Shewanella competent cell preparation method based on thermodynamic compensation strategy

Abstract

The invention is applicable to the field of biotechnology and microbial engineering, and particularly relates to a shewanella competent cell preparation method based on a thermodynamic compensation strategy, and the method comprises the following steps: culturing shewanella to the middle stage of the logarithmic phase, and immediately putting the shewanella in an environment of 0 DEG C for quenching; centrifugally collecting the cooled bacterial liquid by using a centrifugal rotor pre-cooled to 0-4 DEG C; after centrifugation, carrying out ice bath standing for heat exchange offset; and dual-ion echelon washing: resuspending the washed thalli by using a glycerol-containing CaCl2 solution aiming at the thalli precipitate, packaging, quickly freezing by using a liquid nitrogen gas phase layer, and preserving at-80 DEG C. The method gets rid of dependence on a refrigerated centrifuge and an electrotransduction instrument, so that common laboratories and even family laboratories with basic conditions can perform shewanella genetic engineering operation, and the temperature of thalli in the whole process can be strictly controlled below 10 DEG C through a TCIC strategy under the condition of no active refrigeration, so that the activity of competent cells is effectively guaranteed.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology and microbial engineering, and particularly relates to a method for preparing Shewanella competent cells based on a thermodynamic compensation strategy. Background Technology

[0002] Shewanella is a group of Gram-negative facultative anaerobic bacteria widely distributed in marine, freshwater, and sediment environments. Due to its unique extracellular electron transfer (EET) capability, Shewanella has great application potential in fields such as microbial fuel cells (MFCs), bioremediation of heavy metal pollution (such as the reduction of uranium and chromium), and the synthesis of bionanomaterials.

[0003] However, the genetic manipulation system for Shewanella has long suffered from high technical barriers and low accessibility. Existing technologies mainly include: Conjugation: This is currently the most commonly used method, but it is cumbersome, requiring the construction of specific E. coli donor strains (such as WM3064), co-culturing of donor and recipient cells, and multiple rounds of antibiotic screening, typically taking 3-5 days. Electroporation: While highly efficient, it relies on expensive electroporators. More importantly, Shewanella cell membranes are highly mobile, resulting in extremely high cell death rates after electroporation, making parameter optimization difficult. Traditional chemical transformation: The conventional E. coli CaCl2 method is extremely ineffective when directly applied to Shewanella (transformation efficiency is typically <10 CFU / µg). This is mainly because the outer membrane lipopolysaccharide (LPS) structure of Shewanella is more compact and extremely sensitive to temperature fluctuations. Traditional methods require strict maintenance of 0-4°C throughout the process, which necessitates expensive high-speed refrigerated centrifuges.

[0004] Many basic laboratories, teaching laboratories, or temporary field stations are often only equipped with ordinary benchtop centrifuges at room temperature. Because the centrifuge rotor generates a large amount of heat through friction with the air during high-speed rotation ("aerodynamic heating effect"), the internal temperature of a centrifuge can rapidly rise above 30°C after just 10 minutes of centrifugation at room temperature. This drastic temperature change disrupts the "quasi-crystalline" membrane structure formed by Shewanella bacteria at low temperatures, leading to complete failure in the preparation of competent cells.

[0005] Therefore, developing a simple and efficient Shewanella transformation method that does not rely on a refrigerated centrifuge is a key technical issue for breaking down hardware barriers in this field and promoting the penetration of synthetic biology research. Summary of the Invention

[0006] The purpose of this invention is to provide a method for preparing Shewanella competent cells based on a thermodynamic compensation strategy, which aims to solve the problems of existing transformation methods relying on high-speed refrigerated centrifuges, which limit the preparation conditions and result in high transformation costs.

