Pseudoalteromonas sp. and application thereof

A host bacteria, cold-adaptive technology, applied to cold-adaptive host bacteria strains and applications, marine biology field

Inactive Publication Date: 2011-03-16
SHANDONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

There are no reports or patents on cold-adapted bacterial expression systems in China

Method used

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  • Pseudoalteromonas sp. and application thereof
  • Pseudoalteromonas sp. and application thereof
  • Pseudoalteromonas sp. and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] Example 1: Preparation method of plasmid deletion strain SM20429

[0047] Pseudoalteromonas BSi20429 strain was eliminated by SDS-high temperature treatment, and the plasmid-deleted strain SM20429 of the strain was obtained. This strain is characterized by the complete loss of the plasmid, which is not integrated into the strain genome and is genetically stable. Compared with the original strain, the strain did not change significantly in character. Specific steps are as follows:

[0048] 1) Pick a single colony of Pseudoalteromonas BSi20429 strain on the plate, inoculate it in 5ml 2216E liquid medium, and culture it at 25°C with shaking at 160rpm until OD 600 is 1.0, the primary bacterial liquid is obtained;

[0049] 2) Transfer 100 μl of bacterial liquid to fresh 5ml 2216E liquid medium, culture at 33°C with shaking at 160rpm until OD 600 about 1.0.

[0050] 3) Take 100 μl of the bacterial liquid and transfer it to 5ml 2216E liquid medium added with 25 μl of 1% S...

Embodiment 2

[0055] Example 2: Construction of autonomously replicating expression vector pWD

[0056] 1. Description

[0057] Size: 7654bp

[0058] E. coli replication elements:

[0059] Origin of replication: origin of replication derived from plasmid pUC19, pUC19 was purchased from Takara Company.

[0060] Selection marker: ampicillin-resistant gene, derived from plasmid pUC19 (purchased from Takara Company).

[0061] Pseudoalteromonas replication elements:

[0062] Origin of replication: origin of replication derived from plasmid pSM429 (GenBank accession number EU627679) extracted from strain Pseudoalteromonas sp.BSi20429.

[0063] Selection marker: chloramphenicol resistance gene, cloned in plasmid pBT (purchased from Stratagene Company)

[0064] Promoter: Lac promoter, derived from plasmid pUC19 (purchased from Takara Company).

[0065] Multiple cloning sites: PstI, XhoI and SalI. See attached figure 2 .

[0066] 2. Construction method

[0067] (i) PCR amplification of a 3...

Embodiment 3

[0076] Embodiment 3: the establishment of transformation system of Pseudoalteromonas cold-adapted

[0077] The present invention adopts the method of electrotransformation, takes SM20429 bacterial strain as host, takes self-replicating plasmid pWD as carrier, establishes the transformation system of Pseudoalteromonas cryotropic, and the steps are as follows:

[0078] 1) Take a cold-adapted host bacterial strain whose strain preservation number is CCTCC M 2010239, inoculate it into 2216E liquid medium, and culture it with shaking at 20°C overnight to obtain a primary bacterial liquid;

[0079] 2) Inoculate the primary bacterial liquid prepared in step 1) into 2216E liquid medium at an inoculum size of 1% by volume, and cultivate to OD at 20°C 600 0.8-1.0, centrifuge at 6000rpm for 5min, collect the bacteria, discard the supernatant, and bathe in ice for 10min to get the bacteria;

[0080] 3) Wash the bacterial cells prepared in step 2) with 20ml of pre-cooled sterile water con...

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Abstract

The invention relates to a strain of Pseudoalteromonas sp. and application thereof, in particular to a strain of Pseudoalteromonas sp. SM20429 and the application thereof, which belong to the technical field of marine organisms. The Pseudoalteromonas sp. SM20429 was collected in China Center for Type Culture Collection (CCTCC) on 19 September, 2010, wherein the address is Wuhan University in China and the strain collection number is CCTCC M 2010239. The invention also relates to an autonomous replication expression vector pWD used for the Pseudoalteromonas sp. SM20429, and a construction method and the application thereof. The nucleotide sequence of the autonomous replication expression vector pWD is shown as SEQ ID NO.1. The strain SM20429 has a typical psychrophilic characteristic and can grow quickly at the low temperature of 15 DEG C. Therefore, the strain SM20429 has a potential value of being developed into a psychrophilic expression host capable of largely expressing foreign proteins to fulfill the aim of preparing psychrophilic protein quickly at the low temperature.

Description

technical field [0001] The invention relates to a strain of cold-adapted host bacteria and its application, in particular to a strain of cold-adapted host bacteria (Pseudoalteromonas sp.) SM20429 and its application, and belongs to the technical field of marine biology. Background technique [0002] In the field of biotechnology and bioengineering, the purpose of expressing and preparing recombinant proteins is achieved by cloning foreign genes into expression vectors and transferring them into a suitable host cell. Applying this expression system to industrial production can be used to prepare a large amount of recombinant protein and reach the industrial level. [0003] An expression system first needs a transformation system suitable for transforming foreign genes, and the transformation system should include 1) a suitable selection marker; 2) a carrier for carrying foreign genes into the host bacterium, which can be carried out in the host bacterium. 3) a transformation...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/20C12N15/63C12N15/66C12N15/56C12N9/42C12R1/01
Inventor 张玉忠陈秀兰赵殿丽于子超解彬彬何海伦周百成
Owner SHANDONG UNIV
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