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Arthrobacter sp. expression plasmid and application thereof

A technology for expressing plasmids and Arthrobacter, which is applied in the fields of molecular biology and bioengineering

Active Publication Date: 2015-10-07
南京趣酶生物科技有限公司 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Of the above-mentioned plasmids, only pART2 and pART3 can be used to express genes in Arthrobacter, and other plasmids are limited to the study of the copy number of the plasmid in Arthrobacter and the electrotransformation experiment of Arthrobacter

Method used

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  • Arthrobacter sp. expression plasmid and application thereof
  • Arthrobacter sp. expression plasmid and application thereof
  • Arthrobacter sp. expression plasmid and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Example 1: Acquisition of Arthrobacter replicon.

[0032] By searching the natural plasmid of Arthrobacter in GenBank, it was found that the pA3 plasmid (GenBank accession number is AJ131246.1) has a full length of 2205bp and contains 5 ORFs (such as figure 1 shown) the full sequence. In order to study the replicon of this plasmid, the gene sequence of pA3 plasmid was synthesized by Suzhou Jinweizhi Biotechnology Co., Ltd. for whole gene synthesis.

[0033] The size of pA3 is 2205bp, its nucleotide sequence is shown in SEQ ID NO: 2, the G+C content is 59.86%, and it contains 5 ORFs, each of which is ORF. The amino acid sequences encoded by the 5 ORFs marked in GenBank were BLASTed in NCBI, and the comparison results are shown in Table 1. The results showed that the ORF1 amino acid sequence had 30% similarity to the replication initiation factor of Propionibacterium acnes (see figure 2 ) shows that ORF1 is most likely the replication initiation factor of pA3, but the ...

Embodiment 2

[0049] Example 2: Construction and functional verification of the novel Arthrobacter plasmid pUAKP.

[0050] The P13-3 promoter (the sequence shown in 152-261 in SEQ ID NO: 1, which was screened by our laboratory) was cloned into the BamHI site of the pUAK4 plasmid ( Figure 7 ), to get the pUAKP plasmid ( Figure 8 ).

[0051] The gfp gene (green fluorescent protein) was inserted into the multiple cloning site of the pUAKP plasmid to obtain the pUAKP-gfp plasmid ( Figure 9 ), using the pARK-gfp (pARK plasmid with gfp gene connected at the multiple cloning site) plasmid as a control, use a fluorescence microscope to observe the fluorescence intensity of each bacterial strain under the same exposure time, as shown in Figure 10 As shown, middle A, B, and C are Arthrobacter empty bacteria respectively (the Arthrobacter used in the present invention is CGMCC3584, and the specific information of this strain has been disclosed in detail in the Chinese patent whose application nu...

Embodiment 3

[0052] Example 3: Application of pUAKP plasmid.

[0053] The hypoxanthine phosphoribosyltransferase gene (abbreviated as hgprt gene, the sequence of which has been disclosed in 201310248615.1) is cloned into the multiple cloning site of pARK (the nucleotide sequence of the plasmid has been disclosed in 201510186254.1) plasmid and pUAKP plasmid, The pARK-hgprt plasmid and the pUAKP-hgprt plasmid were respectively obtained. The above two plasmids were transformed into Arthrobacter CGMCC 3584 respectively, and the obtained strains were named pAK-Arth and pUK-Arth respectively, and cAMP was produced by fermentation as follows:

[0054] 1. Strain activation: three strains of Arthrobacter CGMCC 3584, pAK-Arth, and pUK-Arth were respectively cultured on Arthrobacter solid medium (glucose 10g / L, peptone 10g / L, yeast extract 10g / L, beef extract 10g / L, Agar 20g / L pH7.2, 121 ℃ high pressure steam sterilization 15min) on the streak activation 36h.

[0055] 2. Seed liquid: pick single co...

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Abstract

The invention discloses an Arthrobacter sp. expression plasmid, and belongs to the technical field of gene engineering. The plasmid disclosed in the invention is annular in shape, and the nucleotide sequence of the plasmid is represented by SEQ ID NO:1, comprises an Arthrobacter sp. promoter sequence, a multiple cloning site sequence, a replicating sequence in Arthrobacter sp., a kanamycin resistant gene sequence, an Escherichia coli replicating sequence and an amicillin resistant gene sequence, and can autonomously replicate in Escherichia coli and Arthrobacter sp.. The plasmid is used to express ghprt gene in the Arthrobacter sp. of CGMCC3584, and the cAMP output of the plasmid is 65.2% higher than that of original strain.

Description

technical field [0001] The invention belongs to the technical field of molecular biology and bioengineering, and specifically relates to an Arthrobacter expression plasmid, a construction method and application of the plasmid. Background technique [0002] Arthrobacter is an obligate aerobic microorganism widely distributed in soil and belongs to the actinomycetes Micrococcaceae (Micrococcaceae) in systematic taxonomy. Arthrobacter cells will produce significant morphological changes during the growth process. The cells in the early culture are irregular thin rods, and some bacilli are arranged at a certain angle. After entering the stationary phase, the cells are spherical. Transfer them to fresh In the culture medium, the cells will branch again and form irregular rods. This cycle is the most typical morphological feature of Arthrobacter. Arthrobacter can live not only in ordinary soil, but also in various extreme environments with complex conditions, such as organic solv...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/74C12N15/113C12R1/06
Inventor 谢婧婧王浩绮宋天顺
Owner 南京趣酶生物科技有限公司
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