7.16 Experimenters: Yangxin Zhang, Yixuan Xie

Summary: In this experiment, we used gene sequences related to flagellar movement, including CheA, CheY, and CheZ, and designed relevant PCR primers using SnapGene software. Overnight shaking culture was performed for E. coli BL21 and E. coli DH5α stored in the laboratory.

1.Experimental Materials

Table 1 Main Plasmids and Strains Used in This Experiment

Table2 The primers used in this study

2. Experimental Methods and Results

2.1 Plasmid Maps

Figure 1 Map of Plasmid PBBR1MCS-5-CheA

Figure 2 Map of Plasmid PBBR1MCS-5-CheY

Figure 3 Map of Plasmid PBBR1MCS-5-CheZ

2.2 Preparation of LB Medium (150 mL × 9, including 3 batches of solid medium)

Each liter of LB medium contains 10 g sodium chloride (NaCl), 5 g yeast extract, and 10 g tryptone. The medium was sterilized using an autoclave at 115°C for 20 minutes. For solid LB medium, 2% (w/v) agar powder was added as a coagulant, followed by autoclaving at 115°C for 20 minutes. After cooling to room temperature, a gel-like solid medium was formed.

2.3 Bacterial Shaking Culture

DH5α and BL21 were taken out of the refrigerator; 100 μL of each strain was added to a culture flask containing 6 mL of LB medium, followed by shaking culture at 37°C for 12–16 hours (overnight).

Notes: All operations should be performed near a Bunsen burner. Do not place the flask lid on the workbench. After use, seal the medium and pipette tips in their original packaging.

7.18 Experimenters：Yangxin Zhang Yixuan Xie

Summary: The plasmid backbone of pBBR1MCS-5 was amplified, and plasmids were constructed via seamless cloning and restriction enzyme ligation. PCR amplification and electrophoresis verification of relevant genes and vectors revealed errors in the amplification of the plasmid backbone.

1. Experimental Methods and Results

1.1 Plasmid Extraction (Using a Trial Kit)

1. Take the overnight-cultured bacterial solution, centrifuge at 10,000×g for 1 minute, and discard the supernatant (aspirate as much as possible). For large volumes of bacterial solution, centrifuge in multiple batches to collect the bacterial pellet.
2. Add 500 μL of colorless Solution RB (containing RNase A) and vortex to resuspend the bacterial pellet; no small bacterial clumps should remain.
3. Add 250 μL of blue Solution LB, gently invert and mix 4–6 times to fully lyse the bacteria, forming a clear blue solution. A color change from translucent to clear blue indicates complete lysis (lysis time should not exceed 5 minutes).
4. Add 750 μL of yellow Solution NB, gently mix 5–6 times (the color changes from blue to yellow completely, indicating thorough mixing and neutralization) until a tight yellow aggregate is formed, and let stand at room temperature for 2 minutes.
5. Centrifuge at 12,000×g for 5 minutes, carefully aspirate the supernatant and add it to a spin column. Centrifuge at 12,000×g for 1 minute, and discard the flow-through. If the supernatant volume exceeds 800 μL, add it to the column in multiple batches, centrifuge as above, and discard the flow-through each time.
6. Add 650 μL of Solution WB, centrifuge at 12,000×g for 1 minute, and discard the flow-through.
7. Centrifuge at 12,000×g for 1–2 minutes to completely remove residual WB.
8. Place the spin column in a clean centrifuge tube, add 30–50 μL of Elution Buffer (EB) or deionized water (pH > 7.0) to the center of the column membrane, and let stand at room temperature for 1 minute.(Note: Preheating EB or deionized water in a 60–70°C water bath improves elution efficiency).
9. Centrifuge at 10,000×g for 1 minute to elute DNA. Store the eluted DNA at -20°C.

1.2 PCR

The plasmid backbone, CheA, CheY, and CheZ were amplified using the primers designed on August 4.

Table 1 General PCR Reaction System

Table 2 General PCR Reaction Program

1.3 DNA Purification

The above PCR products were recovered for DNA purification.

1. Add 3 volumes of Buffer DP to the PCR tube containing the PCR product, vortex to mix, and centrifuge briefly.
2. Transfer the mixture to a HiPure DNA column with a collection tube, and centrifuge at 12,000 rpm for 1 minute.
3. Discard the liquid, add 600 μL of Buffer DW2, and centrifuge at 12,000 rpm for 30 seconds.
4. Repeat step (3) once.
5. Discard the liquid, and centrifuge the column empty for 2 minutes.
6. After centrifugation, dry the column in a 65°C metal bath for 7–8 minutes to completely evaporate ethanol.
7. Finally, add 20 μL of preheated ddH₂O to the column, let stand for 2 minutes, and centrifuge at 12,000 rpm for 2 minutes.
8. Re-aspirate the filtrate and pass it through the column again; repeat step (7).
9. Discard the collection tube. Determine the concentration of the purified product using a NanoDrop instrument, and store it at 4°C for later use.

1.4 Electrophoresis and Gel Extraction

The purified products were subjected to gel extraction. Using the E. coli genome as the template, PCR amplification was performed with primers BL21-CheA-pBBR1-F/BL21-CheA-pBBR1-R to obtain a target fragment with a size of 1965 bp. Similarly, the CheY fragment (390 bp) and CheZ fragment (645 bp) were obtained. The theoretical size of pBBR1MCS-5 is 4768 bp.

Figure 1 Electrophoresis Image of the Fragments

1. First, perform 1% agarose gel electrophoresis to separate the target fragment. Under UV light, cut the gel block corresponding to the target fragment and place it in a 1.5 mL EP tube.
2. Weigh the gel block, add an appropriate volume of Buffer GDP according to the corresponding ratio, and melt the gel in a 55°C metal bath.
3. After the gel is completely melted, centrifuge briefly to collect droplets on the tube wall. Assemble the HiPure DNA Mini Column, transfer all the solutions to the column, and centrifuge at 12,000 rpm for 1 minute.
4. Discard the filtrate, reattach the column to the collection tube, add 300 μL of Buffer GDP, let stand, and centrifuge at 12,000 rpm for 1 minute.
5. Discard the filtrate, reattach the column to the collection tube, add 600 μL of Buffer DW2, and centrifuge at 12,000 rpm for 1 minute.
6. Repeat step (5) once.
7. Discard the filtrate, and centrifuge the column empty for 2 minutes.
8. After centrifugation, dry the column in a 65°C metal bath for 7–8 minutes. Add 25 μL of ddH₂O to the center of the column, let stand for 2 minutes, and centrifuge at 12,000 rpm for 2 minutes.
9. Re-aspirate the filtrate and pass it through the column again; repeat step (8).
10. Discard the collection tube. Determine the concentration of the gel-extracted product using a NanoDrop instrument, and store it at 4°C for later use.

