WO2013091443A1

WO2013091443A1 - Device for induced mutation breeding of microorganisms by plasma

Info

Publication number: WO2013091443A1
Application number: PCT/CN2012/083909
Authority: WO
Inventors: 毕鲜荣; 冯权; 葛楠
Original assignee: 北京思清源生物科技有限公司
Priority date: 2011-12-20
Filing date: 2012-11-01
Publication date: 2013-06-27
Also published as: CN102382763A

Abstract

Disclosed is a device for induced mutation breeding of microorganisms by plasma, which belongs to the field of atmospheric pressure plasma technical processing devices. The device for induced mutation breeding of microorganisms by plasma comprises a control system, a plasma generator (7), and an object stage (10). The control system controls the object stage (10) and the plasma generator (7). The plasma generator (7) emits a plasma jet for the induced mutation breeding of a breeding sample on the object stage (10). The plasma generator uses the plasma jet generated under the conditions of atmospheric pressure and low voltage for the induced mutation breeding of biological samples. The plasma jet has high uniformity and low temperature, avoiding the phenomenon of large numbers of microbial samples dying due to excessively high temperatures of the plasma jet. The device has a perfect function, easy operation method, and thus has a good application prospect in the biotechnology field.

Description

Plasma microbial mutation breeding equipment

The invention belongs to the field of atmospheric pressure plasma technology processing equipment, and particularly relates to a plasma microorganism mutation breeding equipment for microbial mutagenesis breeding. Background technique

Microbial strains are the basis and key to the fermentation industry. A good microbial strain can increase the efficiency of the fermentation industry and reduce the cost of the product. Therefore, microbial breeding is becoming more and more important. With the continuous development of research, many different levels of microbial mutagenesis technology have emerged and matured. Mutagenesis breeding has made rapid progress in the field of transforming microbial strains, solving some special problems in breeding work and developing new products, not only overcoming the shortcomings of conventional breeding cycles, slow progress, and difficulty in obtaining highly efficient varieties. Compared with genetic engineering, mutation breeding is simple, especially for the transformation of strains with complex metabolic networks. It is more efficient and cost-effective. Therefore, mutation breeding has always been an important breeding method in the fermentation industry. .

In recent years, research on the application of plasma to microbial mutation breeding has also begun. The plasma is the fourth state of the substance coexisting with the solid, liquid and gaseous states of the substance, and is a partially ionized gas having a density of positive ions and electrons substantially equal. Plasma has special physical and chemical properties such as light, heat, sound and electricity. It has been widely used in many fields such as ozone synthesis, preparation of ultraviolet light source, and manufacture of high-power co ₂ laser. In the past literatures and products, plasma is usually used as a physical sterilization method, which has the advantages of fast, low temperature, easy operation, non-toxicity and good killing effect. However, the use of plasma for biological mutation breeding is still a brand new. There are few related technologies and technologies. There is no biological mutation breeding equipment developed by plasma technology.

Table 1 Comparison of various mutagenesis methods surroundings

Popularization equipment ^ m

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Rate characteristic

Sexuality

(M, E, bowel simple danger

y dye

Wait)

Learning

Simple

Lure simple danger

Dyeing

Change

Simple

-m. simple danger

> dye

Jane 3⁄4«

Uv simple special «preparation, complex

Martyrdom

Miscellaneous (high)

'thing

Special «preparation

Rational

Miscellaneous (high)

Lure

Special Φ3⁄4 preparation, complex

Faster neutron

Miscellaneous (high)

Specialized

- Miscellaneous <high)

Specialized

Seed bundle 3⁄4Λ

Miscellaneous (high)