[0007] This invention is achieved through a method for preparing Shewanella competent cells based on a thermodynamic compensation strategy, the method comprising: Bacterial culture and rapid cooling: Shewanella was cultured to the middle of the logarithmic growth phase and immediately placed in a 0°C environment for rapid cooling; Centrifugation harvest: The cooled bacterial culture was collected by centrifuging using a centrifuge rotor pre-cooled to 0-4℃; the centrifugation process was carried out in an intermittent mode, and after each centrifugation, the culture was placed in an ice bath to allow for heat exchange and offsetting. Dual ion-step washing: For bacterial cell precipitates, the cells are first resuspended and washed with pre-cooled MgCl2 solution, collected by centrifugation, and then resuspended and washed with pre-cooled CaCl2 solution. The centrifugation steps during the washing process are performed in an intermittent mode. Resuspension and cryopreservation: The washed bacterial cells were resuspended in a CaCl2 solution containing glycerol, aliquoted, and then rapidly frozen in liquid nitrogen at -80°C.

[0008] Preferably, the centrifugation process adopts an intermittent mode. The step of allowing the centrifuge tube to stand in an ice bath after each centrifugation to offset the heat exchange includes: pre-cooling the metal rotor in an environment of 4°C or lower for at least 4 hours before centrifugation; the centrifugation time is 7-9 minutes and the rotation speed is 3000-5000 rpm; after the centrifugation is completed, the centrifuge tube is immediately removed and incubated in a 0°C ice water bath for 2-5 minutes.

[0009] Preferably, the mid-logarithmic growth phase refers to the range where the OD600 value of the bacterial culture reaches 0.4-0.6.

[0010] Preferably, during the process of resuspending and washing with pre-cooled MgCl2 solution, centrifuging, and then resuspending and washing with pre-cooled CaCl2 solution, 0.1 M MgCl2 solution is used to remove culture medium residues and utilize Mg. 2+ To stabilize the extracellular membrane lipopolysaccharide structure, a 0.1 M CaCl2 solution was used to replace Mg. 2+ It induces the formation of DNA channels in the cell membrane. After a second wash and resuspending, the bacterial culture is placed in a 0°C ice-water bath and incubated for 25-35 minutes.

[0011] Preferably, in the step of resuspending the washed bacterial cells in a glycerol-containing CaCl2 solution, the glycerol-containing CaCl2 solution is a 0.1 M CaCl2 solution containing 15% glycerol by volume.

[0012] Preferably, the liquid nitrogen gas phase quick-freezing step includes: placing the dispensed bacterial cells in the gas phase region of the liquid nitrogen surface where they immediately evaporate for 60-90 seconds, allowing the sample to pass through the ice crystal formation zone, and then transferring it to an ultra-low temperature freezer for storage.

[0013] Preferably, Shewanella includes at least Shewanella putrefactive, Shewanella oneidae, or Shewanella algae.

[0014] Preferably, the steps for chemical transformation using Shewanella competent cells include: thawing frozen competent cells on ice, adding exogenous DNA, gently mixing, and then incubating on ice for 30 minutes; heat-shocking the mixture in a 42°C water bath for 90 seconds, followed immediately by an ice bath for 2-3 minutes; adding antibiotic-free LB liquid medium and thawing at 30°C and 150-200 rpm for 2-4 hours; centrifuging to concentrate the cells and spreading them on solid plates containing the appropriate antibiotics for culture.

[0015] Preferably, the recovery time is 3 hours.

[0016] Preferably, the centrifugal concentration step involves centrifuging the revived bacterial solution at room temperature for no more than 3 minutes, discarding part of the supernatant, and retaining 100 µL of the remaining solution to resuspend the bacterial cells for coating.