1.5 Restriction Enzyme Digestion of the Plasmid Backbone

Electrophoresis verification showed that the size of the vector plasmid backbone band was incorrect. It was speculated that the reverse PCR primers were designed too close to each other, so Hind III was reselected for restriction enzyme ligation. After digestion, gel electrophoresis still showed incorrect band sizes.

1. Restriction enzyme digestion: The target fragment and plasmid with the same restriction site were subjected to single or double digestion in the corresponding digestion system to obtain DNA fragments with the same sticky ends. Digestion was performed at 50°C for 15 minutes. Taking Hind III digestion as an example, the specific digestion conditions are as follows:

1.6 Re-culture of DH5α (Same as 8.4)

7.20 Experimenter：Yangxin Zhang

Summary: Plasmid pSR43.6r was transformed. Since sequencing of pBBR1MCS-5/DH5α confirmed correctness, it was speculated that the plasmid extraction kit was ineffective. Therefore, the kit was replaced to re-amplify the pBBR1MCS-5 plasmid backbone, CheA, CheY, and CheZ genes. The target fragments (after purification and gel extraction) and the linearized vector pBBR1MCS-5 were ligated using one-step seamless cloning enzyme, followed by transformation into E. coli DH5α to construct recombinant plasmids pBBR1-MCS-CheA, pBBR1-MCS-CheY, and pBBR1-MCS-CheZ.

1. Experimental Materials

Table 1 Main Plasmids and Strains Used in This Experiment

2. Experimental Methods and Results

2.1 LB Resistance Plates

1. Prepare a 50 mg/mL streptomycin stock solution: Dissolve streptomycin in deionized water, then filter-sterilize (aspirate the antibiotic stock solution with a syringe, pass through a sterile filter membrane, collect into a sterile EP tube, and store at -20°C).
2. Heat and melt the solid LB medium, cool to approximately 55°C, and add streptomycin stock solution at a ratio of 1‰ (100 μL per 100 mL medium).

2.2 Procedure for pSR43.6 Transformation

1. Take E. coli DH5α competent cells out of the -80°C refrigerator, and thaw on ice for 5–10 minutes.
2. In a biosafety cabinet, add the ligation product to 100 μL of DH5α, gently tap the bottom of the tube 2–3 times, and incubate on ice for 30 minutes.
3. After ice incubation, heat-shock in a 42°C water bath for 90 seconds.
4. Immediately transfer to ice for 2 minutes after heat shock.
5. In a biosafety cabinet, add 900 μL of LB medium, and incubate in a 37°C shaker for 45 minutes to 1 hour.
6. After incubation, centrifuge at 5000×g for 1 minute, aspirate and discard approximately 900 μL of supernatant. Resuspend the remaining ~100 μL bacterial solution, spread it on LB plates containing the corresponding antibiotic, and incubate inverted in a 37°C incubator overnight.

2.3 Re-amplification of pBBR1MCS-5 Plasmid Backbone, CheA, CheY, and CheZ Genes

Figure 1 Electrophoresis Image of the Fragments

7.24 Experimenters：Yangxin Zhang Yixuan Xie Caijing Wu

Summary: Plasmid pGS58.6 was transformed; primers were redesigned, and the pET28a plasmid was used to amplify the vector backbone. BL21 bacterial solution was pretreated, and three target gene fragments (CheA, CheY, and CheZ) were amplified using corresponding primers. Electrophoresis verification confirmed correct amplification of CheA and CheY; the bands were excised for gel extraction and purification. The target fragments CheA and CheY were ligated to the linearized vector pET28a via seamless cloning, followed by transformation into E. coli DH5α to obtain engineered strains DH5α-pET28a- CheA and DH5α-pET28a- CheY.

1. Experimental Materials

Table 1 Main Plasmids and Strains Used in This Experiment

Table2 The primers used in this study

2. Experimental Methods and Results

2.1 Plasmid Maps

Figure 1 Map of Plasmid pET28a- CheA

Figure 2 Map of Plasmid pET28a- CheY

Figure 3 Map of Plasmid pET28a- CheZ

2.2 Preparation of LB Medium (Same as 8.4)

2.3 Transformation of Plasmid pGS58.6 (Same as 8.7)

2.4 Construction of CheA and CheY Expression Vectors

Figure 1 Electrophoresis Image of the Fragments

Taking pET28a -CheA as an example, PCR amplification was performed with primers BL21-CheA-F/BL21-CheA-R to obtain a target fragment of 1965 bp. Using pET28a plasmid as the template, PCR amplification was performed with pet28a-fan-F/p1 to obtain a plasmid backbone of 5301 bp. Electrophoresis results (as shown in the figure above) showed clear and correctly sized bands, confirming successful amplification of CheA, CheY genes, and pET28a plasmid backbone. These products were purified and subjected to gel extraction for later use.

Seamless Cloning Reaction Conditions: Incubate at 50°C for 15 minutes. The specific reaction system is as follows:

The purified target fragments and plasmid backbone were mixed at a molar ratio of 3:1, and ligated using 2×Hieff Clone Enzyme at 50°C for 15 minutes to obtain the ligation product. The ligation product was transformed into E. coli DH5α competent cells, plated, and incubated overnight.

8.9 Experimenters：Yangxin Zhang Yixuan Xie

Summary: Single colonies were picked from DH5α-pGS58.6, DH5α-pGS43.6, DH5α-pBBR1-MCS-CheA, DH5α-pBBR1-MCS-CheY, DH5α-pBBR1-MCS-CheZ, DH5α-pET28a-CheA, and DH5α-pET28a-CheY plates. After shaking culture, colony PCR was performed on the latter 5 strains. Strains with correct electrophoresis results were sent for sequencing.

1. Experimental Methods and Results

1.1 Colony PCR

1. In a biosafety cabinet, prepare a certain number of sterile EP tubes; add 200 μL of LB medium (containing the corresponding antibiotic) to each tube.
2. Use a sterile white pipette tip to pick 12 single colonies from each plate, add them to the pre-prepared medium, pipette to mix, seal the EP tubes with parafilm, and incubate in a 37°C shaker for more than 2 hours.
3. Prepare the PCR system in the first well, and dispense it into the remaining 11 wells using a multi-channel pipette:

4. Perform colony PCR according to the following program:

5. After PCR, perform 1% agarose gel electrophoresis. Bacterial solutions with correctly sized bands were sent for sequencing. PCR results are shown in the figures below:


Figure 1 Electrophoresis Image of Colony PCR Results



Figure 2 Electrophoresis Image of Colony PCR Results

Taking pBBR1-MCS-CheA as an example, its theoretical length is approximately 2000 bp, so Strain 1 was selected for sequencing.

