CN113801210A - Induced expression and purification method of microbial nanowires - Google Patents
Induced expression and purification method of microbial nanowires Download PDFInfo
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Abstract
The invention provides an inducible expression and purification method of a microorganism nanowire, belonging to the technical field of protein engineering. According to the invention, the microbial nanowires are sheared mechanically or in a shaking way, and then are repeatedly purified by methods of ethanolamine suspension and ammonium sulfate precipitation, so that the finally obtained microbial nanowires are uniform in size and good in shape and structure.
Description
Technical Field
The invention relates to an inducible expression and purification method of a microorganism nanowire, belonging to the technical field of protein engineering.
Background
Microbial nanowires (also known as protein nanowires), originally found in Geobacter sulfurettii, are a flagella-like attachment structure for microorganisms. The microbial nanowires have conductivity, can reach tens of microns in length, and can transfer electrons to an extracellular electron acceptor, so that bacteria can breathe in a severe environment lacking a soluble and permeable membrane electron acceptor (such as oxygen), and the microbial nanowires have a unique effect in microbial life activities.
The microbial nanowires are considered as a green energy material with great application value, have good biocompatibility and biodegradability, and represent an important future development direction of microbial electrochemistry. The excellent conductivity of the microbial nano-wire endows the microbial nano-wire with higher electron transfer efficiency compared with other electron transfer modes, and the special structural form endows the microbial nano-wire with the capability of enabling an electroactive microbe to realize the extracellular electron transfer without directly contacting an electron acceptor. However, the research on the microbial nanowires is still in the initial stage. Firstly, the comparative study of inducing the microorganism to express the nanowire by different methods is rarely reported; secondly, the electron transfer mechanism of the microbial nanowires is still controversial, the ecological function is still to be explored, and the practical application is still to be expanded. The current main technical bottleneck is how to improve the induced expression and purification efficiency of the microorganism nanowires and promote the scale application of the microorganism nanowires.
Disclosure of Invention
The invention aims to provide a method for inducing expression and purifying a microorganism nanowire, which provides a foundation for the structure and function research and application of the microorganism nanowire.
The purified nano-wires produced by Geobacter sulfurfuglucens of the sulfur-reducing geobacillus are formed by tightly stacking and polymerizing cytochrome OmcS or IV-type pili consisting of PilA protein, so that the nano-wires produced by other microorganisms are distinguished.
In the method for inducible expression and purification of the microbial nanowires provided by the invention, the selected microbes comprise all microbes (pure bacteria or mixed bacteria) capable of producing the conductive nanowires, including but not limited to Geobacter sulfureates; shewanella oneidensis; flexistipes sinusarabici DSM 4947; calditerrivibrio nidoreductuses DSM 19672; desuifuriobrio alkaliphilus AHT 2; methanospirillum Hungatherei and Acidithiobacillus ferrooxidans, and the like.
The induction expression method of the microorganism nano-wire provided by the invention is any one of the following 1) to 3):
1) inducing microorganisms to produce microorganism nanowires under the condition of insufficient electron acceptors and suboptimal temperature;
2) inducing a microorganism to produce a microorganism nanowire in the absence of a soluble electron acceptor;
3) inducing microbial production of microbial nanowires in a microbial electrochemical system using an electrode as the sole electron acceptor;
the microorganism is pure bacteria or mixed bacteria capable of generating conductive nanowires, and the mixed bacteria comprise anaerobic sludge, mixed bacteria enriched in Geobacter, pre-acclimated bacteria of a microbial electrochemical system and the like;
the microorganism can be Geobacter sulfureatensend Shewanella oneidensis, and specifically can be Geobacter sulfureatensend;
the 1) can be specifically: inducing Geobacter sulfuriducens to generate nanowires at a sub-optimal temperature of 25 ℃ by taking 40mM fumarate as an electron acceptor;
the 2) can be specifically: ferric citrate or other ferric manganese oxides with poor crystallinity are used as electron acceptors to induce Geobacter sulfuridurens to generate nanowires;
in the 3), the microbial electrochemical system can be a microbial fuel cell or a microbial electrolysis cell;
the electrodes can be graphite, charcoal electrodes, carbon cloth, carbon felt and carbon brushes; or self-made electrodes such as graphene-based electrodes and noble metal-based electrodes.