[0017] The method for preparing Shewanella competent cells based on a thermodynamic compensation strategy provided by this invention eliminates the dependence on refrigerated centrifuges and electroporators, enabling ordinary laboratories and even home laboratories with basic conditions to perform Shewanella genetic engineering operations. Stable transformation efficiency: Experimental data show that the transformation efficiency of the pBBR1MCS series plasmids in this invention is consistently maintained at 2 × 10⁻⁶. 2 CFU / µg DNA or higher, fully meeting the needs of gene knockout, plasmid construction and complementation experiments; High versatility: It is effective not only against Shewanella putrefactive bacteria, but also against Shewanella oneneda and Shewanella algae; Significant temperature control effect: Through the TCIC strategy, the cell temperature can be strictly controlled below 10℃ throughout the process without active cooling, effectively ensuring the activity of competent cells. Attached Figure Description

[0018] Figure 1 This is a schematic flowchart of a method for preparing Shewanella competent cells based on a thermodynamic compensation strategy, provided in an embodiment of the present invention. Figure 2 This is a schematic diagram of plate colonies after transforming pBBR1MCS-2 plasmid using the method of the present invention in an embodiment of the present invention; Figure 3 This is an electrophoresis image of randomly selected transformants for colony PCR verification, provided in an embodiment of the present invention. Detailed Implementation

[0019] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0020] like Figure 1 The diagram shown is a flowchart illustrating a method for preparing Shewanella competent cells based on a thermodynamic compensation strategy according to an embodiment of the present invention. The method includes: Step 1, cell culture and rapid cooling: Cultivate Shewanella to the middle of the logarithmic growth phase and immediately place it in a 0°C environment for rapid cooling.

[0021] In this step, the Shewanella bacteria include, but are not limited to, *Shewanella putrefaciens*, *Shewanella oneidensis*, or *Shewanella algae*. The Shewanella bacteria are cultured to the mid-logarithmic growth phase and immediately placed in a 0°C environment for rapid cooling to halt cell metabolism. The mid-logarithmic growth phase refers to a bacterial culture with an OD600 value between 0.4 and 0.6. The rapid cooling time is 20-30 minutes.

[0022] Step 2, centrifugation harvest: Use a centrifuge rotor pre-cooled to 0-4℃ to centrifuge and collect the cooled bacterial solution; the centrifugation process adopts intermittent mode, and after each centrifugation, the solution is placed in an ice bath to allow for heat exchange and offsetting.

[0023] In this step, the cooled bacterial culture is collected by centrifuging using a centrifuge rotor pre-cooled to 0-4℃. Centrifugation is carried out in an intermittent mode, and the heat generated by a single centrifugation is offset by heat exchange through subsequent static incubation in a 0℃ ice bath. The intermittent mode specifically includes: pre-cooling the metal rotor in an environment at 4℃ or lower for at least 4 hours before centrifugation; setting the single centrifugation time to 7-9 minutes and the rotation speed to 3000-5000 rpm; immediately after a single centrifugation, removing the centrifuge tube and incubating it in a 0℃ ice-water bath for 2-5 minutes to balance the Joule heat generated by friction during centrifugation and ensure that the bacterial temperature remains below 10℃.

[0024] Step 3, Double ion-step washing: For bacterial precipitates, first resuspend and wash with pre-cooled MgCl2 solution, collect by centrifugation, and then resuspend and wash with pre-cooled CaCl2 solution. The centrifugation steps in the washing process are in intermittent mode.

[0025] In this step, the bacterial precipitate is first resuspended and washed with pre-cooled MgCl2 solution, collected by centrifugation, and then resuspended and washed with pre-cooled CaCl2 solution. The centrifugation step in the washing process also adopts the intermittent mode described in step 2. The specific parameters of the double ion gradient washing are as follows: First wash: using 0.1 M MgCl2 solution, aimed at removing culture medium residues and utilizing Mg 2+ Stabilize the extracellular membrane lipopolysaccharide structure; second wash: using 0.1 M CaCl2 solution to displace Mg. 2+ It induces the formation of DNA channels in the cell membrane; after the second wash and resuspending, the bacterial culture needs to be incubated in a 0°C ice-water bath for 25-35 minutes.

[0026] Step 4, Resuspension and cryopreservation: The washed bacterial cells were resuspended in a CaCl2 solution containing glycerol, aliquoted, and then rapidly frozen in liquid nitrogen at -80°C.