7.28 Experimenter：Yangxin Zhang

Summary: Sequencing confirmed correctness of some strains, including DH5α-pBBR1-MCS-CheA, DH5α-pBBR1-MCS-CheY, DH5α-pBBR1-MCS-CheZ, DH5α-pET28a-CheA, and DH5α-pET28a-CheY. Plasmids pGS58.6 and pGS43.6r were extracted, digested with Sal I, and verified by gel electrophoresis.

1. Experimental Methods and Results

1.1 Sequencing Result Alignment

Taking DH5α-pBBR1-MCS-CheA as an example, the sequencing file S22508091596-D1.consensus.insert was imported into the original pBBR1-MCS-CheA file in SnapGene. The inserted sequence was aligned as shown in the figure; consistency with the original sequence indicated correct sequencing.

Figure 1 Schematic Diagram of Sequencing Result Alignment (Sequencing Confirmed Correct in the Figure)

1.2 Plasmid Extraction

1. Column equilibration: Add 500 μL of Equilibration Buffer BL to the adsorption column CP3 (placed in a collection tube), centrifuge at 12,000 rpm (~13,400×g) for 1 minute. Discard the waste liquid in the collection tube, and place the adsorption column back into the collection tube (use columns treated on the same day).
2. Take 1–5 mL of overnight-cultured bacterial solution, add to a centrifuge tube, centrifuge at 12,000 rpm (~13,400×g) for 1 minute using a conventional desktop centrifuge. Aspirate and discard the supernatant as much as possible (for large volumes of bacterial solution, collect the bacterial pellet in a single centrifuge tube via multiple centrifugations).
3. Add 250 μL of Solution P1 (RNase A added) to the centrifuge tube with the bacterial pellet, and thoroughly resuspend the pellet using a pipette or vortex oscillator.
4. Add 250 μL of Solution P2 to the centrifuge tube, and gently invert 6–8 times to fully lyse the bacteria.
5. Add 350 μL of Solution P3 to the centrifuge tube, immediately invert gently 6–8 times to mix thoroughly; white flocculent precipitates will form. Centrifuge at 12,000 rpm (~13,400×g) for 10 minutes.
6. Use a pipette to transfer the supernatant collected in the previous step to the adsorption column CP3 (placed in a collection tube), taking care not to aspirate the precipitate. Centrifuge at 12,000 rpm (~13,400×g) for 30–60 seconds. Discard the waste liquid in the collection tube, and place the adsorption column CP3 back into the collection tube.
7. Optional step: Add 500 μL of Deproteinization Buffer PD to the adsorption column CP3, centrifuge at 12,000 rpm (~13,400×g) for 30–60 seconds. Discard the waste liquid in the collection tube, and place the adsorption column CP3 back into the collection tube.
8. Add 600 μL of Washing Buffer PW (anhydrous ethanol added) to the adsorption column CP3, centrifuge at 12,000 rpm (~13,400×g) for 30–60 seconds. Discard the waste liquid in the collection tube, and place the adsorption column CP3 back into the collection tube.
9. Repeat step (8).
10. Place the adsorption column CP3 into the collection tube, centrifuge at 12,000 rpm (~13,400×g) for 2 minutes to remove residual washing buffer. Open the lid of the adsorption column CP3, place it in an oven for 8 minutes to completely dry the residual washing buffer in the adsorption material.
11. Place the adsorption column CP3 in a clean centrifuge tube, add 55 μL of ddH₂O to the center of the adsorption membrane, let stand at room temperature for 2 minutes, and centrifuge at 12,000 rpm (~13,400×g) for 2 minutes to collect the plasmid solution into the centrifuge tube. To increase plasmid recovery, re-add the eluted solution to the adsorption column, let stand at room temperature for 2 minutes, and centrifuge at 12,000 rpm (~13,400×g) for 2 minutes to collect the plasmid solution. Determine the concentration using a Nanodrop instrument.

1.3 Restriction Enzyme Digestion and Electrophoresis Verification

The theoretical length of pSR43.6r is 6282 bp, and that of pSR58.6 is 3936 bp. Electrophoresis results showed correctly sized bands.

Figure 2 Electrophoresis Image of the Fragments

8.1 Experimenter：Yangxin Zhang

Summary: Overnight shaking culture was performed for DH5α-pBBR1-MCS-CheA, DH5α-pBBR1-MCS-CheY, DH5α-pBBR1-MCS-CheZ, DH5α-pET28a-CheA, and DH5α-pET28a-CheY.

1. Experimental Methods and Results

1.1 Bacterial Shaking Culture

Aspirate 50 μL of the sequenced correct bacterial solution, add to 6 mL of LB medium (containing antibiotics), and culture overnight in a 37°C, 220 rpm shaker.

8.3 Experimenter：Yangxin Zhang

Summary: Glycerol stock was prepared for DH5α-pBBR1-MCS-CheA, DH5α-pBBR1-MCS-CheY, DH5α-pBBR1-MCS-CheZ, DH5α-pET28a-CheA, and DH5α-pET28a-CheY. Plasmids were extracted from the remaining bacterial solution and transformed into BL21, followed by plating and overnight culture.

1. Experimental Methods and Results

1.1 Glycerol Stock Preparation and Plasmid Extraction

1. Aspirate 600 μL of overnight-cultured bacterial solution, mix with 400 μL of 50% glycerol, add to a cryovial, and store at -20°C.(2) Extract plasmids from the remaining bacterial solution, and determine the concentration of the recovered plasmids using a NanoDrop instrument.

1.2 Transformation into BL21

1. Take E. coli BL21 competent cells out of the -80°C refrigerator, and thaw on ice for 5–10 minutes.
2. Add 2.5 μL of plasmid to 100 μL of BL21, gently tap the bottom of the tube 2–3 times, and incubate on ice for 30 minutes.
3. After ice incubation, heat-shock in a 42°C water bath for 90 seconds.
4. Immediately transfer to ice for 2 minutes after heat shock.
5. In a biosafety cabinet, add 900 μL of LB medium, and incubate in a 37°C shaker for 45 minutes to 1 hour.
6. After incubation, centrifuge at 5000×g for 1 minute, aspirate and discard approximately 900 μL of supernatant. Resuspend the remaining ~100 μL bacterial solution, spread it on LB plates containing the corresponding antibiotic, and incubate inverted in a 37°C incubator overnight.