The self-made biochar electrode comprises the following steps:
cutting fresh sugarcane into 2 × 2cm pieces with thickness of 1-2mm, and drying in oven at 60 deg.C overnight;
it was then placed in a tube furnace at N2Heating at 900 deg.C under air for 1 h;
the carbonized sugarcane charcoal is processed into a required size (1X 1cm) and is connected with a titanium wire to prepare a required electrode.
The invention also provides a purification method of the microorganism nano wire.
The purification method of the microorganism nano wire provided by the invention comprises the following steps:
(1) collecting the microorganism expressing the nanowires, and suspending the microorganism in 150mM ethanolamine buffer;
(2) shearing the nanowires, and centrifuging to remove cells;
(3) the nanowires in the supernatant were precipitated overnight with 5-30% ammonium sulfate (based on the saturation of ammonium sulfate in water) and then centrifuged;
(4) resuspending the precipitate in ethanolamine buffer solution, centrifuging to remove impurities;
(5) collecting the nanowires in the supernatant, precipitating with 5-30% ammonium sulfate, and centrifuging;
(6) re-suspending the nanowires in the precipitate in ethanolamine buffer solution, dialyzing with deionized water, and storing at 4 deg.C.
For the microbial nanowires produced by method 1), the operation of step (1) is: 13000g of the well-grown Geobacter sulfureatensection solution is centrifuged, and cells are collected and fully suspended in 150mM ethanolamine buffer (pH 10.5);
for the microbial nanowires produced by method 2), the operation of step (1) is: 13000g of the culture solution was centrifuged, and the cells were collected and suspended in 150mM ethanolamine buffer (pH 10.5) thoroughly;
for the microbial nanowires produced by method 3), the operation of step (1) is: when the electricity generation of the microbial electrochemical system reaches the maximum value, collecting the anode biofilm and suspending the anode biofilm in 150mM ethanolamine buffer (pH 10.5);
in the step (2), the nanowire is sheared from the surface of the microorganism, the method for shearing the nanowire can be a mechanical method or a vibration method,
the mechanical method may be implemented by a tissue mashing and homogenizing machine JJ-2B, and more particularly, may be a method of shearing the nanowires 60s at the highest speed (16000r/min) in the tissue mashing and homogenizing machine JJ-2B,
the oscillation method can be realized by a vortex oscillator, and more specifically, the method can be realized by using the vortex oscillator to violently oscillate and separate the nanowires for 120 s;
in the step (2), the centrifugal force may be specifically 13,000g centrifugal force to remove cells;
in the step (3), the centrifugal force may be specifically 13,000 g;
in the step (4), the precipitate is resuspended in ethanolamine buffer solution by violently shaking the centrifugal tube for 10s by using a vortex oscillator;
the centrifugal force is to remove impurities by 23,000g of centrifugal force;
in the step (5), the centrifugal force may be specifically 13,000 g;
in step (6), the nanowires were resuspended in ethanolamine buffer by vigorously shaking the centrifuge tube for 10s using a vortex shaker.
The purified microorganism nano-wire is characterized by a transmission electron microscope, and the sample preparation method is negative dyeing by using phosphotungstic acid or uranyl acetate and the like.
After the microbial nanowires are sheared and purified, the size and the shape structure of the microbial nanowires are well maintained, and the microbial nanowires are uniformly dispersed.
The invention has the following effects: according to the invention, the microbial nanowires are sheared mechanically or in a shaking way, and then are repeatedly purified by methods of ethanolamine suspension and ammonium sulfate precipitation, so that the finally obtained microbial nanowires are uniform in size and good in shape and structure.
Drawings
FIG. 1 shows a well-grown Geobacter sulffuriduens strain;
FIG. 2 is a microbial nanowire expressed by Geobacter sulffuridunens, a strain of Geobacter sulffuridunens;
FIG. 3 is a schematic diagram of the operation of a microbial fuel cell, wherein R is a resistor; e.g. of the type-An electron;
FIG. 4 is a biofilm mass expression microorganism nano-wire formed by mixed bacteria on an electrode;
FIG. 5 shows the mixed bacteria-induced expression of microorganism nanowires with ferric citrate as the sole electron acceptor;
FIG. 6 shows purified Geobacter sulfurreducensens nanowires after sulfur reduction;
FIG. 7(A) is a graph showing that too long shearing time results in too short microbial nanowires; (B) to resuspend the microbial nanowires that are not completely dispersed.
