iGEM 2025 — Team ecust-china

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### LAB

# NOTEBOOK

### OVERVIEW

* [JULY](#804)
* [AUGUST](#811)
* [SEPTEMBER](#901)

# 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

![table1](https://static.igem.wiki/teams/5972/uploads/page/notebookfolder/table1.webp)

Table2 The primers used in this study

![table2](https://static.igem.wiki/teams/5972/uploads/page/notebookfolder/table2.webp)

### 2. Experimental Methods and Results

#### 2.1 Plasmid Maps

![notebook32](https://static.igem.wiki/teams/5972/uploads/page/notebookfolder/notebook32.webp)

Figure 1 Map of Plasmid PBBR1MCS-5-CheA

![notebook33](https://static.igem.wiki/teams/5972/uploads/page/notebookfolder/notebook33.webp)

Figure 2 Map of Plasmid PBBR1MCS-5-CheY

![notebook34](https://static.igem.wiki/teams/5972/uploads/page/notebookfolder/notebook34.webp)

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

![table3](https://static.igem.wiki/teams/5972/uploads/page/notebookfolder/table3.webp)

Table 2 General PCR Reaction Program

![table5](https://static.igem.wiki/teams/5972/uploads/page/notebookfolder/table4.webp)

#### 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.

![notebook2](https://static.igem.wiki/teams/5972/uploads/page/notebookfolder/notebook2.webp)

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:

![table5](https://static.igem.wiki/teams/5972/uploads/page/notebookfolder/table5.webp)

#### 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

![table6](https://static.igem.wiki/teams/5972/uploads/page/notebookfolder/table6.webp)

### 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

![notebook4](https://static.igem.wiki/teams/5972/uploads/page/notebookfolder/notebook4.webp)

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

![table7](https://static.igem.wiki/teams/5972/uploads/page/notebookfolder/table7.webp)

Table2 The primers used in this study

![table8](https://static.igem.wiki/teams/5972/uploads/page/notebookfolder/table8.webp)

### 2. Experimental Methods and Results

#### 2.1 Plasmid Maps

![notebook5](https://static.igem.wiki/teams/5972/uploads/page/notebookfolder/notebook5.webp)

Figure 1 Map of Plasmid pET28a- CheA

![notebook6](https://static.igem.wiki/teams/5972/uploads/page/notebookfolder/notebook6.webp)

Figure 2 Map of Plasmid pET28a- CheY

![notebook7](https://static.igem.wiki/teams/5972/uploads/page/notebookfolder/notebook7.webp)

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

![notebook8](https://static.igem.wiki/teams/5972/uploads/page/notebookfolder/notebook8.webp)

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:

![table9](https://static.igem.wiki/teams/5972/uploads/page/notebookfolder/table9.webp)

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:
![table10](https://static.igem.wiki/teams/5972/uploads/page/notebookfolder/table10.webp)4. Perform colony PCR according to the following program:
![table11](https://static.igem.wiki/teams/5972/uploads/page/notebookfolder/table11.webp)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:
![notebook9](https://static.igem.wiki/teams/5972/uploads/page/notebookfolder/notebook9.webp)

Figure 1 Electrophoresis Image of Colony PCR Results

![notebook10](https://static.igem.wiki/teams/5972/uploads/page/notebookfolder/notebook10.webp)

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.

![notebook11](https://static.igem.wiki/teams/5972/uploads/page/notebookfolder/notebook11.webp)

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.

![notebook12](https://static.igem.wiki/teams/5972/uploads/page/notebookfolder/notebook12.webp)

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

![table12](https://static.igem.wiki/teams/5972/uploads/page/notebookfolder/table12.webp)

### 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:
![notebook13](https://static.igem.wiki/teams/5972/uploads/page/notebookfolder/notebook13.webp)

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

![table13](https://static.igem.wiki/teams/5972/uploads/page/notebookfolder/table13.webp)

Table 2 Main Plasmids and Strains Used in This Experiment

![table14](https://static.igem.wiki/teams/5972/uploads/page/notebookfolder/table14.webp)

### 2. Experimental Methods and Results

#### 2.1 Plasmid Maps

![notebook14](https://static.igem.wiki/teams/5972/uploads/page/notebookfolder/notebook14.webp)

Figure 1 Map of Plasmid pSR58.6

![notebook15](https://static.igem.wiki/teams/5972/uploads/page/notebookfolder/notebook15.webp)

Figure 2 Map of Plasmid pSR58.6-CheA

![notebook16](https://static.igem.wiki/teams/5972/uploads/page/notebookfolder/notebook16.webp)

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

![notebook17](https://static.igem.wiki/teams/5972/uploads/page/notebookfolder/notebook17.webp)

Figure 4 Map of Plasmid pSR58.6-CheY

![notebook18](https://static.igem.wiki/teams/5972/uploads/page/notebookfolder/notebook18.webp)

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.