8.6 Experimenter：Yangxin Zhang

Summary: Single colonies were picked from overnight-cultured plates of BL21-pBBR1-MCS-CheA, BL21-pBBR1-MCS-CheY, BL21-pBBR1-MCS-CheZ, BL21-pET28a-MCS-CheA, and BL21-pET28a-MCS-CheY, followed by overnight shaking culture at 37°C.

1. Experimental Materials

Table 1 Main Plasmids and Strains Used in This Experiment

2. Experimental Methods and Results

Pick single colonies from the plates, immerse the pipette tip (used for picking colonies) into a shake flask containing 6 mL of LB medium (with antibiotics), and culture overnight.

8.8 Experimenter：Yangxin Zhang

Summary: Glycerol stock was prepared for overnight-cultured bacterial solutions of BL21-pBBR1-MCS-CheA, BL21-pBBR1-MCS-CheY, BL21-pBBR1-MCS-CheZ, BL21-pET28a-CheA, and BL21-MCS-CheY. Plasmids were extracted from the remaining 2 mL bacterial solution for electrophoresis detection.

1. Experimental Methods and Results

1. Aspirate 600 μL of overnight-cultured bacterial solution, mix with 400 μL of 50% glycerol, add to a cryovial, and store at -20°C.
2. Extract plasmids from 2 mL of bacterial solution, and determine the concentration of the recovered plasmids using a NanoDrop instrument.
3. Prepare a 1% agarose gel using a large comb. Mix 18 μL of the above plasmid with 2 μL of 10×loading buffer, load the sample, and run electrophoresis at 150 V for 25 minutes. Electrophoresis results are shown in the figure below:

The plasmid size was consistent with the theoretical size, confirming successful construction of BL21-pBBR1-MCS-CheA, BL21-pBBR1-MCS-CheY, and BL21-pBBR1-MCS-CheZ strains.

8.9-8.15 Experimenters：Yangxin Zhang Yanye Zhang Caijing Wu

Summary: During this period, attempts were made to construct DH5α-pBBR1-MCS-CheA-Flag, DH5α-pBBR1-MCS-CheY-Flag, DH5α-pBBR1-MCS-CheZ-Flag, DH5α-pDawn-CheZ-Flag, DH5α-pDawn-CheZ, and DH5α-pDawn-sfGFP. However, sequencing results of some strains did not match the theoretical sequence. The complete construction process of successfully constructed strains is detailed in subsequent records and is omitted here. The pDawn plasmid was transformed into E. coli DH5α; after shaking culture, the pDawn plasmid was extracted, digested with Sal I, and verified by gel electrophoresis.

8.18 Experimenters：Yanye Zhang Yixuan Xie Caijing Wu Yangxin Zhang

Summary: To construct a green light-induced expression system, CheA and CheY gene fragments (with and without tags) were amplified, verified by electrophoresis, and subjected to gel extraction.

1. Experimental Materials

Table1 The primers used in this study

Table 2 Main Plasmids and Strains Used in This Experiment

2. Experimental Methods and Results

2.1 Plasmid Maps

Figure 1 Map of Plasmid pSR58.6

Figure 2 Map of Plasmid pSR58.6-CheA

Figure 3 Map of Plasmid pSR58.6-CheA-Flag

Figure 4 Map of Plasmid pSR58.6-CheY

Figure 5 Map of Plasmid pSR58.6-CheY-Flag

2.2 PCR Amplification of CheA and CheY Fragments

Mix 20 μL of deionized water with 1 μL of E. coli BL21 bacterial solution, heat-treat, and perform PCR amplification with primers BL21-CheA-pSR58.6-R/BL21-CheA-pSR58.6-F. Electrophoresis results (as shown in the figure below) showed a single band of correct size, confirming successful amplification of the CheA gene. After gel extraction, the concentration was determined using a NanoDrop nucleic acid quantifier, and the product was stored at -20°C for later use.

Plasmids pBBR1-MCS-CheY and pBBR1-MCS-CheA were extracted, and PCR amplification was performed with primers BL21-CheA-pSR58.6-F/BL21-CheA-pSR58.6-Flag-R, BL21-CheY-pSR58.6-F/BL21-CheY-pSR58.6-Flag-R, and BL21-CheY-pSR58.6-F/BL21-CheY-pSR58.6-R. Electrophoresis results (as shown in the figure below) showed single bands: the theoretical size of CheA is 1965 bp, CheA-Flag is 2046 bp, CheY is 390 bp, and CheY-Flag is 411 bp.

Figure 6 Electrophoresis Image of the Fragments

After gel extraction, the concentration was determined using a NanoDrop nucleic acid quantifier, and the products were stored at -20°C for later use.

8.22 Experimenters：Yixuan Xie Yanye Zhang Yangxin Zhang

Summary: The pSR58.6 plasmid backbone was amplified, and CheA, CheA-Flag, CheY, CheY-Flag expression vectors were constructed. The vectors were transformed into E. coli TOP10 competent cells.

1. Experimental Materials

Table 1 Main Plasmids and Strains Used in This Experiment

2. Experimental Methods and Results

2.1 PCR Amplification of the Plasmid Backbone

Using pSR58.6 plasmid as the template, PCR amplification was performed with primers pSR58.6-R/pSR58.6-F. Electrophoresis results (as shown in the figure below) showed a single band of correct size, confirming successful amplification of the plasmid backbone.

2.2 Plasmid Construction via Seamless Cloning (Taking pSR58.6-CheA as an Example)

The purified target fragment CheA and plasmid backbone (0.03 pmol) were mixed at a molar ratio of 3:1, and ligated using 2×MultiF Seamless Assembly Mix at 50°C for 15 minutes to obtain the ligation product. Thaw competent cells for cloning on ice for 5–10 minutes, then add approximately 10 μL of the DNA sample to be transformed to 100 μL of competent cells. After incubating on ice for 30 minutes, heat-shock for 90 seconds. Add 900 μL of LB medium, recover for 45 minutes, collect the bacteria, and spread on LB plates containing chloramphenicol.

8.23 Experimenters：Yangxin Zhang Yixuan Xie

Summary: Single colonies were picked from overnight-cultured plates of TOP10-pSR58.6-CheA, TOP10-pSR58.6-CheA-Flag, TOP10-pSR58.6-CheY, and TOP10-pSR58.6-CheY-Flag for verification. Colony PCR was performed, and positive strains were sent for sequencing.

8.24 Experimenter：Caijing Wu

Summary: sfGFP, CheZ, CheZ-Flag fragments, and pDawn backbone were obtained via PCR. Plasmids pDawn-CheZ, pDawn-CheZ-Flag, and pDawn-sfGFP were further constructed via seamless cloning and transformed into E. coli DH5α for overnight culture.