[0027] In this step, the washed bacterial cells are resuspended in a CaCl2 solution containing glycerol, aliquoted, and then rapidly frozen in liquid nitrogen vapor phase and stored at -80°C. The resuspension solution is a 0.1 M CaCl2 solution containing 15% (v / v) glycerol. Rapid freezing in liquid nitrogen vapor phase means placing the aliquoted bacterial cells in the vapor phase region of liquid nitrogen (1-5 cm above the liquid surface) for 60-90 seconds to allow the sample to quickly pass through the ice crystal formation zone, and then transferring it to an ultra-low temperature freezer for storage.

[0028] The process of chemical transformation using competent cells prepared according to this invention is as follows: Thaw frozen competent cells on ice, add exogenous DNA, gently mix, and incubate on ice for 30 minutes; heat shock the mixture in a 42°C water bath for 90 seconds, followed immediately by an ice bath for 2-3 minutes; add antibiotic-free LB liquid medium and revive at 30°C and 150-200 rpm for 2-4 hours; concentrate the bacterial cells by centrifugation and plate them on solid plates containing the appropriate antibiotics; the preferred revival time is 3 hours. Centrifugation concentration refers to centrifuging the revival bacterial solution at room temperature for no more than 3 minutes, discarding most of the supernatant, and resuspending the bacterial cells in approximately 100 µL of the remaining liquid for plating.

[0029] This invention provides a method for preparing Shewanella competent cells based on a thermodynamic compensation strategy. The method is implemented using a non-refrigerated centrifuge, and its main principle is as follows: Precise growth window control: After activation, Shewanella bacteria were transferred to LB liquid medium and cultured at 30°C with shaking until mid-logarithmic growth phase (OD600 = 0.4-0.6). At this stage, the cells are in mid-logarithmic growth, with vigorous cell wall synthesis but not yet excessive cross-linking, making it most suitable for preparing competent cells.

[0030] Metabolic cessation: Immediately place the bacterial culture in a 0°C ice-water bath for 20-30 minutes to lock in the physiological state of the cells.

[0031] Thermocompensated Intermittent Centrifugation (TCIC): Pre-cool the centrifuge rotor at 4°C or lower (preferably -20°C) for at least 4 hours; use this rotor for centrifugation, setting the parameters to 3000-5000 rpm for 7-9 minutes; after centrifugation, quickly remove the centrifuge tubes and immerse them in an ice-water bath for 2-5 minutes to "recool". This step rapidly absorbs the heat accumulated during centrifugation using an external cold source, preventing the bacteria from renaturing due to temperature rise.

[0032] Stepwise ion washing: Discard the supernatant, gently resuspend the bacterial cells in pre-cooled 0.1 M MgCl2 solution, incubate on ice, and then centrifuge; perform TCIC centrifugation again, discard the supernatant; resuspend the bacterial cells in pre-cooled 0.1 M CaCl2 solution, and incubate on ice for 25-35 minutes. This step is the critical induction period for competent cell formation.

[0033] Gas-phase rapid freezing preservation: Centrifuge again and discard the supernatant. Resuspend the contents in a 0.1 M CaCl2 solution containing 15% glycerol (concentrated approximately 100 times). After aliquoting, place the contents in the gas phase layer at the mouth of the liquid nitrogen tank (not below the liquid surface, 1-5 cm away from the liquid surface) for 60-90 seconds to prevent liquid nitrogen from seeping in and causing the tube to burst, while simultaneously achieving vitrification. Then transfer the contents to an ultra-low temperature freezer for storage.

[0034] The effects of the present invention are illustrated by the following experiments: Preparation of experimental materials and reagents: To ensure the reproducibility of the method of this invention, it is recommended that the reagents and consumables involved in the experiment be prepared according to the following standards: Strain source: The Shewanella putrefaciens BS1 and Shewanella oneidensis MR-1 used in this example are both laboratory-preserved strains.

[0035] Reagent formulation and treatment: 0.1 M MgCl2 solution: Weigh 2.03 g magnesium chloride hexahydrate (MgCl2·6H2O, analytical grade) and dissolve it in 100 mL of deionized water.

