Exogenous plasmid in-vitro methylation modification method used for converting different strains

An exogenous plasmid and methylation technology, applied in the field of microorganisms, can solve the problems of inability to carry out, DNA sequence modification, restriction modification system deletion or mutation and other complicated operations, and achieve the effect of simple operation

Inactive Publication Date: 2015-10-28
GUANGDONG INST OF MICROORGANISM
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  • Abstract
  • Description
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Problems solved by technology

[0007] 3. Deletion or mutation of restriction modification system
[0009] Since the above methods are based on the condition that the genome information of the strain is relatively clear, and the DNA sequence modification or the deletion or mutation of the restriction modification system are relatively complicated, when the research object has not yet undergone whole genome sequencing or there is no corresponding commercial methylase strains, the above method cannot be carried out

Method used

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  • Exogenous plasmid in-vitro methylation modification method used for converting different strains
  • Exogenous plasmid in-vitro methylation modification method used for converting different strains
  • Exogenous plasmid in-vitro methylation modification method used for converting different strains

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] Embodiment 1: the acquisition of bacterial strain cell extract;

[0026] The activated Bacillus subtilis ATCC9372 and Bacillus licheniformis ATCC14580 were respectively transferred to LB liquid medium, cultured to logarithmic growth phase at 30°C, 200r / min; centrifuged at 4°C, 5000r / min for 10 minutes to collect the bacteria, and used Wash twice with pre-cooled deionized water; resuspend the bacteria in TNM (100mM Tris-HCl (pH 7.5), 50mM NaCl, 5mM MgCl) of 1 / 3 the volume of the original bacterial solution 2 ) buffer solution, use the ONE SHOT high-pressure cell disruptor to crush the sample under the condition of a pressure of 30kpsi; centrifuge at 5000r / min at 4°C for 10 minutes to remove cell debris, and collect the centrifuged supernatant, which is the cell extract (Cell free extracts, CFE), use immediately or aliquot and store at -70°C for later use.

Embodiment 2

[0027] Embodiment 2: Analysis of bacterial strain restriction modification system;

[0028] Dilute the obtained cell extracts of Bacillus subtilis ATCC 9372 and Bacillus licheniformis ATCC 14580, respectively, and perform restriction experiments according to the following system: cell extract (CFE) 10μL, plasmid DNA (pKSV7) 400-500ng, ddH 2 O up to 20 μL.

[0029] The above reaction system was well mixed and placed at 37°C for 2 hours, and then detected by gel running. Restriction analysis of plasmid pKSV7 by Bacillus subtilis ATCC9372 and Bacillus licheniformis ATCC14580 figure 1 As shown, the cell extracts of Bacillus subtilis ATCC9372 and Bacillus licheniformis ATCC14580 can degrade the exogenous plasmid pKSV7.

Embodiment 3

[0030] Example 3: In vitro methylation modification of exogenous plasmid DNA;

[0031] The obtained cell extracts of Bacillus subtilis ATCC9372 and Bacillus licheniformis ATCC14580 were respectively configured according to the following system for in vitro methylation modification of plasmid DNA:

[0032] Cell extract (CFE) 30μL

[0033] Plasmid pKSV712μg

[0034] EDTA (500mM, pH 8.0) 1μL

[0035] SAM (s-adenosylmethionine) (200 μM) 3 μL

[0036] DTT (dithiothreitol) (100mM) 1μL

[0037] wxya 2 O up to 100μL (SAM in the system provides methyl function)

[0038] The above reaction system was incubated at 37°C for 12 hours. During this period, SAM (200 μM) was added twice, 3 μL each time, and 1× volume of isopropanol was added, precipitated overnight at -20°C, centrifuged at 4°C for 20 minutes, and the supernatant was discarded. , add 1mL volume fraction of 70% ethanol aqueous solution to wash, dry in ultra-clean bench, redissolve in 20μL ddH 2 O, the methylation-modified...

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Abstract

The invention discloses an exogenous plasmid in-vitro methylation modification method used for converting different strains. According to the method, cell-free extracts of bacillus subtilis or bacillus licheniformis are extracted, the cell-free extracts are used for performing in-vitro methylation modification on exogenous plasmid DNA, and therefore the plasmid DNA where methylation modification is performed can be obtained. The cell-free extracts of the bacillus subtilis or the bacillus licheniformis (the cell-free extracts include strain specificity methylase) perform in-vitro methylation modification on exogenous plasmids, and therefore the plasmid DNA where methylation modification is performed can be shifted into recipient bacteria with a restriction modification system, shear degradation caused by the recipient bacteria is avoided, and the exogenous plasmid in-vitro methylation modification method can be used for different strains and new bacteria where whole genome sequencing is not performed. The method is easy to operate.

Description

technical field [0001] The invention belongs to the field of microorganisms, and in particular relates to a method for in vitro methylation modification of exogenous plasmids used for transforming different bacterial strains. Background technique [0002] In recent years, more and more studies have found that an important obstacle for the recipient bacteria to be difficult or impossible to be transformed is the restriction modification system of the recipient bacteria. The main reason is that the modification restriction system can cut and degrade most or even all of the exogenous DNA that enters the cell before it can perform its function, so transformants cannot be obtained or the number of transformants is very small. At present, there are mainly the following solutions: [0003] 1. Specific methylation modification of DNA [0004] At present, since the whole genome sequencing of more and more strains has been completed, the genetic background of these strains is relati...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/10C12N15/75C12R1/125C12R1/10
Inventor 张嘉冯荣芳宋达许玫英孙国萍郭俊
Owner GUANGDONG INST OF MICROORGANISM
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