Biological mothballing system suitable for synechococcus elongatus, and construction method and application of biological mothballing system

A construction method and technology of Synechococcus, applied in the field of biological storage systems, can solve the problems of strain growth impact, strain death, long time, etc.

Active Publication Date: 2019-11-12
TIANJIN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The existing active lethal system is usually a combination of an inducible promoter and a lethal gene, but due to restrictions such as the stringency of the promoter, the growth of uninduced strains will be affected
The passive system is based on the production of auxotrophic strains by knocking out specific genes, but it often takes a long time for the strains to die due to "starvation"

Method used

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  • Biological mothballing system suitable for synechococcus elongatus, and construction method and application of biological mothballing system
  • Biological mothballing system suitable for synechococcus elongatus, and construction method and application of biological mothballing system
  • Biological mothballing system suitable for synechococcus elongatus, and construction method and application of biological mothballing system

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] (1) In vitro amplification of the target gene

[0023] A bacterial genome extraction kit was used to extract the Synechococcus 7942 genome as a template,

[0024] Take the sequence shown in SEQ ID No. 6 as the upstream primer of the sepA2 gene,

[0025] Take the sequence shown in SEQ ID No. 7 as the downstream primer of sepA2 gene,

[0026] Take the sequence shown in SEQ ID No. 8 as the upstream primer of the sepT2 gene,

[0027] Take the sequence shown in SEQ ID No. 9 as the downstream primer of the sepT2 gene,

[0028] Take the sequence shown in SEQ ID No. 10 as the upstream primer of PisiAB promoter,

[0029] Use the sequence shown in SEQ ID No. 11 as the downstream primer of the PisiAB promoter to perform PCR amplification;

[0030] Using the bacterial genome extraction kit to extract the Synechocystis 6803 genome as a template, the sequences shown in SEQ ID No. 12 and SEQ ID No. 13 are respectively the PpsbA2 promoter upstream and downstream primers for PCR amplification;

[003...

Embodiment 2

[0047] The growth curves of Synechococcus 7942 wild-type and mutant strains in normal liquid medium BG11 and iron-deficiency liquid medium BG11 were determined.

[0048] The Synechococcus 7942 (wild strain WT) and the mutant strain 7942-sepA2 / T2 obtained in Example 1 were cultured in normal liquid medium BG 11 and liquid medium BG 11 containing deferoxamine 100 μM, and the setting parameter of the shaker was light. Intensity 200μmolphotons m -2 s -1 , The speed is 160rpm, and the temperature is 37°C.

[0049] The steps are: take OD during vaccination 750nm Add 5 mL of 0.2 fresh cells to 20 mL of culture medium, make 3 parallel samples for each group, measure the absorbance at 750 nm with a 96-well plate microplate reader, measure it every 24 hours, and draw a growth curve ( figure 2 ).

[0050] From figure 2 It can be observed that in the normal liquid medium BG 11, the growth status of the wild strain and the mutant strain is almost the same, while in the medium induced by iron ...

Embodiment 3

[0052] The growth curves of Synechococcus 7942 wild-type and mutant strains in normal liquid medium BG11 and iron-deficiency liquid medium BG11 were determined.

[0053] The Synechococcus 2973 (wild strain WT) and the mutant strain 2973-sepA2 / T2 obtained in Example 1 were cultured in normal liquid medium BG 11 and liquid medium BG 11 containing deferoxamine 100 μM, and the setting parameter of the shaker was light. Intensity 200μmolphotons m -2 s -1 , The speed is 160rpm, and the temperature is 37°C.

[0054] The steps are: take OD during vaccination 750nm Add 5 mL of 0.2 fresh cells to 20 mL of culture medium, make 3 parallel samples for each group, measure the absorbance at 750 nm with a 96-well plate microplate reader, measure it every 24 hours, and draw a growth curve ( figure 2 ).

[0055] From figure 2 It can be observed that in the normal liquid medium BG 11, the growth status of the wild strain and the mutant strain is almost the same, while in the medium induced by iron ...

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Abstract

The invention discloses a biological mothballing system suitable for synechococcus elongatus, and a construction method and application of the biological mothballing system. The construction method comprises the steps that a virulent gene sepT2, an antitoxic gene sepA2 and an iron-deficiency inducible promoter PisiAB are obtained from the synechococcus elongatus 7942, and a promoter PpsbA2 is obtained from synechocystis 6803; a terminator Trbcl is obtained from integrative plasmid pBA3031 through amplification; pBR322 is taken as a carrier, the virulent gene sepT2 is expressed with the iron-deficiency inducible promoter PisiAB, and the terminator Trbcl is used for terminating transcription; and the antitoxic gene sepA2 is expressed with the promoter PpsbA2, and plasmid is constructed and transferred into the synechococcus elongatus. According to the system, the synechococcus elongatus grows normally under the non-inducing situation and rapidly dies after being induced. The important theoretical and practical significance for development and optimization of the biological mothballing system of the synechococcus elongatus is achieved, and reference is also provided for solving the bio-safety problem of other cyanobacteria.

Description

Technical field [0001] The invention belongs to the field of biosafety, and specifically relates to a bio-storage system suitable for Synechococcus 7942 and Synechococcus 2973, a construction method and application thereof, and can be used to prevent the escape of genetically engineered bacteria. Background technique [0002] As traditional energy sources are increasingly depleted, the greenhouse effect is gradually serious, and the environment is constantly deteriorating, bio-energy has attracted more and more attention due to its green and sustainable characteristics. The development and utilization of alternative new energy sources is imminent. As a photosynthetic cyanobacterium, Synechococcus can directly use light energy and carbon dioxide to produce biofuels and fine chemicals. In recent years, it has been widely used as a new generation of "cell factories". Synechococcus 7942 is a commonly used model strain with a clear genetic background, while Synechococcus 2973 has beco...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12N15/74C12R1/01
CPCC07K14/195C12N15/74
Inventor 陈磊周宇晴孙韬陈子熙宋馨宇张卫文
Owner TIANJIN UNIV
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