[0036] After preparation, the solution was sterilized by filtration through a 0.22 µm filter membrane and then pre-cooled at 4°C for later use. 0.1M CaCl2 solution: Weigh 1.47 g of calcium chloride dihydrate (CaCl2·2H2O, analytical grade) and dissolve it in 100 mL of deionized water. Sterilize by filtration through a 0.22 µm filter membrane and pre-cool at 4°C.

[0037] Cryopreservation solution containing 15% glycerol: Measure 15 mL of glycerol (glycerol, purity ≥99%) and mix with 85 mL of the 0.1 M CaCl2 solution prepared above. After thorough mixing, filter through a 0.22 µm filter membrane for sterilization. This solution has a high viscosity and needs to be filtered slowly.

[0038] Pretreatment of utensils: Centrifuge tubes: Select 1.5 mL polypropylene (PP) centrifuge tubes with uniform wall thickness, and pre-cool them at -20℃ for 30 minutes before use.

[0039] Pipe tip: It is recommended to pre-cool the pipette tip used for resuspending bacteria during the experiment at 4°C to prevent heat from being introduced during liquid aspiration.

[0040] Example 1: Preparation of competent cells of Shewanella putrefactive bacteria: 1. Equipment pre-cooling: Remove the metal rotor from a standard benchtop centrifuge (model Eppendorf 5418, without refrigeration function) and place it in a -20°C freezer overnight (approximately 12 hours or more) to pre-cool it, allowing it to accumulate sufficient cold energy. Prepare a sufficient amount of ice-water mixture.

[0041] 2. Cell Culture and Monitoring: Resuscitate *Shewanella putrefactive* BS1 from -80℃ glycerol tubes onto LB agar plates and incubate overnight at 30℃. Inoculate a single colony into 5 mL of LB liquid medium and incubate overnight at 30℃ as a seed culture. The next day, transfer the seed culture to 50 mL of fresh LB medium at a 1:100 ratio (500 µL) and incubate at 30℃ and 200 rpm. Starting from 2 hours of incubation, sample and monitor the OD600 value every 20 minutes. Stop incubation immediately when the OD600 reaches approximately 0.5 (range 0.4-0.6 is acceptable).

[0042] 3. Rapid Cooling and Harvesting (TCIC Strategy): Quickly place the bacterial culture bottle in an ice-water bath and shake for 1-2 minutes, then allow it to cool for 20 minutes. Aliquot the bacterial culture into 1.5 mL centrifuge tubes and attach them to pre-cooled metal rotors. Centrifuge at 4000 rpm for 8 minutes. Key Operation: After the centrifuge stops, immediately (within 30 seconds) remove the centrifuge tubes and place them in an ice-water bath for 3 minutes. At this point, the centrifuge tube walls should feel cool to the touch; if they feel warm, the centrifugation time has been too long or the rotor has not been sufficiently pre-cooled.

[0043] 4. Double ion-step washing: Discard the supernatant and add 1 mL of pre-cooled 0.1 M MgCl2 solution. The bacteria are fragile at this stage; resuspend them by gently tapping the tube wall (do not vigorous blowing). Incubate on ice for 10 minutes. Reload the rotor and repeat the centrifugation and ice-cooling steps (4000 rpm, 8 min, ice bath for 3 min). Discard the supernatant and resuspend the bacteria in 1 mL of pre-cooled 0.1 M CaCl2 solution. Incubate on ice for 30 minutes. Centrifuge and ice-cool again.

[0044] 5. Aliquoting and cryopreservation: Discard the supernatant, add 200 µL of 0.1 M CaCl2 solution containing 15% (v / v) glycerol to resuspend the bacterial cells. At this point, the bacterial suspension will be milky white and viscous. Aliquot into 50 µL / tube. Place the cryopreservation tubes in the vapor layer above the liquid nitrogen surface (about 2 cm below the liquid surface) for 90 seconds. After observing that the liquid inside the tube turns white and solidifies, quickly place it in a freezer at -80°C for storage.

