Method and special culture medium for SILAC labelling of mycobacterium smegmatis protein

A technology of Mycobacterium smegmatis and culture medium, applied in the directions of microorganism-based methods, biochemical equipment and methods, bacteria, etc., can solve the problem of unknown effect, unsatisfactory labeling effect of Mycobacterium smegmatis, negative effects of labeling effect, etc. question

Active Publication Date: 2019-07-12
BEIJING PROTEOME RES CENT
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, this type of medium is not specifically designed for Mycobacterium smegmatis, and the effect of applying this medium to the SILAC marker of Mycobacterium smegmatis is unknown, and the added amino acids are not suitable for cultivating Mycobacterium smegmatis essential nutrients and may even have a negative impact on labeling
Patent CN201210276080 discloses a kind of SILAC labeling medium for Escherichia coli, according to the research of the inventor of the present application, the labeling effect of Mycobacterium smegmatis in the medium of similar components is not ideal

Method used

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  • Method and special culture medium for SILAC labelling of mycobacterium smegmatis protein
  • Method and special culture medium for SILAC labelling of mycobacterium smegmatis protein
  • Method and special culture medium for SILAC labelling of mycobacterium smegmatis protein

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0058] Example 1, Mycobacterium smegmatis proteome marker efficiency test in efficient prokaryote SILAC marker medium

[0059] The inventors firstly used the full marker medium (Ping L, Zhang H, Zhai L, Dammer EB., Duong DM., Li N, Yan Z, Wu J, XuP. Quantitative proteomics reveals significant changes in cell shape and anenergy shift after IPTG induction via an optimized SILAC approach for Escherichia coli.J Proteome Res.2013,12:5978-5988.) on Mycobacterium smegmatis M.smegmatisMC 2 155 conducted SILAC labeling studies. The Escherichia coli SILAC marker medium SILACE used by the inventors is shown in Table 1.1.

[0060] Table 1.1 Components of SILACE special medium for SILAC-labeled Escherichia coli

[0061]

[0062]

[0063] 1.1. Preparation of SILAC-labeled E. coli medium SILACE

[0064] According to the above-mentioned components and their final concentrations, configure with ultrapure water to obtain Escherichia coli SILAC labeling medium SILACE. Inorganic salts a...

Embodiment 2

[0075] Embodiment 2, the design and labeling efficiency test of Mycobacterium smegmatis SILAC labeling medium ZJL-x

[0076] Since Mycobacterium smegmatis did not achieve high labeling efficiency in the medium SILACE, we chose Middlebrook 7H9, a commonly used medium for mycobacteria [Atlas, Ronald M.; James W. Snyder (2006). Handbook of media for clinical microbiology.CRC Press.ISBN 978-0-8493-3795-6.] as the basal medium (the composition of the basal medium is shown in Table 2.0), and the gluten that affects the biosynthesis of lysine and arginine Amino acid and aspartic acid were removed, and Mycobacterium smegmatis-specific SILAC marker medium ZJL-x was designed, the components of which are shown in Table 2.1.

[0077] Table 2.0 Composition of Mycobacterium smegmatis basal medium Middlebrook 7H9

[0078] components Final concentration (mg / L) ammonium sulfate 500 L-Glutamic Acid (L-Glutamic Acid) 500 Pyridoxine 1 biotin 0.5 Sodium ci...

Embodiment 3

[0097] Embodiment 3, the design and labeling efficiency test of Mycobacterium smegmatis SILAC special marker medium ZJL-y

[0098] Based on the results of Example 2, we doubled the content of heavy-labeled amino acids K6 and R10 on the basis of the medium ZJL-x, and designed the Mycobacterium smegmatis SILAC labeling medium ZJL-y as shown in Table 3.1.

[0099] Table 3.1 Components of ZJL-y special medium for SILAC-labeled Mycobacterium smegmatis

[0100]

[0101]

[0102] 3.1. Configuration of Mycobacterium smegmatis SILAC marker medium ZJL-y

[0103] According to the above-mentioned components of ZJL-y and its final concentration, it was configured with ultrapure water to obtain a culture medium for the SILAC labeling experiment of Mycobacterium smegmatis. Inorganic salts and glucose are sterilized by autoclaving, and amino acids are sterilized by filtration.

[0104] 3.2. Extraction of total protein

[0105] The 4th, 5th, 7th, and 8th generation SILAC-labeled M.sme...

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Abstract

The invention discloses a special culture medium for SILAC labelling of a mycobacterium smegmatis protein. The culture medium for SILAC labeling is composed of inorganic salts, histidine hydrochloride, isoleucine, valine, leucine, phenylalanine, tryptophan, serine, multi-stable isotope labelled lysine, multi-stable isotope labelled arginine, threonine, tyrosine, methionine, adenine sulfate, uraciland glucose. The inorganic salts comprise ammonium sulfate, sodium citrate, disodium hydrogen phosphate, potassium dihydrogen phosphate, ammonium ferric citrate, magnesium sulfate, calcium chloride,zinc sulfate and copper sulfate. Experiments prove that the culture medium for SILAC labelling of the mycobacterium smegmatis protein can label the mycobacterium smegmatis protein efficiently within 10 generations, and creates conditions for preparation of quantitative internal standards and efficient and accurate comparative study of quantitative proteome.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a method for using SILAC to mark the proteome of Mycobacterium smegmatis and a special culture medium thereof. Background technique [0002] Tuberculosis (Tuberculosis, TB) is an infectious disease caused by Mycobacterium tuberculosis. It has a wide range of epidemics and a high degree of harm. In 2016, there were 10.4 million new cases of tuberculosis worldwide and more than 1.8 million deaths, about one-third of which were HIV-positive patients. In my country, there are about 1.3 million new patients and 200,000 deaths every year. The situation is also very grim. The emergence of drug-resistant strains tends to accelerate, which intensifies the difficulty of TB prevention, diagnosis and treatment. Strengthening the research on tuberculosis pathogenic bacteria has important clinical value, but Mycobacterium tuberculosis is a slow-growing mycobacterium, and it takes 4-5 weeks for c...

Claims

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Application Information

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IPC IPC(8): C12N1/20C12R1/34
CPCC12N1/20
Inventor 徐平张俊令王富强张瑶李衍常常蕾高慧英
Owner BEIJING PROTEOME RES CENT
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