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Method of double crossover homologous recombination in clostridia

A technique of homologous recombination and double crossover, applied in other methods of inserting foreign genetic material, recombinant DNA technology, biochemical equipment and methods, etc.

Inactive Publication Date: 2012-02-22
UNIVERSITY OF NOTTINGHAM
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The disadvantage of starting with a mutant strain is the labor involved in generating the initial "starter strain"
Another disadvantage is that the technician must compare the single mutant (parent) strain to the double mutant (offspring) strain in any experiment performed and then have to extrapolate back to glean the significance of the original wild type (clinical) strain

Method used

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  • Method of double crossover homologous recombination in clostridia
  • Method of double crossover homologous recombination in clostridia
  • Method of double crossover homologous recombination in clostridia

Examples

Experimental program
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Embodiment Construction

[0081] Construction of codA expression plasmid

[0082] The codA expression cassette was isolated from the vector used by Fox et al., Gene Therapy. (1996) 3: 173-178 and cloned into pMTL960. This plasmid was used to test the function of the codA gene in E. coli and C. difficile. In E. coli, the construct worked as expected, allowing growth in the absence of FC but not in the presence of FC. Similarly, as in Figure 4 It was shown that when transformed into Clostridium difficile and grown on minimal medium containing 100 μg / ml FC (modified from the recipe described by Karlsson et al. (1999) Microbiology 145:1683-1693), codA mooring The Clostridium difficile cells of the box can be distinguished. That is, cells expressing codA did not grow.

[0083] The medium described by Karlsson et al. contains:

[0084]

[0085] * Amino acids in minimally defined medium (MDM)

[0086] ** Amino acids are added a little more to give supplemental defined medium (SDM)

[0087] *** Ami...

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Abstract

The invention relates to a method of double crossover homologous recombination in a host Clostridia cell comprising: a first homologous recombination event between a donor DNA molecule and DNA of the host cell to form a product of the first recombination event in the host cell, wherein the donor DNA molecule comprises a codA gene and at least two homology arms; and a second recombination event within the product of the first homologous recombination event, thereby to form a product of the second homologous recombination event in the host cell which is selectable by the loss of the codA gene; and a related vector and altered host cell.

Description

technical field [0001] The present invention relates to methods for modifying the nucleic acid of Clostridium cells, in particular by utilizing double crossover homologous recombination. Background technique [0002] The ability to be effective and There is a requirement to reproducibly manipulate microbial genomes. For example, microbial genome-specific alterations can be made to remove ("knock-out") or alter endogenous cellular functions, or to expand function by adding one or more exogenous activities ("knock-ins"). [0003] This host cell recombination machinery is often critical during genome manipulation. Essentially, as long as two independent DNA molecules in a cell share a region of common DNA sequence (ie, a region of homology), they can "recombine" through a Campbell-like mechanism to form a single DNA molecule. Thus, an extrachromosomal element (eg, a plasmid) introduced into a target host organism can "integrate" into the host cell genome to generate a "singl...

Claims

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Application Information

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IPC IPC(8): C12N15/74C12N15/90
CPCC12N15/74C12N15/902
Inventor 斯蒂芬·托马斯·卡特曼纳杰尔·彼得·明顿
Owner UNIVERSITY OF NOTTINGHAM
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