1. Experimental Materials

Table 1 Main Plasmids and Strains Used in This Experiment

Table2 The primers used in this study

2. Experimental Methods and Results

2.1 Plasmid Maps

Figure 1 Map of Plasmid pDawn

Figure 2 Map of Plasmid pDawn-CheZ

Figure 3 Map of Plasmid pDawn-CheZ-Flag

Figure 4 Map of Plasmid pDawn-sfGFP

2.2 Construction of Plasmids pDawn-CheZ, pDawn-CheZ-Flag, and pDawn-sfGFP via Seamless Cloning (Taking pDawn-CheZ as an Example)

1. First, perform PCR amplification: Using the E. coli genome as the template, amplify the CheZ target fragment with primers BL21-CheZ-pDawn-F/BL21-CheZ-pDawn-R (the sfGFP fragment was amplified via PCR from pSR58.6). 1% agarose gel electrophoresis revealed a target fragment of 645 bp (electrophoresis results shown in the figure below).

2. Next, using the pDawn plasmid as the template (plasmid map shown in Figure 1), amplify the plasmid backbone with primers pDawn-F/pDawn-R. 1% agarose gel electrophoresis revealed a plasmid backbone of 7013 bp (electrophoresis results shown in the figure below)
3. After verifying correct band sizes, purify the DNA and perform gel extraction using a DNA purification kit. Determine the concentration of the recovered products using a NanoDrop instrument.
4. Mix the purified target fragment and plasmid backbone at a molar ratio of 3:1, and ligate using 2×Hieff Clone Enzyme at 50°C for 15 minutes to obtain the ligation product.
5. Transform the ligation product into E. coli DH5α competent cells, plate, and incubate overnight.

8.25 Experimenter：Caijing Wu

Summary: Single colonies were picked from DH5α-pDawn-CheZ, DH5α-pDawn-CheZ-Flag, and DH5α-pDawn-sfGFP plates. Strains with positive colony PCR results were sent for sequencing.

1. Experimental Materials

Table1 The primers used in this study

2. Experimental Methods and Results

2.1 Colony Picking and Colony PCR

Prepare 12×3 sterile EP tubes and overnight-cultured plates. Add 200 μL of LB medium (containing antibiotics) to each EP tube. Use a 2.5 μL pipette tip to pick single colonies from the plates, transfer the white pipette tip into the EP tube, and incubate in a 37°C, 220 rpm shaker.

Figure1 Electrophoresis Image of Colony PCR Results

8.27 Experimenters：Caijing Wu Yixuan Xie Yangxin Zhang

Summary: Prepare medium and perform bacterial shaking culture.

1. Experimental Methods and Results

1.1 Preparation of Solid LB Medium and Semi-Solid LB Medium

Formulation: Weigh 10 g tryptone, 5 g yeast extract, and 10 g sodium chloride, add ultrapure water to a final volume of 1 L, and mix well. For solid LB medium, add an additional 3 g of agar powder to 150 mL of liquid medium. For semi-solid LB medium, add an additional 0.375 g of agar powder (0.25% (w/v)) to 150 mL of liquid medium. After aliquoting, sterilize by autoclaving at 115°C for 20 minutes.

8.28 Experimenters：Caijing Wu Yixuan Xie

Summary: Glycerol stocks were prepared for overnight-cultured bacterial solutions of DH5α-pDawn-CheZ, DH5α-pDawn-CheZ-Flag, DH5α-pDawn-sfGFP, TOP10-pSR58.6-CheA, TOP10-pSR58.6-CheA-Flag, TOP10-pSR58.6-CheY, and TOP10-pSR58.6-CheY-Flag. Plasmids were extracted from the remaining bacterial solution and transformed into BL21, followed by plating and overnight culture. BL21-pBBR1-MCS-CheA, BL21-pBBR1-MCS-CheY, and BL21-pBBR1-MCS-CheZ were activated and cultured with shaking overnight.

1. Experimental Materials

2. Experimental Methods and Results

2.1 Glycerol Stock Preparation and BL21 Transformation

1. Aspirate 600 μL of overnight-cultured bacterial solution, mix with 400 μL of 50% glycerol, add to a cryovial, and store at -20°C.
2. Extract plasmids from the remaining bacterial solution, and determine the concentration of the recovered plasmids using a NanoDrop instrument.
3. Take BL21 (DE3) competent cells out of the -80°C refrigerator, thaw on ice. Add 3 μL of the plasmid to be transformed to 100 μL of competent cells, incubate on ice for 30 minutes, heat-shock, recover for 45 minutes, collect the bacteria by centrifugation, spread on plates, and incubate inverted at 37°C overnight.

2.2 Activation and Shaking Culture

Add 50 μL of glycerol-stocked bacterial solution to 6 mL of LB medium (containing antibiotics), and culture with shaking at 37°C, 220 rpm overnight.

8.29 Experimenters：Yixuan Xie Caijing Wu

Summary: Single colonies were picked from overnight-cultured plates of BL21-pDawn-CheZ, BL21-pDawn-CheZ-Flag, BL21-pDawn-sfGFP, BL21-pSR58.6-CheA, BL21-pSR58.6-CheA-Flag, BL21-pSR58.6-CheY, and BL21-pSR58.6-CheY-Flag, followed by overnight shaking culture. BL21-pBBR1-MCS-CheA, BL21-pBBR1-MCS-CheY, and BL21-pBBR1-MCS-CheZ were induced with IPTG and spotted on semi-solid LB medium to verify motility.

1. Experimental Methods and Results

1.1 Colony Picking and Shaking Culture

Select single colonies from the plates. First, transfer the colony-picking pipette tip into an EP tube containing 200 μL of LB medium (with antibiotics) and culture for more than 2 hours. Then aspirate 50 μL of the cultured bacterial solution into a shake flask containing 6 mL of LB medium (with antibiotics), and culture overnight.

1.2 IPTG Induction

1. Inoculate 50 μL of the overnight-cultured seed solution into a vial containing 7 mL of LB medium (with Gm), and culture with shaking at 37°C, 220 rpm.
2. Starting from 2 hours after inoculation, monitor the OD₆₀₀ of the bacterial solution every 30 minutes using a spectrophotometer. Calibrate with 2 mL of deionized water; mix 500 μL of bacterial solution with 1.5 mL of deionized water for measurement (4× dilution). Culture until the OD₆₀₀ of the bacterial solution reaches 0.6–0.8.
3. Add 1 mM IPTG, and culture with shaking at 37°C, 220 rpm for 3 hours.

