC IPC(8): C12N15/65C07K14/00C12N15/11C12Q1/04G01N21/64
Inventor 孔凡静王素莲王绪慧
Owner 生工生物工程(上海)股份有限公司
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