CN115354041A

CN115354041A - Preparation method of M13 phage single-stranded DNA

Info

Publication number: CN115354041A
Application number: CN202211276917.5A
Authority: CN
Inventors: 李辰
Original assignee: Beijing Junquan Zhiyao Biotechnology Co ltd
Current assignee: Beijing Junquan Zhiyao Biotechnology Co ltd
Priority date: 2022-10-19
Filing date: 2022-10-19
Publication date: 2022-11-18

Abstract

The invention relates to the technical field of biology, in particular to a preparation method of M13mp18 phage single-stranded DNA. The method comprises the following steps: culturing host bacteria until OD value is 10 to 20, and inoculating M13 phage according to MOI of 0.1 to 10 for fermentation culture; centrifuging the co-cultured culture, taking supernatant, ultrafiltering, and precipitating with polyethylene glycol; the precipitate was lysed and purified to obtain M13mp18 phage single stranded DNA. The method greatly improves the expression level of M13 phage single-stranded DNA, and makes up for the disadvantage of low expression level of M13 phage single-stranded DNA in the prior art. The invention reduces the production cost of the M13 phage single-stranded DNA, improves the economic benefit of the M13 phage single-stranded DNA, lays a foundation for the popularization and the application of the DNA nanometer folded paper carrier, and has important significance for preparing the DNA nanometer folded paper carrier in a large scale and improving the economic benefit.

Description

Preparation method of M13 phage single-stranded DNA

Technical Field

The invention relates to the technical field of biology, in particular to a preparation method of M13 phage single-stranded DNA.

Background

DNA origami is an important branch of DNA nanotechnology. The invention of the method is that hundreds of short single-stranded DNAs are used as staples in 2006 (Nature 2006) to assist in folding a phage genome single-stranded DNA (also called scaffold chain) with the length of thousands of bases, and the phage genome single-stranded DNA is self-assembled to form a pre-designed structure. Among them, the single-stranded DNA carried by M13 phage is one of the most commonly used scaffold strands. The M13 phage is a filamentous phage that contains a circular single-stranded DNA molecule that contains the genetic information required for DNA replication and phage propagation. Wherein M13mp series are used for modifying wild type M13, and a multiple cloning site and a LacZ gene are inserted, so that the DNA sequence template can accommodate exogenous DNA300-400bp, and can be used for preparing a single-stranded template and a nucleic acid probe used in DNA sequencing.

The M13 phage particle is filamentous and infects only F + (containing the F plasmid, capable of producing sexual pili) E.coli. After infection of a host, the host cells are usually not lysed, but rather the phage particles are secreted from the infected cells, and the host cells continue to grow and divide.

When M13 phage single-stranded DNA is prepared in the prior art, M13 virus particles are mainly precipitated by a polyethylene glycol method, and then M13 single-stranded DNA is extracted by organic reagents such as an ethanol precipitation method and a phenol extraction method, so that in the aspect of expression, industrialization cannot be realized by linear amplification of equipment, and the expression level is low; in the aspect of extraction, a large amount of organic reagents are generally required to be used, and pre-cooling at-20 ℃ is often required, so that the requirements of a production workshop on environmental protection, explosion prevention and the like are increased, and the industrial amplification cost and risk are greatly increased.

Disclosure of Invention

In view of the above, the present invention provides a method for preparing single-stranded DNA of M13 bacteriophage. The method greatly improves the yield of the M13 phage single-stranded DNA, reduces the production cost of the ssDNA, is suitable for large-scale preparation of the DNA nano origami carrier, and effectively improves the economic benefit.

In order to achieve the above object, the present invention provides the following technical solutions:

a method for preparing M13 phage single-stranded DNA, comprising:

culturing host bacteria until the OD value is 10 to 20, and inoculating M13 phage according to the MOI of 0.1 to 10 for fermentation culture;

centrifuging the co-cultured culture, taking supernatant, ultrafiltering to change liquid, and precipitating with polyethylene glycol; the precipitate is lysed and purified to obtain M13 phage single-stranded DNA.

In the present invention, the specific type of M13 phage is not particularly limited, and M13 phage commonly used in the art may be used. In a particular embodiment, specifically the M13mp18 bacteriophage.

In the present invention, the host bacterium is Escherichia coli, specifically JM109 strain in the specific example.

In some embodiments, the host bacteria are cultured to an OD of 15 and M13 phage is inoculated.

In some embodiments, the MOI is 0.1.

In the present invention, the medium for the fermentation culture contains 5 to 15mM Mg ²⁺ And 10 to 15mM Ca ²⁺ 2 XYT medium.