![notebook19](https://static.igem.wiki/teams/5972/uploads/page/notebookfolder/notebook19.webp)

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

![table15](https://static.igem.wiki/teams/5972/uploads/page/notebookfolder/table15.webp)

### 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

![table16](https://static.igem.wiki/teams/5972/uploads/page/notebookfolder/table16.webp)

Table2 The primers used in this study

![table17](https://static.igem.wiki/teams/5972/uploads/page/notebookfolder/table17.webp)

### 2. Experimental Methods and Results

#### 2.1 Plasmid Maps

![notebook20](https://static.igem.wiki/teams/5972/uploads/page/notebookfolder/notebook20.webp)

Figure 1 Map of Plasmid pDawn

![notebook21](https://static.igem.wiki/teams/5972/uploads/page/notebookfolder/notebook21.webp)

Figure 2 Map of Plasmid pDawn-CheZ

![notebook22](https://static.igem.wiki/teams/5972/uploads/page/notebookfolder/notebook22.webp)

Figure 3 Map of Plasmid pDawn-CheZ-Flag

![notebook23](https://static.igem.wiki/teams/5972/uploads/page/notebookfolder/notebook23.webp)

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).
![notebook24](https://static.igem.wiki/teams/5972/uploads/page/notebookfolder/notebook24.webp)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

![table18](https://static.igem.wiki/teams/5972/uploads/page/notebookfolder/table18.webp)

### 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.

![notebook25](https://static.igem.wiki/teams/5972/uploads/page/notebookfolder/notebook25.webp)

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

![table19](https://static.igem.wiki/teams/5972/uploads/page/notebookfolder/table19.webp)

Table 2 Main Plasmids and Strains Used in This Experiment

![table20](https://static.igem.wiki/teams/5972/uploads/page/notebookfolder/table20.webp)

### 2. Experimental Methods and Results

#### 2.1 Plasmid Maps

![notebook26](https://static.igem.wiki/teams/5972/uploads/page/notebookfolder/notebook26.webp)

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

![notebook27](https://static.igem.wiki/teams/5972/uploads/page/notebookfolder/notebook27.webp)

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

![notebook28](https://static.igem.wiki/teams/5972/uploads/page/notebookfolder/notebook28.webp)

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

![table21](https://static.igem.wiki/teams/5972/uploads/page/notebookfolder/table21.webp)

### 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

![table22](https://static.igem.wiki/teams/5972/uploads/page/notebookfolder/table22.webp)

### 2. Experimental Methods and Results

#### 2.1 Plasmid Maps

![notebook29](https://static.igem.wiki/teams/5972/uploads/page/notebookfolder/notebook29.webp)

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

![notebook30](https://static.igem.wiki/teams/5972/uploads/page/notebookfolder/notebook30.webp)

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.

![notebook31](https://static.igem.wiki/teams/5972/uploads/page/notebookfolder/notebook31.webp)

# 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.

{% endblock %}

{% extends "layout.html" %}
{% block page\_content %}

### PROJECT

# ENGINEERING

### OVERVIEW

* [INTRODUCTION](#introduction)
* [CYCLE 1](#cycle1)
* [CYCLE 2](#cycle2)
* [CYCLE 3](#cycle3)

# Introduction

The methodology of synthetic biology is rooted in the fundamental principles of engineering,
among which the engineering cycle stands as one of the most core frameworks. This cycle provides
comprehensive and systematic guidance for the design and testing of biological systems, encompassing
four key phases: design, build, test, and learn.

In this project, we have enabled Escherichia coli
BL21 to exhibit tumbling behavior under green light irradiation and linear movement under blue
light by constructing light-driven micro-nanorobots. Meanwhile, we fully recognize that a standardized
engineering cycle is particularly critical for such interdisciplinary research; therefore,
on the basis of strictly adhering to this framework, we have further conducted innovative explorations
to ensure that the research process consistently adheres to the full-process logic of engineering
problem-solving.

