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Novel method to isolate mutants and to clone the complementing gene

A target gene and amino acid technology, applied in the field of microbial mutation and screening of mutants, can solve complex time-consuming, non-specific binding and other problems

Inactive Publication Date: 2005-08-31
DUPONT NUTRITION BIOSCIENCES APS
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0009] - by purifying the regulatory protein, which is complex and time-consuming as the protein can only be identified by its DNA-binding properties
Some purification methods involve affinity chromatography using the bound DNA fragments as a matrix, a disadvantage of this method of purification is that usually more than one protein binds to said fragments both specifically and non-specifically

Method used

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  • Novel method to isolate mutants and to clone the complementing gene
  • Novel method to isolate mutants and to clone the complementing gene
  • Novel method to isolate mutants and to clone the complementing gene

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0120] Example 1: Construction of plasmids

Embodiment 11

[0121] Example 1.1: Construction of selection plasmid pIM130

[0122] Such as figure 1 Plasmid pIM130 was selected for the construction shown in PCR1 with oligonucleotide 1 (SEQ ID NO: 1) 5'-CACAATGCATCCCCCTTTATCCGCCTGCCGT-3' (Formula 1) and oligonucleotide A (SEQ ID NO: 2) 5'-CAATTGCGACTTGGAGGACATGATGGGCAGATGAGGG -3' (Formula 2) generates a fragment from pIM120 (de Graaff et al., 1994). Oligonucleotide 1 was derived from positions 600-619 (SEQ ID NO: 5) of the Aspergillus tubingensis xlnA promoter (de Graaff et al., 1994) to which 10 nucleotides containing an NsiI site were added. The 3' end of oligonucleotide A was derived from the Aspergillus niger goxC transcription unit (Whittington et al., 1990) (positions 708-723) (SEQ ID NO: 6) ending before the translation initiation site, while its 5' end Derived from the coding region of the A. niger pyrA gene from the translation initiation site (Wilson et al., 1988) (positions 341-359, SEQ ID NO: 7).

[0123] By containing 10μl...

Embodiment 12

[0132] Example 1.2: Construction of plasmid pIM135

[0133] A second plasmid containing the goxC basic transcription unit fused to the pyrA coding and termination regions was constructed from plasmid pIM120. A fragment was generated from plasmid pIM120 in PCR4 using oligonucleotide 3 and oligonucleotide 2 (Formula 3) whose sequence was 5'-CACAATGCATCGTATAAGTAACCTCGTTCG-3' (Formula 5) derived from From the goxC basic transcription unit (positions 640-660, SEQ ID NO: 6), and 10 nucleotides containing an NsiI site were added. The resulting fragment was isolated from the gel as described in Example 1.1, digested with NsiI and EcoRV and cloned together with the 2.2 kb EcoRV / XbaI fragment of pGW635 into the XbaI / NsiI digested plasmid pGEM-7Zf(+) to give the plasmid pIM135.

[0134] Plasmid pIM135 can be used as a construction vector to prepare the UAS vector carrying any desired inducible enhancer sequence or activator sequence, genes involved in metabolism of the present inventio...

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Abstract

The subject invention lies in the field of microorganism mutation and selection of the mutants. In particular, the invention is directed at obtaining metabolic mutants in a simple, direct and specific manner. In a preferred embodiment it is also possible to obtain desired mutants not comprising recombinant DNA, thereby facilitating incorporation thereof in products for human consumption or application, due to shorter legislative procedures. The method according to the invention involves random mutation and specific selection of the desired metabolic mutant. A nucleic acid cassette comprising a nucleic acid sequence encoding a bidirectional marker, said nucleic acid cassette further comprising a basic transcriptional unit operatively linked to the nucleic acid sequence encoding the bidirectional marker and said nucleic acid cassette further comprising an inducible enhancer or activator sequence linked to the basic transcription unit in such a manner that upon induction of the enhancer or activator sequence the bidirectional marker encoding nucleic acid sequence is expressed, said inducible enhancer or activator sequence being derived from a gene associated with activity of part of metabolism, said inducible enhancer.or activator sequence being derived from a gene associated with metabolism is claimed as is application thereof in a selection method for mutants. In addition a regulator gene xlnR encoding an activating regulator of an inducible enhancer or activator sequence and application of said gene and / or its expression product in overexpression of homologous or heterologous protein or peptide is described. Knockout mutants wherein said gene is absent or inactivated and mutants with increased or decreased DNA binding capacity are also claimed.

Description

[0001] This application is a divisional application of the Chinese invention patent application with the application number 96196200.3, the application date is June 24, 1996, and the invention title is "New method for isolating mutants and cloning complementary genes". technical field [0002] The present invention belongs to the field of microbial mutation and screening mutants. Specifically, the present invention relates to a nucleic acid sequence encoding an expression product xylR or an equivalent nucleic acid sequence thereof, and an amino acid sequence encoded by the nucleic acid sequence or an equivalent thereof. The present invention also relates to obtaining metabolic mutants in a simple, direct and specific manner. Background technique [0003] The current industrially applicable and automated method for preparing mutants is a mutation method without a screening process and subsequent analysis of mutants, which needs to be improved. An alternative approach with a s...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/09C07K14/38C07K14/47C12N1/15C12N1/19C12N1/21C12N9/04C12N9/24C12N9/88C12N15/10C12N15/31C12N15/63C12N15/65C12N15/80C12Q1/02C12Q1/68
CPCC12Y302/01003C12Y302/01099C12Y302/01055C12Y302/01037C12N15/65C12Q1/02C12N9/24C12N15/1027C12N9/248C12N15/635C12N15/63C12Y302/01015C12N9/88C07K14/38C12N9/2434C07K14/4705C12N15/80C12N9/0006C12Y302/01008
Inventor 伦德特·亨德里克·德格拉夫亨丽埃特·凯瑟琳娜·范登布勒克雅克·菲瑟
Owner DUPONT NUTRITION BIOSCIENCES APS
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