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Method for improving ammonium salt tolerance of synechocystis PCC6803 and application

A technology of PCC6803 and Synechocystis, which is applied in the field of improving the high-concentration ammonium salt tolerance of Synechocystis PCC6803, and achieves the effect of wide application prospect and improved tolerance.

Active Publication Date: 2018-12-18
SOUTH CHINA UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the literature and patents on improving the ammonium salt tolerance of Synechocystis sp. PCC6803 have not been reported
At the same time, the function and mechanism of S2P (site 2) protease Sll0528 in Synechocystis sp. PCC6803 involved in ammonium stress adaptation have not been reported yet.

Method used

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  • Method for improving ammonium salt tolerance of synechocystis PCC6803 and application
  • Method for improving ammonium salt tolerance of synechocystis PCC6803 and application
  • Method for improving ammonium salt tolerance of synechocystis PCC6803 and application

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Experimental program
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Embodiment 1

[0026] The construction of the recombinant plasmid p3031P0K includes the amplification of the target gene fragment, the enzyme-cut link between the target gene segment and the vector plasmid, and the identification of the recombinant plasmid.

[0027] Use the primer pair SEQ ID NO.1 and SEQ ID NO.2 in the sequence table, and use the Synechocystis PCC6803 wild-type genomic DNA (bacterial genome extraction kit) as a template to amplify the slr2030 fragment by PCR. The fragment sequence is as shown in SEQ ID NO .15 shown. At the same time, the target fragment slr2030 and the vector plasmid pUC118 were cut with restriction endonucleases HindⅢ and PstⅠ, and then the target fragment slr2030 and the vector plasmid pUC118 were connected with ligase to form the recombinant plasmid pUC118-2030. The recombinant plasmid pUC118-2030 was introduced into competent Escherichia coli DH10B for replication, and the Escherichia coli was grown in solid medium and liquid medium successively for 16h...

Embodiment 2

[0033] Obtaining the overexpression algae strain OE0528 includes the homologous double exchange between the recombinant plasmid p3031POK and the wild-type genome of Synechocystis sp. PCC6803, and the screening of the overexpression algae strain OE0528.

[0034] The constructed recombinant plasmid p3031P0K was mixed with the wild-type Synechocystis sp. PCC6803, cultivated in a shaker for 6h, and then spread on the solid BG11 medium covered with mixed fiber filter membrane. After 24h, the mixed fiber membrane was transferred to a medium containing 10 μg / mL kanamycin on the solid BG11 medium, the solid BG11 medium is placed in a light incubator, and after a few weeks, the medium grows a single algae colony, which is the transformant. In this process, the recombinant plasmid p3031P0K enters the wild-type Synechocystis sp. PCC6803, performs homologous double exchange with the help of homology arms slr2030 and slr2031, and overexpresses the element km r +psbA2 promoter+sll0528 inse...

Embodiment 3

[0040] Cultivation of Synechocystis sp. PCC6803 wild type and overexpressed strain OE0528 under high concentration ammonium salt stress.

[0041] by OD 730 = 0.1 as the initial concentration, the wild-type Synechocystis sp. PCC6803 and the overexpressed algae strain OE0528 were respectively inserted into the BG11 liquid medium containing ammonium chloride concentrations of 0 mM, 120 mM, 150 mM, 180 mM, and 210 mM. -2 ·s -1 , temperature 29 ℃, rotating speed 150r / min shaker for 5 days, measure OD every 24h 730 Value, draw the growth curve, and measure the 800nm ​​~ 400nm wavelength scanning absorption value until the 5th day of culture, and draw the whole cell absorption map.

[0042] Figure 5It is a growth curve graph, in which it can be observed that as the concentration of ammonium chloride increases gradually, the growth rate of WT gradually slows down, especially under the conditions of 180mM and 210mM ​​ammonium chloride, the growth of WT is severely restricted. Howe...

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Abstract

Belonging to the field of industrial microorganisms, the invention discloses a method for improving the ammonium salt tolerance of synechocystis PCC6803 and application. By means of the method, a synechocystis PCC6803 overexpression strain OE0528 with significantly improved ammonium salt tolerance can be obtained. The algal strain can be used for construction of a high concentration ammonium saltwastewater purified genetic engineering strain. By overexpression of the sll0528 gene in synechocystis PCC6803, the synechocystis PCC6803 overexpression strain OE0528 has significantly improved ammonium salt tolerance. Under 180-210mM ammonium salt stress, the growth status of the overexpression algal strain is obviously superior that of the wild type. The ammonium salt tolerant overexpression algal strain obtained by the invention has important theoretical and practical significance for further construction and utilization of genetic engineering bacteria of high concentration ammonium salt, and has broad application prospects.

Description

technical field [0001] The invention belongs to the field of industrial microorganisms, and in particular relates to a method and application for improving the high-concentration ammonium salt tolerance of Synechocystis sp. PCC6803. Background technique [0002] Ammonium salt is the main nutrient element of microalgae, and it is also a metabolic intermediate product commonly found in microalgae. However, high concentrations of ammonium salts are toxic to most microalgae, which limits the range of survival and application prospects of microalgae, such as the purification of wastewater with higher concentrations of ammonium salts. Therefore, research on the method and application of improving the tolerance of high-concentration ammonium salts of Synechocystis sp. PCC6803 is of great significance for the development of industrial production and application of Synechocystis sp. [0003] The genetic modification of Synechococcus sp. PCC6301, which belongs to the same cyanobacter...

Claims

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Application Information

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IPC IPC(8): C12N1/13C12N15/63C12N15/66C12N15/90C12R1/89
CPCC07K14/405C12N15/63C12N15/66C12N15/902
Inventor 陈谷刘小芳许白雪
Owner SOUTH CHINA UNIV OF TECH
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