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Modification method of corynebacterium plasmid replicon and product of corynebacterium plasmid replicon

A technology of coryneform bacteria and replicons, applied in the field of bioengineering, can solve problems such as separation instability

Active Publication Date: 2021-11-16
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, segregation instability occurs when the plasmid copy number is small, demonstrating a positive correlation between plasmid copy number and segregation stability

Method used

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  • Modification method of corynebacterium plasmid replicon and product of corynebacterium plasmid replicon
  • Modification method of corynebacterium plasmid replicon and product of corynebacterium plasmid replicon
  • Modification method of corynebacterium plasmid replicon and product of corynebacterium plasmid replicon

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] In vitro transformation of pGA1 Corynebacterium replicons:

[0044] The purpose of this experiment was to obtain a high copy pGA1-Rep-I325S / T398 mutant. The steps are as follows:

[0045] (1) Using the plasmid pEC-XK99E as a template, design primers, obtain the Corynebacterium pGA1 minimal replicon region (1696bp) in the pEC-XK99E plasmid by PCR, i.e. fragment 1, the primers are Replicon_F, Replicon_R and their nucleotide sequences They are shown in sequence as SEQ ID NO.3 and SEQ ID NO.4. Synthesized by Suzhou Jinweizhi Biotechnology Co., Ltd. The PCR reaction conditions were: pre-denaturation at 94°C for 3 min; denaturation at 94°C for 30 s, annealing temperature at 55°C for 30 s, extension temperature at 72°C for 2 min, for 34 cycles; and finally at 72°C for 5 min. The PCR reaction system is shown in Table 1.

[0046] Table 1 PCR reaction system

[0047]

[0048] The product after PCR amplification is subjected to agarose gel electrophoresis, and the agarose ...

Embodiment 2

[0055] Plasmid construction with mutant Rep gene:

[0056] In order to better detect product performance, the present embodiment provides the construction of a plasmid constitutively expressing EGFP protein pEC-XK99E-Rep-T / S, including the following steps:

[0057] (1) Based on the pEC-XK99E plasmid, the original genes of the lacIq promoter, lacI, and lac operon were excised to construct pEC-XK99E-Ptac containing the constitutive tac promoter.

[0058] (2) Amplify EGFP, fragment 7, using primers EcoRI-EGFP_F and BamHI-EGFP_R. The amplification method is PCR using PrimeSTAR polymerase reaction, and the reaction conditions are: pre-denaturation at 98°C for 3 min; denaturation at 98°C for 15s, annealing temperature at 55°C for 15s, extension temperature at 72°C for 1 min, and carry out 30 cycles; the final 72°C reaction is performed for 30 cycles. Hold for 5min.

[0059] (3) Double-enzyme cleavage of fragment 7 through the restriction sites EcoRI and BamHI, first add the enzyme...

Embodiment 3

[0073] Copy ability test of plasmid pEC-XK99E-EGFP-Rep-T / S in Corynebacterium glutamicum:

[0074] Take the pEC-XK99E-EGFP and pEC-XK99E-EGFP-Rep-T / S plasmids and transfer them into the wild-type strain of Corynebacterium glutamicum BZH001 respectively. The steps are as follows: (1) Take 5 μl of pEC-XK99E-EGFP and pEC-XK99E -EGFP-Rep-T / S plasmid was added to 100 μl of Corynebacterium glutamicum BZH001 competent cells, ice bathed for 10 minutes, and then transferred to a frozen electroporation cup; (2) 1800Kv electric shock twice; (3) Immediately add preheated In LBHIS recovery medium at 46℃, water bath at 46℃ for 6min, transfer to 30℃ shaker for recovery culture for 2h; (4) Spread on LBHIS solid medium with 30μg / ml kanamycin resistance, at 30℃ 24h in the incubator.

[0075] Detect the fluorescence value of the strain to reflect the copy number, and the steps are as follows:

[0076] (1) When the initial OD was set to 0.2, the bacteria of Corynebacterium glutamicum BZH001 con...

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Abstract

According to the invention, based on a pGA1 corynebacterium replicon in pEC-XK99E, which is a low-copy plasmid replicon with a copy number of about 30 in corynebacterium glutamicum, 325-site isoleucine of a replicating protein Rep is mutated into threonine, and 398-site amino acid for coding serine is changed into a synonymous codon, so that a replicon pGA1-Rep-I325T / S398 capable of increasing the plasmid copy number of corynebacterium is successfully obtained. The replicon is used for construction of high-copy plasmids, and after a plasmid pEC-XK99E-Rep-T / S carrying the pGA1-Rep-I325T / S398 replicon is replicated in corynebacterium glutamicum, it is proved that the number of plasmids in thalli is increased through plasmid in-vitro extraction. According to the method, pEC-XK99E-EGFP-Rep-T / S plasmids are constructed, fluorescence values under different thallus amounts are measured, and the fluorescence amount is increased by three times compared with that of mutant plasmids, namely, the expression amount of protein is increased by the modified plasmids. Meanwhile, by virtue of a fluorescent quantitative PCR means, the copy number after mutation is determined to be about 245 which is 8 times of that of a plasmid of a wild type replicon.

Description

technical field [0001] The invention belongs to the field of biological engineering, in particular to a modified coryneform bacterium plasmid replicon and a product thereof. Background technique [0002] The commonly used plasmids of Corynebacterium glutamicum are shuttle plasmids of Corynebacterium / Escherichia coli, and the copy number of the plasmid in Escherichia coli is 5-6 times that in Corynebacterium glutamicum. Therefore, the expression of exogenous proteins in C. glutamicum through the shuttle plasmid still has a lot of room for improvement. It is very meaningful to construct a high-copy shuttle plasmid that can be used in C. glutamicum. [0003] The endogenous plasmids that have been isolated from C. glutamicum have been classified into several Corynebacterium plasmid families according to the similarity of replication mode and replication proteins: pBL1, pCG1, pSR1 and pGA1 families all adopt rolling circle replication. It has been reported in a patent that the p...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/77
CPCC12N15/77C12N2800/101C12N2800/22
Inventor 刘秀霞黄丹妮余心宇杨艳坤白仲虎
Owner JIANGNAN UNIV
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