Detailed Description
The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.
Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
Example 1 three methods for inducing microbial nanowire expression
The first method comprises the following steps: geobacter sulfureatedeus expression microorganism nanowires were induced inside the anaerobic bottles using fumarate (40mM) as an electron acceptor.
1. Preparing a culture medium (1.5g/L ammonium chloride, 0.775g/L sodium dihydrogen phosphate dihydrate, 0.1g/L potassium chloride, 2.5g/L sodium bicarbonate, 1.23g/L sodium acetate, 1g/L yeast, 6.4g/L disodium fumarate, 0.5g/L cysteine, 10mL/L trace element solution and 10mL/L vitamin solution), adding into an anaerobic bottle, and aerating and deoxidizing for 1h by using high-purity nitrogen;
2. sealing the anaerobic bottle, and sterilizing in an autoclave at 121 deg.C for 20 min;
3. inoculating Geobacter sulfuriducens in an anaerobic incubator, wherein acetate (15mM) serves as an electron donor and fumarate (40mM) serves as an electron acceptor;
4. geobacter sulfurlowers were cultured in a chemical thermostat at 25 ℃ under strict anaerobic conditions, and the well-grown Geobacter sulfurlowers in pink color as shown in FIG. 1. As shown in FIG. 2, the microorganism nanowires expressed by Geobacter sulfuriduccus.
And the second method comprises the following steps: the electrode is used as a unique electron acceptor in a microbial electrochemical system to induce pure bacteria Geobacter sulfuriducens or mixed bacteria to express microbial nanowires.
1. And (3) constructing a microbial electrochemical reactor (as shown in figure 3).
Wherein the electrode uses a self-made biochar electrode as a unique electron acceptor;
the self-made biochar electrode comprises the following steps:
cutting fresh sugarcane into 2 × 2cm pieces with thickness of 1-2mm, and drying in oven at 60 deg.C overnight;
it was then placed in a tube furnace at N2Heating at 900 deg.C for 1h under air;
processing the carbonized sugarcane charcoal into a required size (1 multiplied by 1cm) and connecting the processed sugarcane charcoal with a titanium wire to prepare a required electrode;
2. adding electrolyte (2g/L sodium acetate, 12.5mL/L microelement solution, 5mL/L vitamin solution, 50mM PBS buffer solution), removing oxygen with high purity nitrogen aeration for 30min, and sterilizing at 121 deg.C for 20 min;
3. inoculating mixed bacteria (pre-acclimatized bacteria by using a microbial electrochemical system), wherein 2g/L acetate is used as an electron donor;
4. and monitoring the voltage change of the microbial electrochemical system by using a voltage data acquisition system. As shown in fig. 4, the biofilm formed on the electrode by the mixed bacteria expresses a large amount of microbial nanowires.
And the third is that: ferric citrate is used as an electron acceptor to induce the Geobacter sulfureatenser or mixed bacteria (anaerobic sludge) to express the microorganism nano-wire.
1. Preparing a culture medium (2g/L sodium acetate, 12.2g/L ferric citrate, 12.5mL/L microelement solution, 5mL/L vitamin solution and 50mM PBS buffer solution), adding into an anaerobic bottle, and using high-purity nitrogen to aerate and remove oxygen for 1 h;
2. sealing the anaerobic bottle, and sterilizing in an autoclave at 121 deg.C for 20 min;
3. inoculating anaerobic sludge, wherein acetate is used as an electron donor, and ferric citrate is used as a sole electron acceptor. As shown in fig. 4, the expression of the microorganism nanowires was induced from the inside of the mixed bacteria using ferric citrate as the sole electron acceptor.