In some embodiments, the fermentation medium is a medium containing 15mM Mg ²⁺ And 15mM Ca ²⁺ 2 XYT medium.

In the invention, the conditions of the fermentation culture are as follows: the temperature is 37 ℃, the pH value is 7.00, the stirring speed is 150 to 650 rpm, and the dissolved oxygen value DO is 20 to 30 percent; the time for fermentation culture is 4h to 5h.

In the present invention, the polyethylene glycol precipitate comprises: taking supernatant, ultrafiltering to obtain filtrate, adding polyethylene glycol and NaCl to make the final concentration of polyethylene glycol be 4-10 wt% and the final concentration of NaCl be 0.5mmol/L, standing, and centrifuging;

the cracking is carried out by alkali; the purification is column chromatography purification, and the adopted purification column is Sephacryl S-500 HR, sepharose 6 FF or Capto Core 700, preferably Capto Core 700;

centrifuging the co-cultured culture, taking supernatant, ultrafiltering, and precipitating with polyethylene glycol; and (5) cracking and purifying the precipitate.

The invention provides a preparation method of M13 phage single-stranded DNA, which comprises the following steps: culturing host bacteria until the OD value is 10 to 20, and inoculating M13 phage according to the MOI of 0.1 to 10 for fermentation culture; centrifuging the co-cultured culture, taking supernatant, ultrafiltering, and precipitating with polyethylene glycol; the precipitate was lysed and purified to obtain M13mp18 phage single stranded DNA. The method greatly improves the expression level of M13 phage single-stranded DNA, and makes up for the disadvantage of low expression level of M13 phage single-stranded DNA in the prior art. The invention reduces the production cost of the M13 phage single-stranded DNA, improves the economic benefit of the M13 phage single-stranded DNA, lays a foundation for the popularization and the application of the M13 phage single-stranded DNA in the DNA nano origami carrier, and has important significance for preparing the DNA nano origami carrier in a large scale and improving the economic benefit.

Drawings

FIG. 1 shows the effect of different infection timings on phage growth;

FIG. 2 shows the effect of different multiplicity of infection (MOI) on phage growth;

FIG. 3 shows the effect of different feeding conditions on phage growth;

FIG. 4 shows the chromatogram of a Capto Core 700 column chromatography purification.

Detailed Description

The invention provides a preparation method of M13 phage single-stranded DNA. Those skilled in the art can modify the process parameters appropriately in view of the disclosure herein. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations and modifications in the methods and applications disclosed herein, or appropriate variations and combinations thereof, may be made to implement and use the techniques of this invention without departing from the spirit and scope of the invention.

Unless otherwise specified, the test materials used in the present invention are all common commercial products and can be purchased in the market.

The invention is further illustrated by the following examples:

example 1 optimization of fermentation parameters for Co-fermentation of M13mp18 phage and host bacteria-timing of infection

1 purpose: and (3) comparing the influence of different infection moments on the growth condition of the phage on a 5L bioreactor, and determining the optimal infection moment.

2 materials and methods

2.1 culture Medium

2 × YT medium: soybean peptone 1.6%, yeast extract 1%, naCl 0.5%, pH7.0.

2.2 Laboratory apparatus

2.2.1 Constant temperature shaking table: shanghai-Heng THI-300C

2.2.2 An ultra-clean workbench: sujingtai SW-CJ-2FD

2.2.3 A pH meter: mettler FE28+ LE438

2.2.4 An ultramicro photometer: denovix DS-11+

2.2.5 An electronic balance scale: shanghai Yueping 3-0.01

2.2.6 An autoclave: shanghai Boxun YXQ-100A

2.2.7 5L fermentation tank: shanghai high-altitude machine B1OF-6005B1G

2.3 test strains

2.3.1 Host bacteria: escherichia coli JM109 Escherichia coli (Migua) Castellani and Chalmers, purchased from American Type Culture Collection (ATCC) and numbered 53323, and stored as Crop Genetics International N.V., and having the genotype F' traD36 proA + proB + lacIq delta (lacZ) M15 delta (lac-proAB) supE44 hsdR17 recA1 gyrA96 thi-1 end A1 relA1 e 14-lambda-.