Special «preparation

Mm

Miscellaneous

Specialized, simple ^ 3⁄4ΐΜ

Wrap

Change <fl£>

At present, there are many devices that use plasma technology to treat solid surfaces, and research on microbial mutation breeding has been in the laboratory stage, and no mature products can be used. Because the physical process of plasma is complex and has the characteristics of nonlinearity, strong coupling, and variable load, it needs to be monitored and controlled by a complete automation system in practical applications, especially for normal pressure conditions. The product of low temperature plasma thus limits the application of this technology in the field of microbial breeding. At present, with the continuous expansion of the application field of plasma technology, especially the emergence of atmospheric pressure low temperature plasma and technological advancement, it has greatly overcome the need for vacuum equipment for conventional cold plasma. The shortcomings will be vigorously promoted in the application and development of microbial breeding, disinfection, chemical reaction, material processing and other fields. Therefore, the need to develop an atmospheric pressure plasma microbial breeding device has become more and more urgent. Summary of the invention

It is an object of the present invention to provide a plasma microbial mutagenesis breeding apparatus. The plasma microbial mutagenesis breeding apparatus includes a control system, a plasma generator, and a stage. The control system controls the stage and the plasma generator. The plasma generator emits a plasma jet to induce mutagenesis of the breeding sample on the stage. The device has perfect functions and simple operation, and can excite gas under normal pressure to generate plasma, which has a good application prospect in the field of biotechnology.

According to one aspect of the invention, the control system includes a controller, a power supply system, and a gas flow control system. The controller is connected to a power system, a stage, and a gas flow control system. The power system and gas flow control system are coupled to the plasma generator. The controller controls the power system and the gas flow control system, thereby controlling the plasma generator. With the control system of the present invention, the processing time, processing power, and working gas flow required for processing can be accurately controlled, and the plasma jet temperature can be controlled by adjusting the power of the RF power source loaded onto the plasma generator.

According to one aspect of the invention, the controller employs a programmable controller. The controller controls the parameters of the plasma generator by controlling the parameters of the power system and the gas flow control system. This precisely controls the parameters required for the operation.

According to one aspect of the invention, the plasma microbial mutagenesis breeding device further comprises a touch screen control panel. The touch screen control panel communicates with the controller to cause the controller to execute operational commands input by the touch screen control panel, control the mutagenesis breeding process, and adjust and deliver control parameters based on the plasma operating state.触摸The touch screen control panel makes it easy for the operator to operate and intuitively enter the corresponding operating conditions.

According to one aspect of the invention, the power supply system includes a radio frequency power supply and a radio frequency power supply matching module. The RF power source is connected to the plasma generator via the RF power matching module to control the operation of the plasma generator by controlling the opening and closing of the RF power source.

According to one aspect of the invention, the flow of gas to the plasma generator is controlled by a controller that controls the flow of gas to the gas flow control system.

According to an aspect of the invention, the controller is controlled according to a position sensor for monitoring the stage The lifting and lowering of the stage causes the distance between the nozzle of the plasma generator and the stage to be adjusted. By controlling the distance between the nozzle and the stage, environmental conditions such as the temperature of the breeding environment can be accurately achieved.

According to an aspect of the invention, the distance between the nozzle of the plasma generator and the stage is 1 to 20 mm. Preferably, the distance is 1-10 mm.

According to one aspect of the invention, the plasma microbial mutagenesis breeding apparatus further includes a cooler for cooling the plasma generator. The cooler can cool the plasma generator to ensure stable discharge and to make the generated plasma jet temperature lower than the microbial tolerance temperature, thereby adjusting the temperature near the stage.

According to one aspect of the invention, the plasma microbial mutagenesis breeding device further comprises an operating cabinet. The stage is disposed inside the operating cabinet, and the nozzle of the plasma generator passes through the wall of the operating cabinet to align with the stage. This ensures the alignment of the nozzle with the stage and the stability of the nozzle.

The invention has the beneficial effects of providing an atmospheric pressure plasma microbial breeding device with the following remarkable features and effects: precise control of processing time, processing power and processing gas flow required for processing, and loading by adjustment The RF power supply on the plasma generator controls the plasma jet temperature. Microorganisms can be mutagenized at room temperature and atmospheric pressure without the need to evacuate the chamber or sterilize the chamber. During the irradiation process, the bacteria, the colonies on the plate or the lyophilized powder are directly placed on the loading platform, and the irradiation of the cells and the plasma generator can be performed by the rotation and lifting operation to complete the irradiation. DRAWINGS

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the embodiments or the description of the prior art will be briefly described below. Obviously, the drawings in the following description are only It is a certain embodiment of the present invention, and other drawings can be obtained from those skilled in the art without any creative work.

