Gluconobacter oxydans shuttle vector for efficient gene expression

A technology for oxidizing glucose and shuttle vectors, applied in the field of genetic engineering, which can solve the problems of limited application potential, poor stability and transformation efficiency of Gluconobacter oxidans, lack of multiple plasmid vectors, etc.

Active Publication Date: 2020-11-24
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] At present, the expression vectors used in Gluconobacter oxidans are mainly pBBRI-MCS series plasmids or their derivative plasmids. This series of plasmids are widely host plasmids, and their stability and transformation efficiency in Gluconobacter...

Method used

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  • Gluconobacter oxydans shuttle vector for efficient gene expression
  • Gluconobacter oxydans shuttle vector for efficient gene expression
  • Gluconobacter oxydans shuttle vector for efficient gene expression

Examples

Experimental program
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Embodiment 1

[0044] Embodiment 1 Construction of Gluconobacter oxidans shuttle plasmid

[0045] According to the results of the genome sequencing of Gluconobacter oxydans WSH-003, Gluconobacter oxidans WSH-004, Gluconobacter oxidans 1.049, and Gluconobacter oxydans 621H genome, through the analysis of the endogenous plasmid gene of Gluconobacter oxydans, such as SEQ ID NO. 1 to 15 gene sequences encoding replication proteins shown in SEQ ID NO.15.

[0046] Primer pairs (shown in Table 1) were designed to amplify the replicating protein sequence. Using the total genomic DNA of Gluconobacter oxydans as a template, the primer pair was used for PCR amplification, and Primer Star MasterMix (Takara Company) high-fidelity enzyme was selected for PCR amplification. 95°C, 15s, 56°C, 15s, 72°C, 1min-3min (determined according to the size of different replication proteins); extension 72°C, 5min. The PCR product was subjected to product purification. The specific steps are:

[0047] Using the comm...

Embodiment 2

[0067] Example 2 Construction of Fluorescent Protein Expression Vectors Using Shuttle Vectors

[0068] Design the primer pair (as shown in Table 2) for amplifying the fluorescent sequence, use the shuttle vector constructed in Example 1 as a template, carry out PCR amplification with the primer pair in Table 3, and select Primer Star MasterMix (Takara Company) high-fidelity enzyme Carried out, the conditions were pre-denaturation at 95°C, 3min; amplification stage 30 cycles, according to 95°C, 15s, 56°C, 15s, 72°C, 1min; extension at 72°C, 5min. The specific steps are:

[0069] Using the p1-Kana constructed in Example 1 as a template, and using T-F / p1-R as primers, amplify the p1-Kana vector fragment; pass the p1-Kana vector fragment and the fluorescent protein fragment shown in SEQ ID NO.16 through Gibson The assembly method is constructed as a fluorescent protein expression vector p1-mcherry;

[0070] Using the p2-Kana constructed in Example 1 as a template, and using T-F / ...

Embodiment 3

[0093] Example 3 Construction of shuttle vectors resistant to different antibiotics

[0094] The antibiotic resistance gene sequences shown in SEQ ID NO.21-SEQ ID NO.25 were synthesized respectively. A primer pair (as shown in Table 5) for amplifying the fluorescent sequence was designed, and the expression vector containing the fluorescent protein was amplified with the primers shown in Table 5. Choose Primer Star MasterMix (Takara Company) high-fidelity enzyme, the conditions are pre-denaturation at 95°C, 3min; the amplification stage is 30 cycles, according to 95°C, 15s, 56°C, 15s, 72°C, 1min; extension at 72°C, 5min .

[0095]Using the p5-k-EGFP constructed in Example 2 as a template, and using T-Res-F / T-Res-R as amplification primers, the T carrier fragment with fluorescent protein was amplified; the synthesis shown in SEQ ID NO.22 The DNA fragment encoding antibiotic resistance was connected with the T carrier fragment with fluorescent protein and the DNA fragment of a...

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Abstract

The invention discloses a gluconobacter oxydans shuttle vector for efficient gene expression, and belongs to the field of gene engineering. Possible replication proteins of endogenous plasmids of gluconobacter oxydans are screened, a series of shuttle vectors are constructed by using the endogenous replication proteins, and the shuttle vectors are transferred into the gluconobacter oxydans for verification, so that the conversion efficiency of the shuttle vectors is found to be greatly improved compared with that of broad-host plasmids pBBR-MCS; and the shuttle vector is used for protein expression, so that the L-sorbose yield of the gluconobacter oxydans is increased by 7.8% compared with the expression quantity of a broad-host vector, and the substrate conversion time is shortened by 27.2%. The genetic modification efficiency of the gluconobacter oxydans is greatly improved, and different requirements for genetic modification of the gluconobacter oxydans are met.

Description

technical field [0001] The invention relates to a gluconic acid bacteria shuttle vector used for high-efficiency gene expression, and belongs to the field of genetic engineering. Background technique [0002] Gluconobacter oxidans is a strictly aerobic Gram-negative bacteria belonging to the genus Glucobacterium, Acetobacteriaceae, which mainly inhabit high-sugar environments, such as fruits, flowers and honey. Gluconobacter has the following characteristics that make it widely used in industry: (1) can quickly and incompletely oxidize sugar and alcohol substrates to generate corresponding aldehydes, ketones and acids; (2) membrane-bound dehydrogenase catalytic activity The site is generally located in the periplasmic space, and the substrate does not need to be transported across the membrane during the catalytic reaction, while the corresponding product is directly secreted into the medium; (3) The catalytic reaction is highly regioselective and stereoselective. The chara...

Claims

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Application Information

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IPC IPC(8): C12N15/74C12N15/70C12N1/21C12R1/01C12R1/19
CPCC12N15/74C07K14/195
Inventor 周景文陈坚刘立曾伟主堵国成李江华
Owner JIANGNAN UNIV
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