Cycle 1 demonstrates the process of constructing plasmids using seamless cloning, along with the subsequent
generation of plasmid-containing engineered strains. Building upon this, Cycle 2 validates the
functionality of the constructed pathways, while Cycle 3 elaborates on the efforts undertaken to verify
the motility of the engineered strains.

# CYCLE 1:Construction of Recombinant Plasmids and Engineered Strains

## 1.1 Construction of Recombinant Plasmids

### Design

To verify the functions of the CheA, CheY, and CheZ genes in Escherichia coli, we first selected
pBBR1MCS-5 as the vector to construct three plasmids, namely pBBR1-MCS-CheA, pBBR1-MCS-CheY, and
pBBR1-MCS-CheZ. Subsequently, we chose the blue light-responsive system pDawn as the vector to construct
pDawn-CheZ, and selected pSR58.6 (from the green light-responsive dual-plasmid system) as the vector to
construct pSR58.6-CheA and pSR58.6-CheY.

![engineering1](https://static.igem.wiki/teams/5972/uploads/page/engineering/engineering1.webp)

Figure 1.Map of the Recombinant Plasmid Using pBBR1MCS-5 as the Vector

![engineering2](https://static.igem.wiki/teams/5972/uploads/page/engineering/engineering2.webp)

Figure 2.Map of the Recombinant Plasmid Using pDawn as the Vector

![engineering3](https://static.igem.wiki/teams/5972/uploads/page/engineering/engineering3.webp)

Figure 3.Map of the Recombinant Plasmid Using pSR58.6 as the Vector

### Build

Plasmids were constructed via seamless cloning. Taking the pBBR1-MCS-CheA plasmid as an example: first,
PCR amplification was performed. Using the Escherichia coli BL21 genome as the template, primers
BL21-CheA-pBBR1-F/BL21-CheA-pBBR1-R were selected to amplify the target CheA fragment. Subsequently,
using the pBBR1MCS-5 plasmid as the template, primers pBBR1MCS-Che-F/pBBR1MCS-Che-R were chosen to amplify
the plasmid backbone.

The sizes of the target fragment and plasmid backbone bands were confirmed by 1% agarose gel
electrophoresis. If the band sizes were verified to be correct, gel extraction was conducted. After the
extraction process, the concentration of the recovered products was determined using a NanoDrop
instrument. The target fragment and plasmid backbone were mixed at a 3:1 ratio, and the ligation
reaction was carried out at 50°C for 15 minutes under the action of seamless cloning enzyme to obtain
the ligation product.

Table1    The primers used in this study

![engineering_table1](https://static.igem.wiki/teams/5972/uploads/page/engineering/engineering-table1.webp)

![engineering4](https://static.igem.wiki/teams/5972/uploads/page/engineering/engineering4.webp)

Figure 4.Schematic Diagram of pBBR1-MCS-CheA Construction via Seamless Cloning

### Test

Taking the construction of the pBBR1-MCS-CheA plasmid as an example: Using the Escherichia coli BL21
genome as the template, PCR amplification was performed with the primers
BL21-CheA-pBBR1-F/BL21-CheA-pBBR1-R, yielding a target fragment with a band size of 2009 bp.
Using the pBBR1MCS-5 plasmid as the template, PCR amplification was conducted with
pBBR1MCS-Che-F/pBBR1MCS-Che-R, resulting in a plasmid backbone with a band size of 4769 bp.

As shown in the electrophoresis image below, single electrophoresis bands with the correct
sizes were clearly observed. Thus, it was confirmed that the CheA gene and the pBBR1MCS-5 plasmid
backbone were successfully amplified.