Figure 1 is an overall schematic view of a plasma microbial breeding device.

Figure 2 is a schematic view of the structure inside the operating room.

Figure 3 is a control structure diagram of a plasma microbial breeding device.

Figure 4 is a typical working flow chart of a plasma microbial breeding equipment.

Figure 5 is a plot of input power versus plasma temperature, where the temperature probe is 2 mm from the generator outlet, 10 liters per helium, and 1 min.

Figure 6 is a graph of processing time versus plasma temperature, where the temperature probe is from the generator outlet 2 mm, helium flow rate 10 l/min, RF power input power 100 W. detailed description

The technical solutions in the embodiments of the present invention are clearly and completely described in the following with reference to the accompanying drawings in the embodiments of the present invention. It is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments obtained by a person of ordinary skill in the art based on the embodiments of the present invention without creative efforts are within the scope of the present invention.

The present invention provides a plasma microbial mutagenesis breeding apparatus. The objects, the technical solutions and the advantages of the present invention will become more apparent from the following detailed description.

In the schematic diagram of the plasma microbial mutagenesis breeding apparatus shown in Figures 1-3, the breeding apparatus includes a housing and a plasma generator for processing the sample disposed within the housing. On the left side of the housing 1 is an operation chamber 2 in which a load platform 10 on which a sample is placed is disposed, and a nozzle 12 connected to the plasma generator 7 extends through the side wall of the operation chamber into the operation chamber, the temperature sensor 13 and the position sensor 11 are fixed on the inner wall of the operation room 2, the breeding sample 14 is placed on the load platform 10; on the right side of the housing, the plasma generator 7, the radio frequency power source 8 and the controller 9 are placed outside the operation chamber; The device 9 is respectively connected to the RF power source 8, the gas flow control module 16, the temperature sensor 13, the position sensor 11, the load platform 10, the cooler 5 and the touch screen control board 4; the plasma generator 7 and the RF power source 8 pass the RF power supply The matching module 15 is connected; the gas flow control module 16 is connected to the plasma generator 7. The cooler 5 is mounted at any position within the casing 1 as long as the cooling function of the plasma generator 7 can be achieved. The wall of the casing 1 is provided with a touch screen control panel 4 and a power control button 3 for the user to control the controller. The bottom of the housing 1 is provided with at least four anchor bolts 6, so that the housing can be controlled to maintain balance.

The controller is a programmable controller, and performs real-time communication with the touch screen control board 4 through a serial cable or a field bus, and is used to execute an operation instruction input by the touch screen control panel 4, control the mutation breeding process, and according to the plasma working state. Adjust and deliver control parameters.

The parameters of the plasma generator and the feedback of the operating state parameters thereof are automatically or manually controlled by the RF power source 8, the RF power matching module 15 and the gas flow control module 16, respectively; automatically or manually adjusting the plasma generator The flow rate of the working gas provided by the nozzle; controlling the opening and closing and power of the RF power source 8, thereby controlling whether the plasma generator 7 operates and operating power, automatically or manually adjusting the RF power matching module, optimizing the control circuit of the plasma generator Working parameters, increasing the incident power to reduce the reflected power, improving the energy utilization efficiency; the distance between the nozzle and the load platform is fed back to the controller by the temperature sensor 13 and the position sensor 11 for the temperature and position signals of the collected load platform. The lift of the load platform is programmed by the controller, or by manually controlling the lift platform 10 to adjust its vertical distance from the nozzle 12 of the plasma generator 7. And to control the start and stop of the cooler, and the coolant circulation speed, a cooling jacket is provided outside the plasma generator 7. The cooler is cooled by a cooling liquid, and the coolant is circulated in a jacket of the plasma generator to cool the plasma generator. The coolant does not need to be replaced during operation. The controller 9 also includes a module that controls the start and stop of the cooler. The cooler can cool the plasma generator to ensure stable discharge and make the generated plasma jet temperature lower than the tolerance temperature of the microorganism, thereby adjusting the temperature near the load platform; and achieving the purpose of controlling the temperature of the breeding environment.

