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A biostorage system, construction method and application suitable for Synechococcus

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

Active Publication Date: 2022-03-11
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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  • A biostorage system, construction method and application suitable for Synechococcus
  • A biostorage system, construction method and application suitable for Synechococcus
  • A biostorage system, construction method and application suitable for Synechococcus

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

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

[0023] Extraction of the polyolite 7942 genome as a template using a bacterial genomic extract kit.

[0024] Sepa2 gene upstream primers in SEQ ID NO.6,

[0025] Subsequently, SEQ ID NO.7 is listed as a Sepa2 gene downstream primer,

[0026] Sepa ID No. 8 is listed as a Sept2 gene upstream primer,

[0027] Subsequently, sept2 gene downstream primers in SEQ ID NO.9,

[0028] Since SEQ ID NO.10 is listed as a Pisiab promoter upstream primer,

[0029] PCR amplification is performed as a Pisiab promoter downstream primer in SEQ ID NO.11.

[0030] Extraction of the epitaxichol 6803 genome as a template was extracted with a bacterial genome extraction kit, and sequences shown in SEQ ID NO.12, SEQ ID NO.13 were respectively PPSBA2 promoters, respectively, PCR amplification;

[0031] Using the integrated plasmid pBA3031 (Sun T, Li S, Song X, et al.Re-direction of carbonflux to key precursor malonyl-CoA via artificial small RNAs in ph...

Embodiment 2

[0047] The growth curve of the polyolite 7942 wild type and mutant strain was determined in normal liquid medium BG 11 and the iron deficiency liquid medium BG11.

[0048] The mantra 7942 (wild strain WT) was cultured in the normal liquid medium BG 11 and the liquid medium BG 11 containing the deteric amine 100 μm, and the shaker is provided. Strength 200μmolPhotons M -2 s -1 , The speed is 160 rpm, and the temperature is 37 ° C.

[0049] Step is: Take OD when inoculation 750nm For 5 ml of fresh cells of 0.2, a 20 ml medium was added, and 3 parallel samples were made each group, and the absorbance value of the wavelength of 750 nm was measured by a 96-well plate enzyme syndrometer, and the growth curve was measured once every 24 hours. figure 2 .

[0050] from figure 2 In the normal liquid medium BG 11, the growth status of wild strains and mutant strains is almost consistent, and in the induced medium induced by iron deficiency, the growth condition of the mutant strain is signif...

Embodiment 3

[0052] The growth curve of the polyolite 7942 wild type and mutant strain was determined in normal liquid medium BG 11 and the iron deficiency liquid medium BG11.

[0053] The mutant strain 2973-Sepa2 / T2 obtained from the first Example 1 was cultured in the normal liquid medium BG 11 and the liquid medium containing the decamine 100 μm, and the shaker setting parameters were illuminated. Strength 200μmolPhotons M -2 s -1 , The speed is 160 rpm, and the temperature is 37 ° C.

[0054] Step is: Take OD when inoculation 750nm For 5 ml of fresh cells of 0.2, a 20 ml medium was added, and 3 parallel samples were made each group, and the absorbance value of the wavelength of 750 nm was measured by a 96-well plate enzyme syndrometer, and the growth curve was measured once every 24 hours. figure 2 .

[0055] from figure 2 In the normal liquid medium BG 11, the growth status of wild strains and mutant strains is almost consistent, and in the induced medium induced by iron deficiency, the...

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Abstract

The invention discloses a biological storage system suitable for Synechococcus 7942, a construction method and an application. The promoter PpsbA2 was obtained from algae 6803; the terminator Trbcl was amplified from the integrated plasmid pBA3031; the toxic gene sepT2 was expressed with the iron deficiency inducible promoter PisiAB using pBR322 as the vector, and the transcription was terminated with the terminator Trbcl; the expression was expressed with the promoter PpsbA2 The antiviral gene sepA2 was constructed and transformed into Synechococcus sp. The system of the present invention grows normally without induction, and dies rapidly after induction. This has important theoretical and practical significance for the development and optimization of the biostorage system of Synechococcus, and also provides a reference for solving the biosafety problems of other cyanobacteria.

Description

Technical field [0001] The present invention belongs to the field of biological safety, and in particular to a biological storage system suitable for polyolis 7942 and polyolite 2973, a method and application of a construction method, which can be used to prevent the escape of the genetic engineering. Background technique [0002] With the increasing depletion of traditional energy, the greenhouse effect is gradually serious, the environment is deteriorated, and the biological energy is increasingly received by its green environmental protection, and the development and utilization of replacement new energy is imminent. As a photosynthetic blue bacterium, polyol is directly utilized to produce biofuels and fine chemicals, which have been widely used as a new generation of "cytoplasma" in recent years. The polyolis 7942 is a commonly used model strain, which has clear genetic background, and the characteristics of polyolina 2973 have become a highly production potential due to its...

Claims

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Application Information

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Patent Type & Authority Patents(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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