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Cyanobacteria Engineering Bacteria System Expressing Exogenous Genes Without Antibiotics and Its Preparation and Application

An expression system and a technology for exogenous genes are applied in the field of preparation of Synechococcus PCC7002 engineering bacteria system, which can solve the problems of reduced growth rate of engineering bacteria, environmental pollution, increased cyanobacterial culture time, etc., so as to reduce environmental pollution and insecurity , the effect of efficient expression

Active Publication Date: 2019-09-13
PEKING UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The use of antibiotics has many disadvantages: (1) the pressure of antibiotic cultivation will reduce the growth rate of engineering bacteria, increase the cultivation time of cyanobacteria, and increase the cost of fermentation; (2) the use of antibiotics itself also requires a certain cost; (3) antibiotics Will cause pollution to the environment; (4) antibiotics will also affect the safety of products
Therefore, the use of antibiotics not only increases the cost of the fermentation industry, but also causes environmental pollution and unreliable product safety.

Method used

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  • Cyanobacteria Engineering Bacteria System Expressing Exogenous Genes Without Antibiotics and Its Preparation and Application
  • Cyanobacteria Engineering Bacteria System Expressing Exogenous Genes Without Antibiotics and Its Preparation and Application
  • Cyanobacteria Engineering Bacteria System Expressing Exogenous Genes Without Antibiotics and Its Preparation and Application

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Example 1. Preparation of engineered bacteria system

[0035] (1) Construction of a homozygous pbsC gene deletion mutant △psbC

[0036] The principle and flow of the construction of the psbC gene deletion mutant △psbC are as follows figure 1 Shown in A. The cyanobacteria wild-type Synechococcus sp.PCC 7002 (presented in June 2003 by Professor Donald A. Bryant, Professor of the Department of Biochemistry and Molecular Biology, Pennsylvania State University, USA, from the cyanobacteria collection of the Pasteur Institute in Paris, France (Pasteur Culture Collection), isolated in 1962 by C. Van Baalen in marine fouling) genomic DNA was used as a template, P1 / P2 was PCR amplified with specific primers to obtain the upstream fragment T1 of psbC (A1559) gene; P3 / P4 was amplified by PCR with specific primers to obtain the downstream fragment T2 of the psbC (A1559) gene; using the pBS plasmid as a template, primers P5 / P6 were used to amplify a kan gene fragment with a size of abou...

Embodiment 2

[0041] Example 2. Using egfp gene to identify the feasibility of the engineered bacteria of the present invention

[0042] Using the primer pair P21 / P22, the egfp gene was amplified. Connect the egfp gene into the pAQ3-Ex integration vector with NdeⅠ and BamHI to obtain the test vector pAQ3-egfp (the plasmid diagram is shown as figure 2 Shown in C), pAQ3-egfp (the schematic diagram of double exchange between test vector and pAQ3 is shown in figure 2 Middle D. The test vector pAQ3-egfp was transformed into the psbC deletion mutant △psbC. + Screen positive clones on the plate. Positive clone in A + After the plate was streaked for 3 generations, it was verified to be homozygous by PCR (see image 3 Middle A, verify the primer pair P23 / P24), and name the homozygous mutant pAQ3-EGFP.

[0043] After the pAQ3-EGFP strain was continuously cultured in non-resistant medium, the protein was purified and detected by SDS-PAGE and Westren blotting ( image 3 Middle B), it can be proved tha...

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Abstract

The invention discloses an antibiotic-free blue-green algae engineering bacterium system capable of expressing exogenous genes, preparation and applications thereof. The blue-green algae engineering bacterium system comprises [delta]psbC mutant of synechococcus PCC7002 and an integrative vector. The integrative vector comprises a core element FlanKA-P1-MCS-P2-psbC-FlanKB. The homologous sections FlanKA and FlanKB can integrate the P1-MCS-P2-psbC section into the intergenetic region of C006 and C007 of indigenous plasmid pAQ3 of synechococcus PCC7002; the exogenous genes are inserted into a polyclonal site MCS, the promoter P1 regulates the expression; and P2-psbC is used to reply to the psbC gene deletion. The provided system has the advantages that blue-green algae can efficiently express exogenous genes without depending on the antibiotic culture pressure; the cost of bioreactors prepared from blue-green algae is largely reduced, and moreover, the environment pollution and danger caused by antibiotic are reduced.

Description

Technical field [0001] The invention relates to a preparation method and application of a cyanobacteria engineering bacteria system, in particular to a preparation method and application of a Synechococcus sp. PCC7002 engineering bacteria system without antibiotic screening and continuous high-efficiency expression of foreign genes, which is a cyanobacteria In the industrialization of fermentation, low-cost and safer large-scale expression of foreign genes provides a way. Background technique [0002] Due to the photosynthetic autotrophic characteristics of cyanobacteria, easy genetic manipulation, and fast growth, there are many examples in the literature and industry that use cyanobacteria as a bioreactor to express foreign genes, such as mosquito venom protein genes, fatty acid unsaturated Enzyme gene, hydroxybutyrate polymerase gene, dehydrogenation gene that catalyzes the synthesis of eicosapentaenoic acid (EPA), cellulose gene. There are currently two main ways to transfer...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/74C12N15/66C12N15/65
Inventor 郑正高董春霞赵进东
Owner PEKING UNIV