[0045] Example 2: Conversion effect verification: Take 100 µL of competent cells prepared in Example 1, thaw them on ice, add 500 ng of pBBR1MCS-2 plasmid (carrying the tceA gene fragment, kanamycin resistant), mix gently, and incubate on ice for 30 minutes. Heat shock the mixture in a 42°C water bath for 90 seconds, then immediately incubate on ice for 2-3 minutes. Add 900 µL of antibiotic-free LB medium and incubate at 30°C and 180 rpm for 3 hours. Centrifuge the bacterial culture at 5000 rpm for 2 minutes at room temperature, discard approximately 900 µL of supernatant, resuspend the cells in the remaining approximately 100 µL of liquid, and spread on LB agar plates containing 50 µg / mL kanamycin. Incubate upside down at 30°C for 48-65 hours. Results: Approximately 100-120 single colonies (e.g., ...) grew on the plates. Figure 2 (As shown). Five single colonies were randomly selected for colony PCR verification, and all of them amplified a specific band of approximately 1500 bp (as shown). Figure 3 As shown in the figure, the positive rate is 100%. The calculated transformation efficiency is approximately 2.2 × 10^ 2 CFU / µg DNA.

[0046] Example 3: Preparation and transformation of *S. oneidensis* MR-1: To verify the universality of the method of the present invention, the model strain Oneida Shewanella MR-1 was used for testing.

[0047] 1. Preparation process: Except for a slight difference in cell culture time (MR-1 grows slightly faster, taking about 2.5 hours to reach OD 0.5), the other steps of rapid cooling, TCIC centrifugation and double ion washing are completely consistent with those in Example 1.

[0048] 2. Transformation Validation: The prepared MR-1 competent cells were transformed into pBBR1MCS-2 plasmid according to the method described in Example 2. The recovery conditions were adjusted to 30℃ and cultured for 4 hours.

[0049] 3. Results: The transformation efficiency, determined by kanamycin plate screening, was 1.8 × 10^ 2 CFU / µg DNA.

[0050] 4. Conclusion: The results show that the thermodynamically compensated preparation strategy described in this invention has good applicability and stability for different species of Shewanella bacteria.

[0051] Comparative Example 1: No thermodynamic compensation strategy (TCIC) was used: Except for the following steps, the remaining steps were the same as in Example 1: the centrifuge rotor was not pre-cooled (room temperature approximately 25°C), and no ice bath was used for recooling after centrifugation; instead, the next step was performed directly. Results: After centrifugation, the tube wall felt noticeably warm to the touch (measured temperature approximately 32°C). After transformation using the same method as in Example 2, no colonies grew on the plate (0 CFU / µg DNA). Conclusion: This indicates that in a room-temperature centrifuge, if the TCIC strategy of this invention is not employed, centrifugation-induced heat generation will lead to failure in competent cell preparation.

[0052] Comparative Example 2: No MgCl2 pre-washing: Except for the following steps, the remaining steps are the same as in Example 1: In the washing step, the first 0.1 M MgCl2 washing step was omitted, and 0.1 M CaCl2 was used for two washes directly. Results: After transformation using the same method as in Example 2, only about 5-10 small colonies grew on the plate, and the transformation efficiency was approximately 1.5 × 10^ 1 The bacterial count was CFU / µg DNA, and the colony growth was poor. Conclusion: This indicates that the CaCl2 method alone is ineffective for Shewanella, and MgCl2 pretreatment is crucial for stabilizing the outer membrane structure and improving transformation efficiency.

[0053] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for preparing Shewanella competent cells based on a thermodynamic compensation strategy, characterized in that, The method includes: Bacterial culture and rapid cooling: Shewanella was cultured to the middle of the logarithmic growth phase and immediately placed in a 0°C environment for rapid cooling; Centrifugation harvest: The cooled bacterial culture was collected by centrifuging using a centrifuge rotor pre-cooled to 0-4℃; the centrifugation process was carried out in an intermittent mode, and after each centrifugation, the culture was placed in an ice bath to allow for heat exchange and offsetting. Dual ion-step washing: For bacterial cell precipitates, the cells are first resuspended and washed with pre-cooled MgCl2 solution, collected by centrifugation, and then resuspended and washed with pre-cooled CaCl2 solution. The centrifugation steps during the washing process are performed in an intermittent mode. Resuspension and cryopreservation: The washed bacterial cells were resuspended in a CaCl2 solution containing glycerol, aliquoted, and then rapidly frozen in liquid nitrogen at -80°C.

