Rsf1010 derivative mob' plasmid containing no antibiotic resistance gene, bacterium comprising the plasmid and method for producing useful metabolites

1. The technology of RSF1010 and metabolites, applied in the field of mutant vectors, can solve the problems of restricting recombinant strains, not describing the stability of RSF1010 and its derivatives, and not being able to identify plasmid stability determinants, etc.

Inactive Publication Date: 2007-05-30
AJINOMOTO CO INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] No studies on the stability of RSF1010 and its derivatives have been described to date
In addition, although the sequence of RSF1010 is known, it has not been possible to identify the plasmid stability determinant, either by functional or molecular analysis
[0007] Biosafety constraints greatly limit recombinant strains biologically

Method used

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  • Rsf1010 derivative mob' plasmid containing no antibiotic resistance gene, bacterium comprising the plasmid and method for producing useful metabolites
  • Rsf1010 derivative mob' plasmid containing no antibiotic resistance gene, bacterium comprising the plasmid and method for producing useful metabolites
  • Rsf1010 derivative mob' plasmid containing no antibiotic resistance gene, bacterium comprising the plasmid and method for producing useful metabolites

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0079] Example 1. RSF1010mob - Plasmid construction

[0080] By including the autonomous regulatory element P lacUV5 - DNA fragment "Red-driven integration" of lacI (Datsenko K.A. and Wanner B.L., Proc. Natl. Acad. Sci. USA, 2000, 97:12:6640-45) into the RSF1010 plasmid to replace the mob locus, to Conduct RSF1010Mob - Construction of the plasmid, the autonomous regulatory element P lacUV5-lacI is marked by the chloramphenicol resistance gene (cat gene).

[0081] First, with primers P1 (SEQ ID NO: 29) and P2 (SEQ ID NO: 30) and pMW-P lacUV5 - lacI-118 plasmid (Skorokhodova, A.Y. et al., Biotechnologiya (rus), No.5, (2004)) was used as a template to amplify P lacUV5 DNA fragment with the structural part of the lacI gene under the control of the promoter. P lacUV5 The nucleotide sequence of the promoter is disclosed in Genbank Accession No. Y00412 (nucleotides 7-100). The nucleotide sequence of lacI is disclosed in Genbank Accession No. NP_414879. In addition, the P use...

Embodiment 2

[0096] Example 2. mob of RSF1010 plasmid with increased copy number - Construction of Derivatives

[0097] According to our data, during the quiescent period, RSF1010mob - Has the same copy number as RSF1010. In the logarithmic phase of growth, the copy number of the obtained derivative was approximately two-fold lower than that of the RSF1010 plasmid. In logarithmic growth phase, addition of IPTG to the medium resulted in RSF1010mob - Plasmid copy number increases because the repB gene involved in replication of RSF-like plasmids is in the P lacUV5 -under the transcriptional control of the lacI autonomous regulatory element. Therefore, proposed from RSF1010mob - Elimination of the lacI gene in the plasmid can increase the copy number of the plasmid. by using XbaI and BamHI restriction enzymes from RSF1010mob - Plasmid excision of the lacI gene to obtain RSF1010mob without the lacI gene - corresponding derivatives. Then, the sticky ends of the resulting DNA fragments ...

Embodiment 3

[0101] Example 3. RSF1010Mob lacking any antibiotic resistance genes and containing thyA gene as selectable marker - Plasmid construction.

[0102] First, two strains were constructed based on the wild-type E. coli strain MG1655; one strain lacked the thyA gene, and the other lacked the tdk gene. The so-called "Red-driven integration" method (Datsenko K.A. and Wanner B.L., Proc.Natl.Acad.Sci.USA, 2000, 97:12 :6640-45) for the inactivation of target genes. The chloramphenicol resistance gene from plasmid pACYC184 was used to disrupt the thyA gene (Cm r ), the kanamycin resistance gene from plasmid pACYC177 was used to disrupt the tdk gene (Km r ). The two obtained mutants carrying antibiotic resistance markers can be used as donors to transduce ΔthyA and Δtdk deleted P1 into another E. coli strain.

[0103] Strains with a thyA deletion were used in the present invention to screen for functionally active copies of the thyA gene cloned on different plasmids.

[0104] The se...

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Abstract

A Mob- plasmid having a RSF1010 replicon, comprising a gene coding for Rep protein and said plasmid has been modified to inactivate gene related to mobilization ability. The present invention also describes a bacterium having an ability to produce useful metabolites, comprising the plasmid and said bacterium lack active thymidylate synthase coded by thyA gene and thymidine kinase coded by tdk gene, and a method for producing useful metabolites, such as native or recombinant proteins, enzymes, L-amino acids, nucleosides and nucleotides, organic acid, vitamins, using the bacterium.

Description

technical field [0001] The present invention relates to mutant vectors and uses thereof, more particularly, the present invention relates to broad host range RSF1010 derived Mobs that do not contain antibiotic resistance genes - plasmid. The invention also relates to bacteria comprising said plasmid and methods of using said bacteria to produce useful metabolites. Background technique [0002] RSF1010 is a well-known IncQ group plasmid that is mobilizable but not self-transmissible and has a remarkable ability to replicate in a broad bacterial host range, including most Gram-negative bacteria (Frey, J. and Bagdasarian, M. The molecular biology of IncQ plasmamids. In: Thomas, C.M. (eds.), Promiscuous Plasmids of Gram NegativeBacteria. Academic Press, London, 1989, p.79-94). The nucleotide sequence of the RSF1010 plasmid is known (Scholz, P. et al., Gene, 75(2), 271-288 (1989); accession number GenBank M28829, gi:152577) and this plasmid has been studied very thoroughly fun...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/68C12N15/74C12N15/70
CPCC12N15/74C12N15/70
Inventor 乔安娜·Y·卡塔什基纳阿雷克桑德拉·Y·斯科罗科多瓦丹尼拉·V·齐门科夫安德雷·Y·格莱维奇洛佩斯·L·埃雷斯艾里纳·V·比尔尤科瓦阿雷克桑德尔·S·米罗诺夫瑟盖·V·马什克奥
Owner AJINOMOTO CO INC
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