Example 2: method for purifying microorganism nano-wire
The method comprises the following steps of collecting microorganisms for expressing the microorganism nanowires, wherein three modes correspond to three methods for inducing the microorganisms to express the nanowires:
the first method comprises the following steps: 13000g of the well-grown Geobacter sulfureatenserin solution is centrifuged by using a centrifuge, and cells are collected and fully suspended in 150mM ethanolamine buffer (pH 10.5);
and the second method comprises the following steps: when the electricity generation of the microbial electrochemical system reaches the maximum value, collecting the anode biofilm and suspending the anode biofilm in 150mM ethanolamine buffer (pH 10.5);
and the third is that: 13000g of the cell suspension was centrifuged using a centrifuge, and the cells were collected and sufficiently suspended in 150mM ethanolamine buffer (pH 10.5);
shearing the nanowires (shearing nanowires from cells at a maximum speed of 16000r/min for 60s in a tissue mashing homogenizer JJ-2B, or separating nanowires by vigorous shaking using a vortex shaker for 120s), centrifuging at 13,000g to remove cells;
the nanowires in the supernatant were precipitated overnight with 10% ammonium sulfate (based on the saturation of ammonium sulfate in water) and then centrifuged at 13,000 g;
resuspending the pellet in ethanolamine buffer and removing other debris with a centrifugal force of 23,000 g;
the nanowires were collected, precipitated with 10% ammonium sulfate, and then centrifuged at 13,000 g;
the final nanowires were resuspended in ethanolamine buffer, dialyzed against deionized water, and stored at 4 ℃. Fig. 6 is the purified microorganism nanowires.
Example 3: microbial nanowire purification parameter optimization
3.1 nanowire shear time optimization:
short shear times (30-60 s): shearing the nanowires for 30s at the highest speed of 16000r/min in a tissue mashing and homogenizing machine JJ-2B, or violently oscillating and separating the nanowires for 60s by using a vortex oscillator, so that the nanowires cannot be sheared from the surfaces of microorganisms, and the purification yield is reduced, and the method comprises the following specific steps:
1. the well-grown bacterial suspension of Geobacter sulfurfuglucens was centrifuged at 13000g using a centrifuge, and the cells were collected and sufficiently suspended in 150mM ethanolamine buffer (pH 10.5);
2. shearing the nanowires (30 s of shearing nanowires from cells at a maximum speed of 16000r/min in a tissue mashing homogenizer JJ-2B, or 60s of separating nanowires by vigorous shaking using a vortex shaker), removing cells by centrifugation at 13,000 g;
3. the nanowires in the supernatant were precipitated overnight with 10% saturated ammonium sulfate and then centrifuged at 13,000 g;
4. resuspending the pellet in ethanolamine buffer and removing other debris with a centrifugal force of 23,000 g;
5. the nanowires were collected, precipitated with 10% saturated ammonium sulfate, and centrifuged at 13,000g, failing to obtain a precipitate.
Moderate shear time (60-120 s): shearing the nanowires for 60s at the highest speed of 16000r/min in a tissue mashing and homogenizing machine JJ-2B, or violently oscillating and separating the nanowires for 120s by using a vortex oscillator, wherein the purified nanowires are shown in FIG. 6 and comprise the following specific steps:
1. the well-grown bacterial suspension of Geobacter sulfurfuglucens was centrifuged at 13000g using a centrifuge, and the cells were collected and sufficiently suspended in 150mM ethanolamine buffer (pH 10.5);
2. shearing the nanowires (shearing nanowires from cells at a maximum speed of 16000r/min for 60s in a tissue mashing homogenizer JJ-2B, or separating nanowires by vigorous shaking using a vortex shaker for 120s), centrifuging at 13,000g to remove cells;
3. the nanowires in the supernatant were precipitated overnight with 10% saturated ammonium sulfate and then centrifuged at 13,000 g; resuspending the pellet in ethanolamine buffer and removing other debris with a centrifugal force of 23,000 g;
4. collecting the nanowires, precipitating with 10% ammonium sulfate, and centrifuging at 13,000 g;
5. the final nanowires were resuspended in ethanolamine buffer, dialyzed against deionized water, and stored at 4 ℃.