The plasma generator 7 of the present invention uses a plasma jet generated under atmospheric pressure and low voltage conditions to mutagenize a biological sample, which has high hooking property and low temperature, and avoids a large amount of microorganisms caused by excessive jet temperature. The phenomenon of lethality of the sample; the variety of discharge gases used in the method of the invention greatly enriches the types of active particles in the excited state, and because of its richer physical and chemical effects, a more abundant mutant strain can be obtained. The plasma generator 7 can be fabricated using existing techniques.

Figure 4 is a schematic flow chart showing the operation of the plasma microbial breeding equipment. The complete operating procedure for a typical breeding sample is as follows:

First step, preferred operation of the strain

According to the kind of the breeding sample 14, the strain of the corresponding species is preferably selected, and the target product amount required for the starting strain is used as the screening strain for the later screening of the strain. The present invention is carried out by the model strain Escherichia coli DH5α (purchased from the Institute of Microorganisms of the Chinese Academy of Sciences). Mutation breeding

Step 2 Preparation of sample slides to be processed

(1) Treatment of Escherichia coli DH5 α or spores: a. Preparation of bacterial suspension: Centrifuge the cultured cells in logarithmic phase, wash 2-3 times with normal saline, and dilute with physiological saline. Prepare the bacterial suspension (OD600=0.8-1); b. Prepare the spore suspension: Pick the spore colonies on the solid medium to the test tube containing the appropriate amount of normal saline, shake the spores to prepare the spore suspension, or in the bevel In a test tube, physiological saline was shaken to disperse the spores to prepare a spore suspension (106 under the microscope);

(2) Preparation of breeding sample 14: Under aseptic conditions, the bacteria prepared in the above a or b Suspension or spore solution, added dropwise to the fungus slide, the amount of addition is 20 ML;

(3) Air-drying treatment of breeding sample 14: Under aseptic conditions, the liquid slide prepared in the above step (2) was air-dried under cold air for 20 minutes until the surface was wet and no obvious droplets were optimal.

The third step is mutagenic operation

(1) The prepared breeding sample 14 is placed on the positioning recess on the loading platform 10 with tweezers, and the position and height of the loading platform 10 are adjusted so that the sample to be processed is located directly below the outlet of the nozzle 12 of the plasma generator 7. , and the distance is appropriate (reference distance is 2 - 3 mm). According to the pre-experiment results, the processing time is set in the touch screen control panel 4 (bacterial reference processing time l-3 min), the temperature sensor probe is 2 mm from the generator outlet, the helium flow rate is 10 liters/min, and the processing time is 1 min; The "Start Processing" button of the touch panel control panel 4 starts automatic processing, the touch screen control panel 4 prompts "Processing" with a yellow-black flashing light, and the time column on the touch screen control panel 4 displays the remaining time; Test plasma input power and The relationship between plasma jet temperature (as shown in Figure 5);

(2) After the timer is finished (or after clicking the touch panel control panel 4 "interrupt processing" button), the plasma generator 7 automatically stops discharging; open the operating room door to lower the stage, remove the sample carrier with tweezers, and place it in preparation A good test tube or centrifuge tube with a suitable amount of sterile saline.

(3) Strongly shake the test tube or tube 3 times, 2 minutes each time, at intervals of 5 minutes. The cells on the slide were thoroughly washed and suspended in physiological saline.

Figure 5 is a graph showing the relationship between the input power of the device and the plasma jet temperature. Test conditions: The temperature sensor probe is 2 mm from the generator outlet, the helium flow rate is 10 liters/min, and the treatment time is 1 min.