![engineering5](https://static.igem.wiki/teams/5972/uploads/page/engineering/engineering5.webp)

Figure 5.Electrophoresis Image of the CheA, CheY and CheZ Fragments

![engineering6](https://static.igem.wiki/teams/5972/uploads/page/engineering/engineering6.webp)

Figure 6.Electrophoresis Image of the Vector Backbone

### Learn

Initially, when constructing recombinant plasmids using pBBR1MCS-5 as the vector, we observed that
the plasmid size was inconsistent with the expected value. We then attempted restriction enzyme ligation,
but the result remained unsatisfactory.Subsequently, we replaced the vector with pET28a, redesigned
primers for seamless cloning, and simultaneously sent the pBBR1MCS-5 vector for sequencing while
repeating the experiment with a new kit. Eventually, we successfully constructed pBBR1-MCS-CheA,
pBBR1-MCS-CheY, and pBBR1-MCS-CheZ using pBBR1MCS-5 as the vector, and these plasmids were used for
subsequent studies.

Additionally, we found that the current design failed to determine whether the target proteins
were successfully expressed in the engineered strains, prompting further design iterations.

### Iteration 1: Construction of Flag-tagged Plasmids for Western Blot

Building on the work in Section 1.1, the following Flag-tagged plasmids were designed: pBBR1-MCS-CheA-Flag,
pBBR1-MCS-CheY-Flag, pBBR1-MCS-CheZ-Flag, pDawn-CheZ-Flag, pSR58.6-CheA-Flag, and pSR58.6-CheY-Flag.

After introducing the aforementioned plasmids into engineered strains and inducing expression, Western
blot (WB) can be used to detect whether the target proteins are successfully expressed. This lays a
foundation for subsequent functional verification.

![engineering7](https://static.igem.wiki/teams/5972/uploads/page/engineering/engineering7.webp)

Figure 7.Maps of the Recombinant Plasmid Using pBBR1MCS-5 as the Vector

![engineering8](https://static.igem.wiki/teams/5972/uploads/page/engineering/engineering8.webp)

Figure 8.Map of the Recombinant Plasmid Using pDawn as the Vector

![engineering9](https://static.igem.wiki/teams/5972/uploads/page/engineering/engineering9.webp)

Figure 9.Maps of the Recombinant Plasmid Using pSR58.6 as the Vector

Taking pSR58.6-CheY-Flag as an example:

Table2    The primers used in this study

![engineering_table2](https://static.igem.wiki/teams/5972/uploads/page/engineering/engineering-table2.webp)

![engineering10](https://static.igem.wiki/teams/5972/uploads/page/engineering/engineering10.webp)

Figure 10.Schematic Diagram of pSR58.6-CheY-Flag Construction via Seamless Cloning

![engineering11](https://static.igem.wiki/teams/5972/uploads/page/engineering/engineering11.webp)

Figure 11.Electrophoresis Image of the CheY Fragment with a Flag tag

![engineering12](https://static.igem.wiki/teams/5972/uploads/page/engineering/engineering12.webp)

Figure 12.Electrophoresis Image of the Vector Backbone

### Iteration 2: Construction of Light-Inducible Plasmids Expressing sfGFP for Fluorescence Detection

We consider that confirming the successful expression of target proteins solely through Western blot does
not meet our research needs. In the light-inducible expression system, the correlation between induction
efficiency and protein expression level can be intuitively reflected by GFP fluorescence intensity, which is
particularly suitable for subsequent system optimization.

Since the pSR58.6 plasmid harbors the sfGFP gene, we constructed the pDawn-sfGFP plasmid. Fluorescence
intensity was used to characterize the activity of the promoter, thereby analyzing the regulatory effects of
different conditions on gene transcription and optimizing the induction conditions.

![engineering13](https://static.igem.wiki/teams/5972/uploads/page/engineering/engineering13.webp)

Figure 13.Maps of the Green Light-Responsive Dual-Plasmid Expressing sfGFP

![engineering14](https://static.igem.wiki/teams/5972/uploads/page/engineering/engineering14.webp)

Figure 14.Map of the pDawn-sfGFP Plasmid

Table3    The primers used in this study

![engineering_table3](https://static.igem.wiki/teams/5972/uploads/page/engineering/engineering-table3.webp)

![engineering15](https://static.igem.wiki/teams/5972/uploads/page/engineering/engineering15.webp)

Figure 15.Schematic Diagram of pDawn-sfGFP Construction via Seamless Cloning

![engineering16](https://static.igem.wiki/teams/5972/uploads/page/engineering/engineering16.webp)

Figure 16.Electrophoresis Image of the sfGFP Fragment

![engineering17](https://static.igem.wiki/teams/5972/uploads/page/engineering/engineering17.webp)

Figure 17.Electrophoresis Image of the Vector Backbone

### Iteration 3: Construction of Fusion Expression Systems for sfGFP with CheA, CheY, and CheZ

In previous studies, we used Edwardsiella piscicida into which GFP was introduced to infect
zebrafish, and took photos under a fluorescence microscope. As shown in the images, green fluorescent
signals could be observed in multiple parts of the zebrafish body, especially concentrated in the head and
the visceral region of the trunk. This indicated that Edwardsiella piscicida successfully invaded the
zebrafish and spread to different tissues or organs, verifying the pathogenicity of Edwardsiella piscicida
to zebrafish and providing intuitive evidence for studying the infection process.