Long shear time (90-180 s): in the tissue mashing and homogenizing machine JJ-2B, the nanowires are sheared for 90s at the highest speed of 16000r/min, or the nanowires are separated by vigorous oscillation for 180s by using a vortex oscillator, and the shearing time is too long, which causes the shearing of the nanowires to be too short, thus being not beneficial to further purification application, as shown in fig. 7A, the specific steps are as follows:
1. the well-grown bacterial suspension of Geobacter sulfurfuglucens was centrifuged at 13000g using a centrifuge, and the cells were collected and sufficiently suspended in 150mM ethanolamine buffer (pH 10.5);
2. shearing the nanowires (shearing nanowires from cells at a maximum speed of 16000r/min for 90s in a tissue mashing homogenizer JJ-2B, or separating nanowires by vigorous shaking using a vortex shaker for 180s), centrifuging at 13,000g to remove cells;
3. the nanowires in the supernatant were precipitated overnight with 10% saturated ammonium sulfate and then centrifuged at 13,000 g;
4. resuspending the pellet in ethanolamine buffer and removing other debris with a centrifugal force of 23,000 g; collecting the nanowires, precipitating with 10% ammonium sulfate, and centrifuging at 13,000 g;
5. the final nanowires were resuspended in ethanolamine buffer, dialyzed against deionized water, and stored at 4 ℃.
Final optimization time: the nanowires were sheared at the highest speed for 60s in the tissue mashing homogenizer JJ-2B, or separated by vigorous shaking for 120s using a vortex oscillator.
3.2 nanowire resuspension optimization:
resuspending without dispersion: as shown in fig. 7B, the microorganism nanowires are not completely dispersed by shaking the centrifugal tube upside down for 10 times, which results in agglomeration, and the specific steps are as follows:
1. centrifuging the well-grown Geobacter sulfurturedeucens bacterial solution by 13000g centrifugal force by using a centrifugal machine, collecting cells, and shaking the centrifugal tube upside down for 10 times to suspend the cell in 150mM ethanolamine buffer solution (pH 10.5);
2. shearing the nanowires (shearing nanowires from cells at a maximum speed of 16000r/min for 60s in tissue mashing homogenizer JJ-2B), centrifuging at 13,000g to remove cells;
3. the nanowires in the supernatant were precipitated overnight with 10% saturated ammonium sulfate and then centrifuged at 13,000 g; the pellet was shaken upside down to resuspend the tube in ethanolamine buffer 10 times and the other debris was removed with a centrifugal force of 23,000 g;
4. collecting the nanowires, precipitating with 10% ammonium sulfate, and centrifuging at 13,000 g;
5. the final nanowires were resuspended in ethanolamine buffer, dialyzed against deionized water, and stored at 4 ℃.
Resuspending and dispersing: as shown in fig. 6, the centrifugal tube was vigorously shaken by a vortex oscillator for 10s, and the microbial nanowires were completely dispersed, the specific steps were as follows:
1. the well-grown bacterial suspension of Geobacter sulfurfuglucens was centrifuged at 13000g using a centrifuge, and the cells were collected and suspended in 150mM ethanolamine buffer (pH 10.5) by vigorously shaking the centrifuge tube 10s using a vortex shaker;
2. shearing the nanowires (shearing nanowires from cells at a maximum speed of 16000r/min for 60s in tissue mashing homogenizer JJ-2B), centrifuging at 13,000g to remove cells;
3. the nanowires in the supernatant were precipitated overnight with 10% saturated ammonium sulfate and then centrifuged at 13,000 g;
4. resuspend the pellet in ethanolamine buffer using a vortex shaker shaking vigorously the centrifuge tube for 10s and remove other debris with a centrifugal force of 23,000 g;
5. collecting the nanowires, precipitating with 10% ammonium sulfate, and centrifuging at 13,000 g;
6. and (3) suspending the final nanowire in ethanolamine buffer solution by vigorously shaking the centrifugal tube for 10s by using a vortex oscillator, dialyzing by using deionized water, and storing at 4 ℃.
And (3) final resuspension optimization scheme: and (3) violently oscillating the nanowires for 10s by using a vortex oscillator during resuspension of the nanowires, so that the nanowires are fully dispersed.
Comparing the purified microorganism nano-wire with the original nano-wire:
as shown in fig. 2, 4, and 5, the generation of nanowires by microorganisms was successfully induced by different methods.