1. Heat and melt the semi-solid medium, cool to approximately 55°C, add Gm, pour into petri dishes with an inner diameter of 6 cm, and let stand in a biosafety cabinet with air flow until the medium solidifies completely.
2. Take 2 μL of the induced bacterial solution, carefully spot it on the center of the prepared semi-solid LB medium plate (avoid piercing the medium surface), and set up 3 biological replicates for each group.
3. Incubate upright at 37°C for 15 hours, observe, and take photos.

8.30 Experimenters：Yixuan Xie Caijing Wu Yanye Zhang

Summary: Glycerol stocks were prepared for overnight-cultured bacterial solutions of BL21-pDawn-CheZ, BL21-pDawn-CheZ-Flag, BL21-pDawn-sfGFP, BL21-pSR58.6-CheA, BL21-pSR58.6-CheA-Flag, BL21-pSR58.6-CheY, and BL21-pSR58.6-CheY-Flag. Plasmids were extracted from part of the bacterial solution for electrophoresis detection; the remaining bacterial solution was temporarily stored at 4°C for later use. To construct 3 plasmids with Flag tags, CheA, CheY, CheZ, and pBBR1MCS-5 backbone were amplified using primers, verified by electrophoresis, and correctly sized bands were excised for gel extraction.

1. Experimental Materials

Table1 The primers used in this study

Table 2 Main Plasmids and Strains Used in This Experiment

2. Experimental Methods and Results

2.1 Plasmid Maps

Figure 1 Map of Plasmid PBBR1-MCS-CheA-Flag

Figure 2 Map of Plasmid PBBR1-MCS-CheY-Flag

Figure 3 Map of Plasmid PBBR1-MCS-CheZ-Flag

2.2 Glycerol Stock Preparation and Plasmid Electrophoresis Detection

1. Aspirate 600 μL of the overnight-cultured bacterial solution, mix it with 400 μL of 50% glycerol, add the mixture to a cryovial, and store it at -20°C.
2. Extract plasmids from 2 mL of the bacterial solution, and determine the concentration of the recovered plasmids using a NanoDrop instrument.
3. Prepare a 1% agarose gel with a large comb. Mix 18 μL of the above plasmid with 2 μL of 10×loading buffer, load the sample onto the gel, and run electrophoresis at 150 V for 25 minutes. The electrophoresis results are shown in the figure below, confirming that the plasmids have been successfully introduced into BL21.

8.31 Experimenters：Yixuan Xie

Summary：Plasmid combinations of pSR58.6-CheA & pSR43.6, pSR58.6-CheA-Flag & pSR43.6, and pSR58.6-CheY & pSR43.6 were respectively co-transformed into BL21 to construct dual-plasmid engineered strains, which were plated and cultured overnight. The amplified fragments were subjected to seamless cloning, followed by transformation into DH5α and plating for overnight culture.

1. Experimental Materials

Table 1 Main Strains Used in This Experiment

2. Experimental Methods and Results

2.1 Transformation into BL21

Take BL21 (DE3) competent cells out of the -80°C refrigerator and thaw on ice. Add 2 μL of each of the two plasmids to be co-transformed into 100 μL of competent cells, incubate on ice for 30 minutes, then heat-shock at 42°C for 90 seconds. Subsequently, perform recovery culture for 45 minutes. After recovery, collect the bacterial cells by centrifugation and spread them on LB plates containing antibiotics (Sm and Cm), followed by inverted culture at 37°C overnight.

9.1 Experimenter：Yixuan Xie

Summary: Single colonies were picked from the overnight-cultured plates of BL21-pSR43.6-pSR58.6-CheA, BL21-pSR43.6-pSR58.6-CheA-Flag, and BL21-pSR43.6-pSR58.6-CheY, followed by overnight shaking culture at 37°C. Plasmids pSR58.6-CheY-Flag and pSR43.6 were co-transformed into BL21 to construct a dual-plasmid engineered strain, which was plated and cultured overnight. Single colonies were picked from the overnight-cultured plates of DH5α-pBBR1-MCS-CheA-Flag, DH5α-pBBR1-MCS-CheY-Flag, and DH5α-pBBR1-MCS-CheZ-Flag for colony PCR verification. Bacterial solutions with positive verification results were sent for sequencing.

9.2 Experimenters：Yixuan Xie Caijing Wu Yanye Zhang

Summary: Overnight shaking culture was performed for DH5α-pBBR1-MCS-CheA-Flag, DH5α-pBBR1-MCS-CheY-Flag, and DH5α-pBBR1-MCS-CheZ-Flag with correct sequencing results. Plasmids pBBR1-MCS-CheA-Flag, pBBR1-MCS-CheY-Flag, and pBBR1-MCS-CheZ-Flag were transformed into BL21, followed by plating and overnight culture.

9.3 Experimenters：Yixuan Xie Yanye Zhang

Summary: Bacterial solutions of BL21-pSR43.6-pSR58.6-CheA-Flag and BL21-pSR43.6-pSR58.6-CheY-Flag were induced with green light to express Flag-tagged CheA and CheY proteins. After induction, Western Blotting (WB) samples were prepared and stored at -20°C for later use. Bacterial solution of BL21-pSR43.6-pSR58.6-CheY spotted on semi-solid medium was induced with green light. Plasmids pSR58.6 and pSR43.6 were co-transformed into BL21 to construct a dual-plasmid engineered strain, which was plated and cultured overnight. The semi-solid culture plates induced the previous day were observed, photographed, and measured. Bacterial solutions of BL21-pBBR1-MCS-CheA-Flag, BL21-pBBR1-MCS-CheY-Flag, and BL21-pBBR1-MCS-CheZ-Flag were induced with IPTG to express Flag-tagged CheA, CheY, and CheZ proteins.

1. Experimental Methods and Results

1.1 Green Light Induction of Flag-Tagged Bacterial Solutions

1. Aspirate 100 μL of the corresponding bacterial solution and inoculate it into 10 mL of LB medium (supplemented with Sm and Cm antibiotics), followed by shaking culture at 37°C and 220 rpm.
2. Starting from 2 hours after inoculation, monitor the OD₆₀₀ of the bacterial solution every 30 minutes using a spectrophotometer. It was speculated that the slow growth of the engineered strain was due to low viability caused by prolonged storage of the bacterial solution or toxicity from dual resistance. The OD₆₀₀ of the bacterial solution reached 0.6–0.8 after approximately 5 hours of culture.
3. Dispense the remaining bacterial solution into two Petri dishes (with a specific diameter). One dish was set as the light group (induced with a green flashlight for 6 hours), and the other as the non-light group (wrapped with aluminum foil for light protection). Both groups were cultured in a 37°C incubator.

