Reporting system for detecting cyclic diguanylic acid (c-di-GMP) content in living cells and application of reporting system

A cyclic diguanylic acid and reporting system technology is applied in the field of reporting systems for detecting the content of cyclic diguanylic acid in living cells, and achieves the effects of authentic and credible detection results, simple and easy operation methods, and convenient detection.

Inactive Publication Date: 2013-06-26
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

How to detect the content of intracellular c-di-GMP is very important, and there is no method in the prior art that can be used to detect c-di-GMP in living cells

Method used

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  • Reporting system for detecting cyclic diguanylic acid (c-di-GMP) content in living cells and application of reporting system
  • Reporting system for detecting cyclic diguanylic acid (c-di-GMP) content in living cells and application of reporting system
  • Reporting system for detecting cyclic diguanylic acid (c-di-GMP) content in living cells and application of reporting system

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Embodiment 1 Contains the construction of the recombinant plasmid of reporter system

[0042] 1 PCR amplification of sequence fragment containing promoter-riboswitch-SD

[0043] Using the genomic DNA of Shewanella oneidensis MR-1 as a template, primers were used for PCR amplification to obtain a fragment (SEQ ID No.4) containing the promoter-riboswitch-SD sequence;

[0044] The primer sequences are:

[0045] Upstream primer 1072p-5': 5'-GGAATTCTGGCATTTGACATAAGCACA-3'; the underline is the EcoR I restriction site (SEQ ID No.6);

[0046] downstream primer

[0047] The underline is the Hind III restriction site, and the boxed sequence is the first 14 bp of the coding sequence of the lac Z gene (SEQ ID No.7); it was synthesized by Shanghai Sangon Bioengineering Technology Co., Ltd.

[0048] The PCR system is:

[0049]

[0050] The PCR conditions were: pre-denaturation at 95°C for 2 min; denaturation at 95°C for 10 s, annealing at 57°C for 20 s; extension at 72°C fo...

Embodiment 2

[0064] Example 2 Obtain the conversion relationship between intracellular c-di-GMP content and reporter gene expression level

[0065] (1) Convert WM3064

[0066] Take out the competent cell WM3064 prepared in advance and stored in -80℃ refrigerator, put it on ice for 5-10min; add 5μL pHGR01-1072p recombinant plasmid, mix well; put it on ice for 30min, heat shock at 42℃ for 90s, add Add 1mL of liquid LB medium to a final concentration of 300μM diaminopimelic acid (DAP), shake slowly at 37°C for 60min; spread onto LB plates containing 50μg / mL kanamycin (KM) and 300μM DAP, at 37°C Culture overnight;

[0067] (2) Identification of positive clones

[0068] Pick a single clone and place it in 20 μL ddH 2 In O, cook at 100°C for 5 minutes, and use pHGR01 universal primers as templates for PCR amplification. The sequence of the pHGR01 universal primers is as follows:

[0069] Upstream primer R01-F: 5'-CAGGCATTTGAGAAGCACACG-3' (SEQ ID No.8);

[0070] Downstream primer R01-R: 5'-C...

Embodiment 3

[0091] Example three specificity analysis

[0092] S pHGR01-1072 was cultured in LB liquid medium containing c-di-GMP structural analogue c-di-AMP (20 μM, 500 μM) to detect whether the reporter system of the present invention specifically binds to intracellular c-di-GMP, and the results are as follows Figure 4 shown.

[0093] Depend on Figure 4It can be seen that the reporter system of the present invention can specifically bind intracellular c-di-GMP and indicate the content of intracellular c-di-GMP.

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Abstract

The invention discloses a reporting system for detecting the cyclic diguanylic acid (c-di-GMP) content in living cells and an application of the reporting system. The reporting system comprises a promoter, a riboswitch, an SD (Shine-Dalgarnosequence) sequence and a reporter gene which are connected in sequence. The application is characterized in that the reporting system is transferred into cells to be detected, reconstitution cells are placed into a culture solution for culture until the OD600 (Optical Density) of the culture solution reaches 0.4 to 0.6, then the expression quantity of the reporter genes in the recombinant cells is detected, and the cyclic diguanylic acid content in the cells to be detected is calculated according to the following formulas: cyclic diguanylic acid content=-1072ln (miller unit-CK) +793.35, wherein the miller unit represents the expression quantity of the reporter genes in the recombinant cells; and the CK represents the background level expression quantity of the reporter genes in the cells to be detected. By adopting the reporting system disclosed by the invention, the c-di-GMP content in the living cells can be conveniently detected; the operation method is simple, convenient and easy to implement; and the detection result is real and reliable.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, in particular to a reporting system for detecting the content of cyclic diguanylic acid in living cells and its application. Background technique [0002] Cyclic bis(3'-5') diguanylic acid (c-di-GMP) is a ubiquitous second messenger molecule in bacteria, which is involved in the regulation of various physiological functions of bacteria, especially in the formation of bacterial biofilms. It plays a crucial regulatory role in the production of pathogenic factors. [0003] Bacterial biofilm is a film-like substance attached to the surface of the bacterial community formed by the adhesion of polymers such as exopolysaccharides produced by bacteria to resist the external adverse environment. Bacterial biofilm can be used for water purification, and it also plays an important role in maintaining the balance of the ecological environment, especially the aquatic environment, and can promote ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/02C12N15/63
Inventor 高海春张海燕
Owner ZHEJIANG UNIV
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