Ultimately, we hope to construct a co-fusion plasmid of sfGFP and the target protein and
introduce it into Edwardsiella piscicida, so as to characterize the bacterial infection ability through
fluorescence.

![engineering18](https://static.igem.wiki/teams/5972/uploads/page/engineering/engineering18.webp)

Figure 18.Fluorescence Microscopic Images of Zebrafish Infected with GFP-Labeled Edwardsiella Piscicida

Building on the aforementioned experiments, we aim to better leverage the localization and
tracking function of sfGFP. Specifically, we will insert sfGFP in series with CheA, CheY, and CheZ
respectively into plasmids via gene cloning, enabling their expression as a single polypeptide chain in
cells.

This design ensures that the luminescent property of the fluorescent protein remains unaffected
by fusion, while the native function of the target proteins is typically preserved—thus achieving the
coupling of "fluorescent labeling-protein tracking". It allows not only the characterization of target
protein expression levels via fluorescence intensity (quantified using instruments like a fluorescence
spectrophotometer or software such as ImageJ) but also the direct observation of the movement status of
engineered strains under a fluorescence microscope.

Currently, this part of the work is under construction.

![engineering19](https://static.igem.wiki/teams/5972/uploads/page/engineering/engineering19.webp)

Figure 19.Maps of the Co-fusion Plasmids of sfGFP and the Target Protein

## 1.2 Construction of the Engineered Strains

### Design

To align with this study, two strains of Escherichia coli (E. coli) were selected as chassis strains for
functional division of labor:

E. coli DH5α: Used as a cloning strain for the construction and amplification of recombinant plasmids. It
lacks the recA recombinase and endA nuclease, which prevents the loss and degradation of plasmids due to
homologous recombination, thus ensuring the stability of recombinant plasmids.

E. coli BL21 (DE3): Used as an expression strain for the induced expression of target proteins. It lacks the
outer membrane protease ompT and protease lon, which reduces the degradation of target proteins.

The final target strain of this study is Edwardsiella piscicida—a Gram-negative bacterium pathogenic to
aquatic organisms, with application potential in the fields of aquaculture disease research and biological
control. However, considering biosafety risks, E. coli was used for functional simulation and technical
verification in the preliminary experimental stage. As a conventional model strain in biological
laboratories, E. coli has a biosafety level of 1, requiring no special protective equipment for operation.
Additionally, it has a clear genetic background and mature genetic manipulation tools, enabling efficient
simulation of core processes of Edwardsiella piscicida (such as plasmid transformation and target gene
expression). This provides a technical reference for the subsequent engineering modification of the actual
pathogenic bacterium.

Table4    Main Strains Used in This Experiment

![engineering_table4](https://static.igem.wiki/teams/5972/uploads/page/engineering/engineering-table4.webp)

### Build

Taking pBBR1-MCS-CheA, pBBR1-MCS-CheY, and pBBR1-MCS-CheZ as examples:

The aforementioned recombinant plasmids were transformed into E. coli DH5α competent cells,
plated, and cultured overnight. Colonies were selected for verification using the primers
pBBR1MCS-Che-CHECK-F/pBBR1MCS-Che-CHECK-R. Strains with correct verification results were sent for
sequencing, and those with confirmed correct sequencing results were named DH5α-pBBR1-MCS-CheA,
DH5α-pBBR1-MCS-CheY, and DH5α-pBBR1-MCS-CheZ.