As shown in fig. 6, after the microbial nanowires are sheared and purified, the size and the morphology structure of the microbial nanowires are kept well, and the microbial nanowires are uniformly dispersed, which indicates that the microbial nanowires are successfully purified.
Claims (7)
1. An induction expression method of microorganism nano-wires comprises the following steps: any one of the following 1) to 3):
1) inducing microorganisms to produce microorganism nanowires under the condition of insufficient electron acceptors and suboptimal temperature;
2) inducing a microorganism to produce a microorganism nanowire in the absence of a soluble electron acceptor;
3) electrodes are used as the sole electron acceptor in microbial electrochemical systems to induce microbial production of microbial nanowires.
2. The method of claim 1, wherein: the microorganism is pure bacteria or mixed bacteria capable of generating the conductive nano-wire,
the microorganism can be specifically Geobacter sulfureatensen, Shewanella oneidensis;
the mixed bacteria comprise anaerobic sludge, mixed bacteria enriched with Geobacter and pre-domesticated bacteria of a microbial electrochemical system.
3. The method according to claim 1 or 2, characterized in that: the 1) is as follows: inducing Geobacter sulfuriducens to generate nanowires at a sub-optimal temperature of 25 ℃ by taking 40mM fumarate as an electron acceptor;
the 2) is as follows: using ferric citrate or ferric manganese oxide with poor crystallinity as an electron acceptor to induce Geobacter sulfuridurons to generate nanowires;
in the 3), the microbial chemical system is a microbial fuel cell or a microbial electrolysis cell;
the electrode is graphite, a biochar electrode, carbon cloth, a carbon felt, a carbon brush, a graphene-based electrode or a precious metal-based electrode.
4. A method for purifying microbial nanowires, comprising the steps of:
(1) collecting the microorganism expressing the nanowires, and suspending the microorganism in 150mM ethanolamine buffer;
(2) shearing the nanowires, and centrifuging to remove cells;
(3) precipitating the nanowires in the supernatant with 5-30% ammonium sulfate overnight, and centrifuging;
(4) resuspending the precipitate in ethanolamine buffer solution, centrifuging to remove impurities;
(5) collecting the nanowires in the supernatant, precipitating with 5-30% ammonium sulfate, and centrifuging;
(6) re-suspending the nanowires in the precipitate in ethanolamine buffer solution, dialyzing with deionized water, and storing at 4 deg.C.
5. The method of claim 4, wherein: for the microbial nanowires produced by the method 1) of claim 1, the operation of step (1) is: 13000g of the well-grown Geobacter sulfureaterucens bacterial liquid is centrifuged, and cells are collected and fully suspended in 150mM ethanolamine buffer solution;
for the microbial nanowires produced by the method 2) of claim 1, the operation of step (1) is: 13000g of the culture solution was centrifuged, and the cells were collected and suspended in 150mM ethanolamine buffer (pH 10.5) thoroughly;
for the microbial nanowires produced by the method 3) of claim 1, the operation of step (1) is: when the electricity generation of the microbial electrochemical system reaches the maximum value, the anode biofilm is collected and suspended in 150mM ethanolamine buffer.
6. The method of claim 4, wherein: in the step (2), the method for shearing the nanowires is a mechanical method or a vibration method,
the mechanical method is realized by a tissue mashing and homogenizing machine JJ-2B, more particularly, the method can be realized by shearing the nanowire 60s at 16000r/min in the tissue mashing and homogenizing machine JJ-2B,
the oscillation method is realized by a vortex oscillator, and more specifically, the nanowires can be separated by vigorous oscillation for 120s by using the vortex oscillator.
7. The method according to any one of claims 4-6, wherein: in the step (2), the centrifugation is a centrifugal force of 13,000g to remove cells;
in the step (3), the centrifugal force is 13,000 g;
in the step (4), the precipitate is resuspended in ethanolamine buffer solution by violently shaking the centrifugal tube for 10s by using a vortex oscillator; the centrifugation is to remove impurities by a centrifugal force of 23,000 g;
in the step (5), the centrifugal force is 13,000 g;
in step (6), the nanowires were resuspended in ethanolamine buffer by vigorously shaking the centrifuge tube for 10s using a vortex shaker.
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