2. The method for preparing Shewanella competent cells based on a thermodynamic compensation strategy according to claim 1, characterized in that, The centrifugation process adopts an intermittent mode. After each centrifugation, the step of allowing the centrifuge tube to stand in an ice bath to offset the heat exchange includes: pre-cooling the metal rotor in an environment of 4°C or lower for at least 4 hours before centrifugation; the centrifugation time is 7-9 minutes and the rotation speed is 3000-5000 rpm; after the centrifugation is completed, the centrifuge tube is immediately removed and incubated in a 0°C ice water bath for 2-5 minutes.

3. The method for preparing Shewanella competent cells based on a thermodynamic compensation strategy according to claim 1, characterized in that, The mid-logarithmic growth phase refers to the period when the OD600 value of the bacterial culture reaches the range of 0.4-0.

6.

4. The method for preparing Shewanella competent cells based on a thermodynamic compensation strategy according to claim 1, characterized in that, Using pre-chilled MgCl2solution for resuspension washing, centrifugation collection, and then using pre-chilled CaCl2solution for resuspension washing, 0.1 M MgCl2solution was used to remove residual culture medium and utilize Mg 2+ Stabilize the extracellular membrane lipopolysaccharide structure, 0.1 M CaCl2solution was used to replace Mg 2+ and induce the cell membrane to form DNA channels, after resuspension and washing for the second time, the bacterial solution was placed in an ice water bath at 0°C and incubated for 25-35 minutes.

5. The method for preparing Shewanella competent cells based on a thermodynamic compensation strategy according to claim 1, characterized in that, In the step of resuspending the washed bacterial cells in a CaCl2 solution containing glycerol, the CaCl2 solution containing glycerol is a 0.1 M CaCl2 solution containing 15% glycerol by volume.

6. The method for preparing Shewanella competent cells based on a thermodynamic compensation strategy according to claim 1, characterized in that, The steps of liquid nitrogen gas phase layer quick-freezing include: placing the dispensed bacterial cells in the gas phase region of liquid nitrogen that immediately evaporates for 60-90 seconds, allowing the sample to pass through the ice crystal formation zone, and then transferring it to an ultra-low temperature freezer for storage.

7. The method for preparing Shewanella competent cells based on a thermodynamic compensation strategy according to claim 1, characterized in that, Shewanella includes at least Shewanella putrefactive, Shewanella oneidae, or Shewanella algae.

8. The method for preparing Shewanella competent cells based on a thermodynamic compensation strategy according to any one of claims 1-7, characterized in that, The steps for chemical transformation using Shewanella competent cells include: thawing frozen competent cells on ice, adding exogenous DNA, gently mixing, and then incubating on ice for 30 minutes; heat-shocking the mixture in a 42°C water bath for 90 seconds, followed immediately by an ice bath for 2-3 minutes; adding antibiotic-free LB liquid medium and thawing at 30°C and 150-200 rpm for 2-4 hours; centrifuging to concentrate the cells and spreading them on solid plates containing the appropriate antibiotics for culture.

9. The method for preparing Shewanella competent cells based on a thermodynamic compensation strategy according to claim 8, characterized in that, The recovery time is 3 hours.

10. The method for preparing Shewanella competent cells based on a thermodynamic compensation strategy according to claim 8, characterized in that, The centrifugation concentration process involves centrifuging the revived bacterial culture at room temperature for no more than 3 minutes, discarding part of the supernatant, and resuspending the bacterial cells in 100 µL of the remaining liquid for plating.

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