Table5    The primers used in this study

![engineering_table5](https://static.igem.wiki/teams/5972/uploads/page/engineering/engineering-table5.webp)

The plasmids from the sequencing samples were transformed into E. coli BL21 competent cells,
plated, and cultured overnight. The following day, single colonies were picked and cultured in shaking
flasks for a certain period, after which plasmids were extracted. These plasmids were subjected to 1%
agarose gel electrophoresis. The engineered strains with plasmid bands of the expected size were named
BL21-pBBR1-MCS-CheA, BL21-pBBR1-MCS-CheY, and BL21-pBBR1-MCS-CheZ.

### Test

After transformation into E. coli DH5α, colony PCR was performed on 12 randomly picked
colonies. The results of PCR electrophoresis verification are shown in Figure 20. Bands with lengths similar
to the positive control fragment have been marked in the electrophoresis image. Positive transformants were
selected for sequencing, and all sequencing results were correct (Figure 21-23).

![engineering20](https://static.igem.wiki/teams/5972/uploads/page/engineering/engineering20.webp)

Figure 20.Electrophoresis Result Image of Colony PCR

![engineering21](https://static.igem.wiki/teams/5972/uploads/page/engineering/engineering21.webp)

Figure 21.Sequence Alignment of DH5α-pBBR1-MCS-CheA Sequencing Results

![engineering22](https://static.igem.wiki/teams/5972/uploads/page/engineering/engineering22.webp)

Figure 22.Sequence Alignment of DH5α-pBBR1-MCS-CheY Sequencing Results

![engineering23](https://static.igem.wiki/teams/5972/uploads/page/engineering/engineering23.webp)

Figure 23.Sequence Alignment of DH5α-pBBR1-MCS-CheZ Sequencing Results

### Learn

During the construction of engineered strains, initially, colonies were too dense to pick single colonies
after transformation and plating. Therefore, the protocol was adjusted from "centrifuging the bacterial
culture, discarding the supernatant, and then plating" to "directly pipetting the bacterial culture for
plating." After this adjustment, the number of colonies became moderate.

Since the green light-inducible system is a dual-plasmid system, plasmids of the pSR43.6r and pSR58.6 series
were co-transformed into BL21, and two types of antibiotics were added to the medium. We observed that the
dual-plasmid engineered strains exhibited a slower growth rate compared to the single-plasmid engineered
strains. This is presumably due to resource competition for plasmid replication and metabolic burden, though
the specific reasons require further investigation.

# CYCLE 2: Expression Verification and Intensity Analysis of CheA, CheY, and CheZ Proteins

## 2.1 Western blot

### Design

We aim to demonstrate that the constructed engineered strains can express the target proteins
of interest under corresponding induction conditions. For instance, the CheZ protein is expressed under blue
light induction, while the CheA or CheY proteins are expressed under green light induction. In the absence
of induction, little or no target protein is expressed. To this end, engineered strains harboring
Flag-tagged plasmids were used, and Western blot was employed to visualize the expression levels of the
target proteins.

### Build

Taking the blue light-driven micro-nanorobot—BL21-pDawn-CheZ-Flag—as an example: Blue light induction was
initiated when the bacterial culture reached an OD₆₀₀ of approximately 0.6. A flashlight emitting blue light
with a wavelength of 470 nm–480 nm was used, and the induction was conducted in a 37 °C incubator for 6
hours. For the dark control group, bacterial cultures were wrapped in aluminum foil and incubated under the
same conditions.

After induction, bacterial cells were washed with PBS and uniformly diluted to a bacterial culture with an
OD₆₀₀ of 1.5. The diluted culture was mixed with SDS-PAGE Sample Loading Buffer, and the mixture was boiled
in boiling water for 10 minutes. Subsequently, SDS-PAGE electrophoresis was performed, followed by membrane
transfer. The membrane was blocked overnight at 4 °C. On the next day, the PVDF membrane was incubated with
HRP-conjugated Mouse anti-Flag-Tag antibody, then with goat anti-mouse secondary antibody. After the
incubation steps, development was carried out.

### Test

![engineering24](https://static.igem.wiki/teams/5972/uploads/page/engineering/engineering24.webp)

Figure 24.WB Development Result Image of BL21-pDawn-CheZ-Flag

The development results are shown in the figure above, and the protein bands generally matched
the expected size. BL21-pDawn-CheZ-Flag induced by blue light expressed more CheZ protein, while the band of
the control group cultured in the dark was thinner. However, target protein bands were still detected in the
non-illuminated group, which deviated from the expected light-responsive regulatory effect.