2.3.2 Phage display: m13mp18

2.4 test methods

2.4.1 The main experimental parameters of the co-fermentation are as follows: the infection time is as follows: the OD values of JM109 E.coli were 10OD, 15 OD and 20OD

2.4.2 Other parameters of the co-fermentation:

2.4.2.1 Multiplicity of infection (MOI): 0.1

2.4.2.1 Temperature: 37 deg.C

2.4.2.2 Stirring speed: 150 to 650 rpm

2.4.2.3 pH value: 7.00

2.4.2.4 Dissolved oxygen value (DO): 25% +/-5%

The temperature, stirring revolution and pH value parameters are automatically adjusted by setting parameters on the interface of the fermentation tank control system. The ventilation volume is determined by a manual regulating valve and a flowmeter and is 2.0 to 8.0L/min. During the culture period, the stirring speed and the aeration rate of the fermentation tank were adjusted. The pH value in the tank is kept at a set value by automatically controlling the acid and alkali supplementation amount in the fermentation process. And sampling every hour to detect the OD600 value of the bacterial liquid, extracting M13 ssDNA according to the extraction method in the molecular cloning experimental guidance, and recording related data.

3 results of the test

The results are shown in Table 1 and FIG. 1.

TABLE 1 Effect of different infection timings on phage growth

Duration of fermentation	1H	2H	3H	4H	5H
						OD
10	1.29	4.28	8.83	9.42	10.15
						OD 15	5.22	15.08	18.83	19.81	21.18
OD 20	5.77	11.41	11.42	11.63	-

Note: the values in the table are cumulative ssDNA yields in mg/L, which is the amount of ssDNA produced per liter of fermentation broth.

4 analysis of results

As shown in Table 1 and FIG. 1, when the infection timing is OD15 OD of JM109 Escherichia coli, the cumulative ssDNA yield is significantly higher than that of the 10OD and 20OD groups, so the infection timing is selected as OD15 to be optimal.

Example 2 optimization of fermentation parameters for Co-fermentation of M13mp18 phage and host bacteria-multiplicity of infection (MOI)

1 purpose: the effect of different plural infection numbers (MOI) on the growth of the phage on a 5L bioreactor was compared and the optimum plural infection number (MOI) was determined.

2 materials and methods

2.1 culture Medium

2 × YT medium: soybean peptone 1.6%, yeast extract 1%, naCl 0.5%, pH7.0.

2.2 Laboratory apparatus

2.2.1 Constant temperature shaking table: shanghai-Heng THI-300C

2.2.2 Superclean bench: sujingtai SW-CJ-2FD

2.2.3 A pH meter: mettler FE28+ LE438

2.2.4 Ultramicro photometers: denovix DS-11+

2.2.5 An electronic balance scale: shanghai Yueping 3-0.01

2.2.6 An autoclave: shanghai Boxun YXQ-100A

2.2.7 5L fermentation tank: shanghai high machine B1OF-6005B1G

2.3 test strains

2.3.2 Phage display: m13mp18

2.4 test methods

2.4.1 The main experimental parameters of the co-fermentation are as follows: multiplicity of infection (MOI): 0.1, 1, 5, 10

2.4.2 Other parameters of the co-fermentation:

2.4.2.1 The infection time is as follows: the OD value of JM109 Escherichia coli was 15 OD

2.4.2.2 Temperature: 37 deg.C

2.4.2.3 Stirring speed: 150 to 650 rpm

2.4.2.4 pH value: 7.00

2.4.2.5 Dissolved oxygen value (DO): 25% +/-5%

The temperature, stirring revolution and pH value parameters are automatically adjusted by setting parameters on the interface of the fermentation tank control system. The ventilation volume is determined by a manual valve regulation and a flowmeter and is 2.0 to 8.0L/min. During the culture period, the number of stirring revolutions of the fermenter and the amount of aeration were adjusted. The pH value in the tank is kept at a set value by automatically controlling the acid and alkali supplementation amount in the fermentation process. And sampling every hour to detect the OD600 value of the bacterial liquid, extracting M13 ssDNA according to the extraction method of molecular cloning experimental guidance, and recording related data.

Test results

Note: the values in the table are cumulative ssDNA yields in mg/L, i.e., the amount of ssDNA produced per liter of fermentation broth

4 analysis of results

As shown in Table 2 and the data in FIG. 2, cumulative ssDNA yields were significantly higher for 0.1 and 1 infectivity than for 5 and 10 groups, with yields of 0.1 and 1 groups being essentially the same, but with an MOI value of 0.1 being selected as the optimum given that the library size of the phage seed library would be greatly reduced after an order of magnitude reduction in MOI.

Example 3 optimization of fermentation parameters for Co-fermentation of M13mp18 phage and host bacteria-feed conditions

1 purpose: the effect of different feeding conditions on the growth of phage on a 5L bioreactor was compared and the optimal feeding conditions were determined.

