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Shuttle plasmid and derivative plasmid thereof

A technology of shuttle plasmids and plasmids, applied in the direction of using vectors to introduce foreign genetic material, recombinant DNA technology, etc.

Inactive Publication Date: 2008-08-27
INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there are few reports on the construction of cloning vectors from Corynebacterium glutamicum plasmids with theta replication mechanism

Method used

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  • Shuttle plasmid and derivative plasmid thereof
  • Shuttle plasmid and derivative plasmid thereof
  • Shuttle plasmid and derivative plasmid thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Example 1. Construction of plasmid pAK6

[0034] The construction process of plasmid pAK6 is as follows image 3 As shown, the specific method is as follows:

[0035]Taking the Corynebacterium glutamicum plasmid pXZ10145 (Shen Tianxiang et al. 1993 Journal of Bioengineering 9.216-222) (Institute of Microbiology, Chinese Academy of Sciences) as the starting plasmid, the plasmid pXZ10145 was digested with EcoRI / SpeI, separated by agarose gel electrophoresis, and recovered Large fragment with the starting point of replication. The E. coli plasmid pUC119-KanR (Guoqing Niu et al 2006 Metabolic Engineering 8.183-195) (Institute of Microbiology, Chinese Academy of Sciences) was digested with EcoRI / XbaI double enzymes, and small fragments with KanR gene were recovered. Connect the two fragments to construct a recombinant plasmid. The recombinant plasmid was transformed into Corynebacterium glutamicum ATCC 13032, and the plasmid was extracted and named pAK0. In order to introduce th...

Embodiment 2

[0036] Example 2. Construction of plasmid pAKC6

[0037] The construction process of plasmid pAKC6 is as follows Figure 7 As shown, the specific method is as follows:

[0038] To construct a promoter detection vector, the reporter gene chloramphenicol acetyltransferase gene CAT and the transcription terminator T of the Corynebacterium glutamicum LeuB gene were introduced into the plasmid pAK6 leuB . First, the coding region and SD of the chloramphenicol acetyltransferase gene (GenBank No.AT219685) were amplified from the plasmid pXC99E (Kirchner O and Tauch A 2003 J. Biotechnology 104: 287-299) (Institute of Microbiology, Chinese Academy of Sciences) by PCR sequence. The primers used are

[0039] P1: 5′-GGG AAGCTT AAGTAATTAACAGGAGCTAAGGAAGCTAAA-3′ and

[0040] P2: 5′-AAC TCTAGA TAAGGGATTTTGGTCATGCG-3', the underlined parts are the recognition sites of HindIII and XbaI, respectively.

[0041] The reaction system is: total volume 50μL, 2×GC bufferII 25μL, dNTP 7μL, LA Taq DNA pol...

Embodiment 3

[0049] Example 3. Plasmids pAK6 and pAKC6 are replicated in theta mode

[0050] 1. Stability experiment of plasmid pAK6 and pAKC6

[0051] Plasmid pUL340 is a rolling circle replicating plasmid. With plasmid pUL340 as a control, the stability of plasmids pAK6 and pAKC6 was determined. Methods as below:

[0052] 1) Using Corynebacterium glutamicum ATCC 13032 as the host of plasmid pUL340 (Ramon I. Santamaria et al 1985 J. Bacteriology Vol. 162 No. 1. p. 463-467) (Institute of Microbiology, Chinese Academy of Sciences), pAK6 and pAKC6.

[0053] PUL340, pAK6 and pAKC6 were respectively introduced into Corynebacterium glutamicum ATCC 13032 to obtain recombinant bacteria pUL340 / 13032 containing pUL340, recombinant bacteria pAK6 / 13032 containing pAK6 and recombinant bacteria pAKC6 / 13032 containing pAKC6.

[0054]Single colonies of pUL340 / 13032, pAK6 / 13032 and pAKC6 / 13032 were respectively inoculated into LB medium containing 50μg / ml kanamycin and cultured at 30°C to logarithmic phase, a...

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Abstract

The invention discloses a shuttle plasmid and a derivative plasmid thereof. A nucleotide sequence of the shuttle plasmid which is provided by the invention is a sequence 1 in a sequence table and a 4872-5664th deoxyribonucleotide from 5'end of the sequence 1 in the sequence table is a kanamycin resistance gene and a promoter thereof. The invention also discloses the derivative plasmid thereof, which is obtained through inverting the kanamycin resistance gene of the 4872-5664th deoxyribonucleotide from the 5'end of the sequence 1 in the sequence table and the promoter of the kanamycin resistance gene and through inserting transcription terminators of a reporter gene and a corynebacterium glutamate LeuB gene in multiple cloning sites, the direction of the reporter gene is opposite to the direction of the kanamycin resistance gene and the promoter of the kanamycin resistance gene, and the transcription terminators of the corynebacterium glutamate LeuB gene are located between the reporter gene and the kanamycin resistance gene. The two plasmids have high stability and can obtain wide application on stic engineering.

Description

Technical field [0001] The invention relates to a shuttle plasmid and its derivative plasmid. Background technique [0002] Plasmids are extrachromosomal genetic factors capable of autonomous replication. It is not a genetic element necessary for the life activities of the host cell, but it can give the host cell some special properties, such as drug resistance, degradation or utilization of certain compounds. In molecular biology research, plasmids can be used as vectors for cloning foreign genes. The research on plasmids has been quite in-depth. On the one hand, people continue to discover new plasmids from nature, and on the other hand, they continue to construct derivative plasmids on the basis of existing plasmids as vectors for genetic engineering. [0003] In the field of bacterial genetic engineering, most of the plasmid vectors come from the Gram-negative bacterium Escherichia coli, and many have been commercialized. The research on the plasmid of the Gram-positive bacte...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/70C12N15/77
Inventor 丁久元李开刘桂明赵智王宇张英姿
Owner INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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