1.2 Bacterial Harvesting and WB Sample Preparation

1. Harvest the bacteria: Take 2 mL of the bacterial solution, centrifuge at 8000×g for 2 minutes, and discard the supernatant.
2. Add 1 mL of PBS to the EP tube, resuspend and mix well, then centrifuge at 8000×g for 2 minutes and discard the supernatant. Repeat the washing step 3 times.
3. Add 800 μL of PBS to the EP tube, resuspend and mix well.
4. Take 100 μL of the above bacterial solution, mix it with 900 μL of PBS, and add the mixture to a cuvette for OD measurement. (The spectrophotometer should be preheated in advance; calibrate with 1 mL of PBS before measurement. The OD value measured at this time is for the 10× diluted sample.)
5. Determine the final diluted OD value based on the measured OD. An OD value of 1.5 was used in this experiment. Take 500 μL of the bacterial solution from Step (3), and calculate the volume of PBS required for dilution to adjust the OD of the diluted sample to 1.5.
6. Take 80 μL of the above sample solution, add it to a new EP tube, mix it with 20 μL of loading buffer using a vortex mixer, and briefly centrifuge with a microcentrifuge to collect the liquid on the tube wall.
7. Preheat a boiling water bath. Once the water boils, place the EP tube on a floating plate and boil for 10 minutes. Store the sample at -20°C for later use.

9.4 Experimenters：Caijing Wu Yanye Zhang Yixuan Xie

Summary: WB samples were prepared from the IPTG-induced bacterial solutions of BL21-pBBR1-MCS-CheA-Flag, BL21-pBBR1-MCS-CheY-Flag, and BL21-pBBR1-MCS-CheZ-Flag. SDS-PAGE electrophoresis, membrane transfer, and blocking were performed, followed by overnight primary antibody incubation at 4°C. Bacterial solution of BL21-pDawn-CheZ-Flag was induced with blue light to express Flag-tagged CheZ protein. After induction, WB samples were prepared and stored at -20°C for later use. Bacterial solution of BL21-pDawn-CheZ spotted on semi-solid medium was induced with green light. Single colonies were picked from the overnight-cultured plate of BL21-pSR43.6-pSR58.6, followed by overnight shaking culture.

1. Experimental Methods and Results

1.1 Western Blot

1. Prepare WB samples from the induced bacterial solutions.
2. Take out the precast 12% SDS-PAGE gel cassette, place the long plate facing outward (placing the short plate outward will cause an open circuit), and fix it with a clamp. Pour an appropriate amount of MOPS electrophoresis buffer between the two plates; after confirming no leakage, place the cassette into the electrophoresis tank.
3. Pull out the comb, and pour MOPS electrophoresis buffer into the gap between the plates until the liquid level inside the plates is slightly higher than that outside (otherwise, no potential difference can be formed). Load 7.5 μL of sample into each well, use a tricolor pre-stained protein molecular weight marker as the marker, and run electrophoresis at 120 V for 50 minutes.
4. After electrophoresis, check if the marker bands are fully separated. Pry open the plastic plates to take out the gel, cut off the required part, soak the gel in electrotransfer buffer, and cut a PVDF membrane and filter papers of appropriate size.
5. Soak the PVDF membrane in absolute methanol for 30 seconds first, then transfer it to electrotransfer buffer and soak for 2 minutes. Take out the sandwich electrotransfer clamp, and arrange the components in the order of "black plate → sponge → filter paper → gel → PVDF membrane → filter paper → sponge → red plate" before placing it into the electrophoresis tank.
6. Pour 1000 mL of electrotransfer buffer into the tank, place the electrophoresis tank in an ice box, and perform electrotransfer at 100 V for 1.5 hours.
7. Preparation of 20×PBS-T: Mix one package of PBS powder (which should be dissolved in 2000 mL of deionized water) with 100 mL of deionized water, then add 1 mL of Tween.
8. Preparation of blocking solution: 10% non-fat milk powder dissolved in PBS-T. (Add 5 g of non-fat milk powder to 50 mL of 1×PBS-T, mix well in a centrifuge tube; store at -20°C if not used immediately.)
9. Place the PVDF membrane into the blocking solution and incubate at room temperature for 2 hours.
10. Primary antibody incubation: Dilute the primary antibody in blocking solution at a ratio of 1:1000 (10 μL of mouse-derived primary antibody diluted in 10 mL of blocking solution). Transfer the PVDF membrane to the primary antibody dilution and incubate overnight at 4°C.

9.5 Experimenters：Caijing Wu Yanye Zhang Yixuan Xie

Summary: Bacterial solutions stored in a 4°C refrigerator were inoculated into a bioreactor at a ratio of 1% (v/v) with 1‰ (v/v) antibiotics, and cultured until the OD value reached 0.6–0.8. Induction was performed with blue light for 3 hours and 6 hours, and the results were observed using a multi-mode microplate reader. Secondary antibody incubation and color development were carried out.

1. Experimental Methods and Results

1.1 Western Blot

11. After primary antibody incubation, wash the membrane with PBS-T 3 times, 10 minutes each time.
12. Secondary antibody incubation: Dilute the secondary antibody in blocking solution at a ratio of 1:1000 (10 μL of goat anti-mouse secondary antibody diluted in 10 mL of blocking solution). Transfer the PVDF membrane to the secondary antibody dilution and incubate at 37°C and 75 rpm for 1 hour.
13. After secondary antibody incubation, wash the PVDF membrane with PBS-T 3 times, 10 minutes each time.
14. Aspirate 1 mL of ECL FemtoLight Substrate and 1 mL of ECL FemtoLight Oxidant, mix them well to prepare the developing solution. Blot the residual liquid on the PVDF membrane with filter paper, then perform development and photography.

1.2 Multi-Mode Microplate Reader Detection

1. After harvesting the bacteria, wash them with pure water 3 times, and dilute the bacterial solution to an OD value of 1 for later use.
2. Use a BioTek multi-mode microplate reader: First, turn on the instrument for self-test. Open the Gen 5 software, select "Immediate Detection", choose "Fluorescence Detection" as the detection method, and set the optical component type to "Monochromator".
3. Select 488 nm as the excitation wavelength and 509 nm as the emission wavelength, with the optical component position set to "Bottom".
4. Use a black microplate: First detect the empty plate, then load pure water, and samples induced for 0 hour, 3 hours, and 6 hours at 100 μL per well. Set up 3 biological replicates for each group.
5. Download the data and compare the fluorescence intensity using relative fluorescence intensity.

9.19 Experimenters：Caijing Wu Yixuan Xie Yanye Zhang

Summary: Preparation of SDS-PAGE gel.