### Learn

The Western blot experiment confirmed that the engineered strains we constructed can express
the corresponding proteins and are regulated by induction signals. For subsequent experiments, we plan to
conduct them in a light-shielded environment. Additionally, we will add an internal reference protein group,
which can correct for systematic errors such as differences in sample loading volume, uneven membrane
transfer efficiency, and variations in protein extraction efficiency. This group serves as a critical
control for quantitative analysis in WB experiments.

## 2.2 Detection of sfGFP Fluorescence Intensity

### Design

Building on the Western blot results, we further utilized sfGFP as a "reporter gene," whose expression is
regulated by promoters. A higher fluorescence intensity typically indicates higher transcription and
translation efficiency of the target gene, allowing direct quantification of the target gene expression
levels across different strains or under different culture conditions. Additionally, since induction
conditions directly affect target gene expression, fluorescence intensity detection can serve as a rapid and
non-invasive optimization indicator to guide subsequent experiments.

### Build

The engineered strains used in this experiment are the blue light-driven BL21-pDawn-sfGFP and the green
light-driven BL21-pSR43.6r&pSR58.6. Three groups were set up: a dark group, a 3-hour light exposure group,
and a 6-hour light exposure group, all cultured at 37 °C. At the corresponding time points, aliquots of the
bacterial culture were taken, washed with water, and diluted to an OD₆₀₀ of 1. The diluted samples were
loaded into a 96-well microplate, and fluorescence data were measured using a multimode microplate reader.

For data processing, the background value of the empty microplate was first subtracted. Then, the
fluorescence intensity of the water group was used as a reference for normalization, and the relative
fluorescence intensity of the other groups was calculated.

### Test

![engineering25](https://static.igem.wiki/teams/5972/uploads/page/engineering/engineering25.webp)

Figure 25.Relative Fluorescence Intensity of Bacterial Culture under Blue Light
Induction

![engineering26](https://static.igem.wiki/teams/5972/uploads/page/engineering/engineering26.webp)

Figure 26.Relative Fluorescence Intensity of Bacterial Culture under Green Light
Induction

The Water group, serving as a blank control, exhibited the lowest relative fluorescence intensity. This
indicates that water itself produces almost no fluorescence, providing a background reference for
fluorescence detection in subsequent experimental groups

The bacterial culture group without light treatment showed a significantly higher relative fluorescence
intensity than the Water group. This suggests that even in the absence of light, the engineered strains
harboring sfGFP-containing plasmids still exhibit a certain level of fluorescence expression.

After 3 hours of light treatment, the relative fluorescence intensity increased further, being significantly
higher than that of the No Light group. This demonstrates that light can induce the expression of sfGFP in
the engineered strains, and the induction effect is already quite significant with a 3-hour light duration.

When the light duration was extended to 6 hours, the relative fluorescence intensity reached its highest
level, showing a further increase compared to the 3-hour group. This indicates that as the light duration
increases, the inductive effect on sfGFP expression continues to strengthen; a longer light duration
promotes the engineered strains to produce more sfGFP, thereby exhibiting higher fluorescence intensity.

### Learn

Overall, the experimental results clearly show that light can effectively induce the expression of sfGFP in
engineered strains harboring sfGFP-containing plasmids, and the induction effect strengthens with the
extension of light duration. At the same time, the results also indicate that the engineered strains exhibit
a certain level of basal sfGFP expression in the absence of light.

Initially, the experimental results were consistently unsatisfactory. After replacing the transparent
microplate with an opaque black microplate and repeating the experiment, the aforementioned results were
obtained. Through this experiment, the induction time was determined to be 6 hours. Due to experimental
constraints, the fluorescence intensity under longer induction durations could not be detected, and this
will be improved in subsequent studies.

# CYCLE 3: Investigation of the Motility of Engineered Strains

### Design

LB semi-solid medium is prepared by adding 0.25% agar to liquid medium. We designed semi-solid medium for
observing bacterial motility primarily based on its specific viscosity and resistance. This resistance not
only allows motile bacteria to move actively but also restricts their random diffusion, making bacterial
motility more distinct and easier to observe.

In our hypothesis, blue light-driven engineered strains tend to move in a straight line, thereby exhibiting
a larger diffusion range with neat edges on the semi-solid plate. In contrast, green light-driven engineered
strains tend to tumble, and their colonies are expected to show blurred edges with an uneven internal
density gradient.