2 materials and methods

2.1 reagents

2.1.1 2 × YT medium: soybean peptone 1.6%, yeast extract 1%, naCl 0.5%, pH7.0.

2.1.2 Mg ²⁺ Ion(s)

2.1.3 Ca ²⁺ Ion(s)

2.2 Laboratory apparatus

2.2.1 Constant temperature shaking table: shanghai-Heng THI-300C

2.2.2 Superclean bench: sujingtai SW-CJ-2FD

2.2.3 A pH meter: mettler FE28+ LE438

2.2.4 Ultramicro photometers: denovix DS-11+

2.2.5 Electronic balance scale: shanghai Yueping 3-0.01

2.2.6 An autoclave: shanghai Boxun YXQ-100A

2.2.7 5L fermentation tank: shanghai high-altitude machine B1OF-6005B1G

2.3 test strains

2.3.1 Host bacteria: escherichia coli JM109 Escherichia coli (Migua) Castellani and Chalmers, purchased from American Type Culture Collection (ATCC) and numbered 53323, stored as Crop Genetics International N.V., and having the genotype F' traD36 proA + proB + lacIq delta (lacZ) M15 delta (lac-proAB) supE44 hsdR17 recA1 gyrA96 thi-1 end A1 relA1 e14-lambda-.

2.3.2 Phage display: m13mp18

2.4 test methods

2.4.1 Main experimental parameters of co-fermentation: see Table 3 for details

2.4.2 Other parameters of the co-fermentation:

2.4.2.2 Multiplicity of infection (MOI): 0.1

2.4.2.3 Temperature: 37 deg.C

2.4.2.4 Stirring speed: 150 to 650 rpm

2.4.2.5 pH value: 7.00

2.4.3.6 Dissolved Oxygen (DO): 25% +/-5%

The temperature, stirring revolution and pH value parameters are automatically adjusted by setting parameters on the interface of the fermentation tank control system. The ventilation volume is determined by a manual valve regulation and a flowmeter and is 2.0 to 8.0L/min. During the culture period, the number of stirring revolutions of the fermenter and the amount of aeration were adjusted. The pH value in the tank is kept at a set value by automatically controlling the acid and alkali supplementation amount in the fermentation process. And sampling every hour to detect the OD600 value of the bacterial liquid, extracting M13 ssDNA according to the extraction method in the molecular cloning experimental guidance, and recording related data.

3 results of the test

TABLE 3 Effect of different multiplicity of infection (MOI) on phage growth

Experiment number	1	2	3	4	5
						Supplementary culture medium	-	-	3L 2×YT	3L	2×YT	3L	2×YT
MgCl ₂ Concentration of	-	15mM	5mM	15mM	15mM
						CaCl ₂ Concentration of	-	15mM	10mM	10mM	15mM
1H	0.12	0.07	0.03	4.80	0.07
						2H	12.36	12.98	13.17	21.84	11.50
3H	24.12	22.63	34.05	39.56	25.97
						4H	29.16	25.96	41.70	44.94	49.78
5H	33.05	32.28	50.20	55.20	58.42

4 analysis of results

As shown in Table 3 and FIG. 3, the fed combination ssDNA used in experiment No. 5 gave the highest yield.

In combination with the above experimental results, it can be determined that the optimal protocol for preparing phage single-stranded DNA of the present invention is the protocol with experiment number 5: the infection time is JM109 Escherichia coli OD value 15, the infection complex number (MOI) is 0.1, and the feeding conditions are as follows: containing 15mmol/LMg ²⁺ 15mmol/L Ca ²⁺ 2 XYT medium.

EXAMPLE 4 column chromatography purification of M13 Single stranded DNA

1. The purpose is as follows: and (3) purifying the cracked M13 single-stranded DNA by using Capto Core 700 column chromatography, removing residual host protein and endotoxin, and replacing the traditional ethanol precipitation method.

2. Materials and methods

2.1 Reagent and consumable

2.1.1 A chromatographic column: capto Core 700

2.1.2 Mobile phase: 20mM Tris-HCl,150mM NaCl, pH 8.0

2.1.3 Reagent:

2.1.3.1 PBB2 (50 mL volume)

0.4g NaOH

0.5g SDS

Adding water to 50mL

2.1.3.2 PBB3 (50 mL volume)

12.3g sodium acetate or 14.72g potassium acetate

5.75mL of glacial acetic acid

Adding water to 50mL

2.1.3.3 75% ethanol (-20 ℃ precooling)

2.1.3.4 Anhydrous ethanol (-20 ℃ precooling)

2.2 Laboratory apparatus

2.2.1 A centrifuge: LYNX 4000 hummer flyer

2.2.2 A purification instrument: saipu SDL 100

2.3 Experimental materials: m13 single-stranded DNA (deoxyribonucleic acid) crudely extracted by PEG (polyethylene glycol) method and alkaline cracking method

2.4 Test method

2.4.1 PEG method for extracting M13 particles

2.4.1.1 The method is described in detail in molecular cloning, experimental Manual 4 th edition (supra), scheme 8 polyethylene glycol precipitation method for preparing M13 phage single-stranded DNA, P24-P26.