Figure 6 is a graph showing the relationship between the processing time of the device and the temperature of the plasma jet. Test conditions: The temperature sensor probe is 2 mm from the generator outlet, the helium flow rate is 10 liters / minute, and the RF power input power is 100 watts.

Table 2 shows the experimental data of the lethality rate of the model strain Escherichia coli DH5α (purchased from the Institute of Microbiology, Chinese Academy of Sciences). The sample slide is 2 mm from the plasma generator outlet, and the helium flow rate is 10 liters/ Minute, RF power input power 100 W)

Table 2 Plasma microbial breeding test results

The device has perfect functions and simple operation, and can excite gas under normal pressure conditions to generate plasma, which has a good application prospect in the field of biotechnology.

The above description is only the preferred embodiment of the present invention and is not intended to limit the scope of the present invention. Any modifications, equivalents, improvements, etc. made within the spirit and scope of the invention are intended to be included within the scope of the invention.

Claims

Claim

A plasma microbial mutagenesis breeding device, wherein the plasma microbial mutagenesis breeding device comprises a control system, a plasma generator (7), a stage (10), and the control system controls the load The stage (10) and the plasma generator (7), the plasma generator (7) emits a plasma jet to perform mutagenesis breeding on the breeding sample on the stage (10).

The plasma microorganism mutagenesis breeding apparatus according to claim 1, wherein the control system comprises a controller (5), a power supply system (8), and a gas flow control system (16), the controller (5) Connecting the power system (8), the stage (10) and the gas flow control system (16), the power system (8) and the gas flow control system (16) connecting the plasma A body generator (7), the controller (5) controls the power supply system (8) and the gas flow control system (16), thereby controlling the plasma generator (7).

The plasma microorganism mutagenesis breeding apparatus according to claim 1, wherein the controller (5) uses a programmable controller, and the controller (5) controls the power system (8) and The parameters of the gas flow control system (16) control the parameters of the plasma generator (7).

The plasma microorganism mutagenesis breeding apparatus according to claim 3, wherein the plasma microorganism mutagenesis breeding device further comprises a touch screen control panel (4), the touch screen control panel (4) and the controller (5) communicating, thereby causing the controller (5) to execute an operation command input by the touch panel control panel (4), control the mutation breeding process, and adjust and deliver the control parameter according to the plasma operating state.

The plasma microbial mutagenesis breeding device according to any of the preceding claims, wherein the power supply system (8) comprises a radio frequency power source (17) and a radio frequency power matching module (15), the radio frequency power source (17) Connected to the plasma generator (7) via the RF power matching module (15) to control the operation of the plasma generator (7) by controlling the opening and closing of the RF power source (17).

The plasma microbial mutagenesis breeding device according to any of the preceding claims, wherein the gas flow rate of the gas flow control system (16) is controlled by the controller (5), thereby controlling access to the The gas flow rate of the plasma generator (7).

The plasma microbial mutagenesis breeding device according to any of the preceding claims, wherein the controller (5) controls the stage according to a position sensor (11) for monitoring the stage The lifting of (10), thereby adjusting the distance between the nozzle (12) of the plasma generator (7) and the stage (10).

The plasma microorganism mutagenesis breeding apparatus according to claim 7, wherein a distance between the nozzle (12) of the plasma generator (7) and the stage (10) is 1 to 20 Millimeter.

The plasma microbial mutagenesis breeding apparatus according to any of the preceding claims, wherein the plasma microbial mutagenesis breeding apparatus further comprises a cooler for cooling the plasma generator (7) .

10. Plasma microbial mutagenesis breeding apparatus according to any of the preceding claims, wherein said plasma microbial mutagenesis breeding apparatus further comprises an operating cabinet (2), said stage

(10) disposed inside the operating cabinet (2), the nozzle (12) of the plasma generator (7) is aligned with the stage through a wall of the operating cabinet (2) ( 10).