1. Experimental Methods and Results

1.1 Preparation of SDS-PAGE Gel

1. Take out the matching glass plates, wash them with tap water, and rinse with deionized water.
2. Place the long plate inward and the short plate outward, align them, clamp them in a holder, and fix the holder vertically on a rack. Pour deionized water to check for leakage; if the liquid level does not drop, it indicates no leakage. Pour off the deionized water and blot the residual water with filter paper.
3. Preparation of separating gel: For a gel thickness of 1 mm, add 2.7 mL of separating gel solution, 2.7 mL of separating gel buffer, and finally 60 μL of modified coagulant. Mix well, use a pipette to add the mixture between the glass plates until the liquid level reaches below the white crossbar. Then fill the gap with isopropanol. Let it stand for about 30 minutes to solidify.
4. After the separating gel solidifies, pour off the isopropanol and blot the residual liquid with filter paper.
5. Preparation of stacking gel: For a gel thickness of 1 mm, add 0.75 mL of stacking gel solution, 0.75 mL of stacking gel buffer, and finally 15 μL of modified coagulant. Mix well, use a pipette to add the mixture between the glass plates until the liquid level is slightly lower than the top of the glass plates. Insert a comb immediately to ensure the liquid fills the gaps without forming air bubbles. Let it stand for about 40 minutes to solidify.
6. Pull out the comb, remove the gel cassette, add an appropriate amount of deionized water to keep it moist, wrap it with plastic wrap, and store it in a 4°C refrigerator for later use.

9.20 Experimenter：Yixuan Xie

Summary: Activation and shaking culture were performed for the glycerol-stocked DH5α-pDawn-CheZ, DH5α-pSR58.6, TOP10-pSR58.6-CheA, TOP10-pSR58.6-CheY, DH5α-pBBR1-MCS-CheA-Flag, DH5α-pBBR1-MCS-CheY-Flag, and DH5α-pBBR1-MCS-CheZ-Flag.

9.21 Experimenters：Yixuan Xie Yanye Zhang

Summary: Secondary bacterial cultures were inoculated from the activated DH5α-pBBR1-MCS-CheA-Flag, DH5α-pBBR1-MCS-CheY-Flag, and DH5α-pBBR1-MCS-CheZ-Flag. When the culture reached an OD value of approximately 0.6, IPTG induction was performed at 37°C for 2 hours, with a control group set up simultaneously. After induction, the bacteria were harvested, diluted to OD₆₀₀ = 1, and WB protein samples were prepared and stored at -20°C for later use.

9.22-9.25 Experimenters：Yixuan Xie Yanye Zhang

Summary: During this period, Western Blot was performed on the above protein samples, but the development results were unsatisfactory. A gradient experiment of annealing temperatures for PCR amplification was conducted to determine the optimal annealing temperature.

9.26 Experimenters：Yixuan Xie

Summary: To construct sfGFP-fused CheA and CheY protein expression vectors, fragments of pSR58.6, CheY, and CheA were amplified. Correctly sized bands were excised for gel extraction.

1. Experimental Materials

Table1 The primers used in this study

2. Experimental Methods and Results

2.1 Plasmid Maps

Figure 1 Map of Plasmid pSR58.6-CheY-sfGFP

Figure 2 Map of Plasmid pSR58.6-CheA-sfGFP

2.2 Fragment Amplification

Taking pSR58.6-CheY-sfGFP as an example, PCR amplification was performed using primers 58.6-Y-linker/58.6-Y-RBS to obtain a target fragment with a size of 424 bp. Using pSR58.6 plasmid as the template, PCR amplification was performed using primers 5-58.6-R/58.6-GFP-linker to obtain a plasmid backbone with a size of 3961 bp. As shown in the electrophoresis figure below, the successful amplification of CheA, CheY genes, and pSR58.6 plasmid backbone was confirmed. These fragments were separately purified and subjected to gel extraction for later use.

9.27 Experimenters: Yixuan Xie Yanye Zhang

Summary:

1. Secondary bacterial cultures were inoculated from the activated BL21-pDawn-CheZ-Flag, BL21-pSR43.6-pSR58.6-CheA-Flag, and BL21-pSR43.6-pSR58.6-CheY-Flag. When the culture reached an OD value of approximately 0.6, induction was performed at 37°C with blue light and green light respectively for 6 hours, with light-shielded control groups set up simultaneously. After induction, the bacteria were harvested, diluted to OD₆₀₀ = 1, and WB protein samples were prepared and stored at -20°C for later use.
2. 2.Secondary bacterial cultures were inoculated from the activated BL21-pDawn-sfGFP and BL21-pSR43.6-pSR58.6. When the culture reached an OD value of approximately 0.6, induction was performed at 37°C with blue light and green light respectively for 6 hours, with light-shielded control groups set up simultaneously. Samples were taken at 3 hours and 6 hours after induction; the bacteria were washed with distilled water and diluted to OD₆₀₀ = 1, then loaded onto a 96-well black microplate at 100 μL per well. Fluorescence intensity was analyzed using a multi-mode microplate reader.
3. 3.The above bacteria were observed under an optical microscope.
4. 4.Glycerol-stocked bacterial solutions of BL21-pDawn-CheZ-Flag, BL21-pSR43.6-pSR58.6-CheA-Flag, and BL21-pSR43.6-pSR58.6-CheY-Flag were activated.
5. 5.The gel-extracted CheA and CheY fragments were separately ligated to the pSR58.6 vector backbone via seamless cloning, then transformed into DH5α and plated for overnight culture.

1. Experimental Methods and Results

1.1 Microscopic Observation

1. Wash the fresh bacterial solution with pure water 3 times, and take out clean glass slides and coverslips.
2. Use a pipette to aspirate 1 μL of the bacterial solution, drop it onto a glass slide, cover with a coverslip, and observe under a microscope with a 40×objective lens.
3. Before using a 100×oil immersion objective, drop cedar oil onto the coverslip. After finding the field of view, take photos and record videos using Image J software.

9.28 Experimenters：Yixuan Xie Yanye Zhang

Summary: SDS-PAGE electrophoresis, membrane transfer, and overnight blocking at 4°C were performed on the protein samples of BL21-pDawn-CheZ-Flag, BL21-pSR43.6-pSR58.6-CheA-Flag, and BL21-pSR43.6-pSR58.6-CheY-Flag induced with blue light, green light, and the light-shielded control groups the previous day.

9.29 Experimenters：Yixuan Xie Yanye Zhang

Summary: After blocking, primary and secondary antibody incubations were carried out. The membrane was washed with PBS-T and then developed. Secondary bacterial cultures were inoculated from BL21-pDawn-CheZ-Flag, BL21-pSR43.6-pSR58.6-CheA-Flag, and BL21-pSR43.6-pSR58.6-CheY-Flag, induced with blue light and green light for 6 hours, and observed and photographed under an optical microscope.