2.4.2 Alkaline cracking method for extracting M13 single-stranded DNA

2.4.2.1 According to the proportion of PBB2: m13 viral particle solution =1:2, adding the PBB2 solution, stirring the mixture evenly and gently, and then respectively standing the mixture at room temperature for 3min.

2.4.2.2 According to the PBB3: m13 viral particle solution =1: adding PBB3 solution according to the proportion of 1.5, stirring the mixture evenly and softly, and standing the mixture for 10min at the temperature of between 2 and 8 ℃.

2.4.2.3 The mixture was centrifuged at 16500g,30min,4 ℃ and the precipitate was discarded. If the supernatant was cloudy, it was centrifuged once more at 16500g,15min,4 ℃. The supernatant was collected.

2.4.3 Control group: ethanol precipitation method

2.4.3.1 The method is described in molecular cloning instruction 4 th edition (upper volume) scheme 8 polyethylene glycol precipitation method for preparing M13 phage single-stranded DNA, P24-P26.

2.4.4 Experimental groups: capto Core 700 column chromatography

2.4.4.1 The supernatant of the cleaved M13 single-stranded DNA was applied to a Capto Core 700 column.

2.4.4.2 And collecting the flow-through liquid according to the light absorption value of 260nm, wherein the flow-through liquid is used as a target object.

2.4.4.3 Ultraviolet concentration determination, scanning wave band: 230-300 nm. And detecting the host protein and endotoxin residue levels.

3. Test results

3.1 chromatographic results

The chromatographic results are shown in FIG. 4.

3.2 M13 single-stranded DNA recovery and impurity removal levels

TABLE 4

Group of	Yield of M13 Single-stranded DNA	Host protein removal rate	Endotoxin levels
				Control group: ethanol precipitation method	62.1%	73.8%	0.00705≤X＜0.07052
Experimental groups: the column chromatography of the invention	85.5%	95.4%	0.00041≤X＜0.00408

4 analysis of results

The results show that the column chromatography method is superior to the traditional ethanol precipitation method in terms of yield, impurity residue level and the like, and the difficulty of the construction of future production workshops is greatly reduced due to the elimination of ethanol.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that it is obvious to those skilled in the art that various modifications and improvements can be made without departing from the principle of the present invention, and these modifications and improvements should also be considered as the protection scope of the present invention.

Claims

1. A method for preparing a single-stranded DNA of M13 phage, comprising:

centrifuging the co-cultured culture, taking supernatant, ultrafiltering, and precipitating with polyethylene glycol; the precipitate is lysed and purified to obtain M13 phage single-stranded DNA.

2. The method of producing according to claim 1, wherein the M13 phage is an M13mp18 phage.

3. The method according to claim 1, wherein the host bacterium is Escherichia coli.

4. The method according to claim 3, wherein the Escherichia coli is JM109 strain.

5. The method according to claim 1, wherein the host bacterium is cultured to an OD of 15, and M13 phage is inoculated.

6. The method of claim 1, wherein the MOI is 0.1.

7. The method according to claim 1, wherein the fermentation culture medium contains 5 to 15mM Mg ²⁺ And 10 to 15mM Ca ²⁺ 2 XYT medium.

8. The method of claim 4, wherein the fermentation medium comprises 15mM Mg ²⁺ And 15mM Ca ²⁺ 2 XYT medium.

9. The method according to claim 1, wherein the conditions of the fermentation culture are:

the temperature is 37 ℃, the pH value is 7.00, the stirring speed is 150 to 650 rpm, and the dissolved oxygen value DO is 20 to 30 percent.

10. The method of claim 1, wherein the polyethylene glycol precipitation comprises: taking supernatant, ultrafiltering to obtain filtrate, adding polyethylene glycol and NaCl to make the final concentration of polyethylene glycol be 4-10 wt% and the final concentration of NaCl be 0.5mmol/L, standing, and centrifuging;

the cracking is carried out by alkali; the purification is column chromatography purification, and the adopted purification column is Sephacryl S-500 HR, sepharose 6 FF or Capto Core 700;