### Build

Three IPTG-inducible expression systems, namely pBBR1-MCS-5-CheZ, pBBR1-MCS-5-CheY, and pBBR1-MCS-5-CheA,
were constructed. Semi-solid medium supplemented with 0.25% agar was dispensed into Petri dishes. For each
system, 2 μL of bacterial culture (either induced for 6 hours or cultured in the dark) was carefully spotted
onto the center of the Petri dish, followed by incubation at 37 °C in an upright position. The
overexpression effect of the target proteins was analyzed by comparing the colony morphology and area of
strains between the induced group (with IPTG) and the control group (without IPTG) on the semi-solid plates.

Two green light-inducible expression systems, pSR43.6&pSR58.6-CheY and pSR43.6&pSR58.6-CheA, were
constructed. It is expected that green light induction will promote the overexpression of CheY and CheA
proteins, thereby inhibiting bacterial diffusion. Additionally, a blue light-inducible expression system,
pDawn-CheZ, was constructed; green light induction is expected to promote the overexpression of CheZ
protein, which in turn enhances bacterial diffusion.

### Test

The results indicate that the current semi-solid culture results cannot fully demonstrate the effect of CheY
and CheA protein overexpression on flagellar tumbling motion, nor can they completely confirm the enhancing
effect of CheZ protein on straight-line motion. Only the potential regulatory trend of these proteins can be
preliminarily inferred.

### Learn

Since no defective strains were constructed, the experimental results were not obvious and were subject to
certain human operation interference. Therefore, the study was optimized in two aspects:

First, the experimental design was modified. Bacterial cultures in the uninduced state were spotted onto
semi-solid plates. After the bacterial cultures infiltrated the medium, light induction (or IPTG induction)
was immediately applied to the plates, ensuring that the bacteria were regulated by the induction signal
while initiating motility and diffusion. This modification guarantees that the bacterial motility is
regulated by the induction condition throughout the process, and the bacteria are under a unified induction
condition from the initial stage of diffusion, resulting in more comparable data. In the original method,
local differences in aeration volume and inducer concentration might exist in the bacterial culture
container. This could lead to some bacteria overexpressing the target protein in advance while others
expressing insufficiently. After spotting, the colony morphology on the plates would be disordered due to
"differences in initial protein levels," making it impossible to distinguish whether the phenomenon was
caused by "induction effect" or "uneven pre-induction." Additionally, during the pre-induction process, the
bacteria might experience metabolic stress due to excessive protein expression, resulting in inconsistent
bacterial activity at the time of spotting.

Second, to more clearly compare the differences in bacterial motility, an oil immersion objective was used
to observe and record videos. The movement trajectories of bacteria in the field of view were analyzed to
improve the accuracy of the experimental results.

### Iteration

From the experimental results in the figure, after culturing the strain BL21-pSR43.6r&pSR58.6-CheA under
dark conditions for 17 hours and green light conditions for 17 hours respectively, the following conclusions
can be drawn:

The colonies in both Petri dishes of the dark-cultured group exhibited relatively regular, small, and
concentrated morphologies. In contrast, the colonies in the two Petri dishes of the green light-cultured
group showed more dispersed morphologies with more irregular edges compared to the dark-cultured group. This
suggests that green light induction may have an impact on the growth or related physiological processes of
the strain.

![engineering27](https://static.igem.wiki/teams/5972/uploads/page/engineering/engineering27.webp)

Figure 27.Comparison Diagram of Semi-Solid Culture Plates With and Without Green
Light Induction

The movement trajectories of the engineered strains were observed using a 100× oil immersion objective, and
videos were recorded for analysis. The figure below shows a freeze-frame image from this field of view.
Rod-shaped Escherichia coli can be observed; however, due to the three-dimensional structure of the
bacteria, clear focusing was not possible in some cases. Nevertheless, this image still indicates that the
bacteria move in different dimensions.

![engineering28](https://static.igem.wiki/teams/5972/uploads/page/engineering/engineering28.webp)

Figure 28.Video Screenshot of Bacterial Movement Trajectories Observed via Oil
Immersion Objective

{% endblock %}

{% extends "layout.html" %}
{% block page\_content %}

### LAB

# EXPERIMENTS

### OVERVIEW

* [PROTOCOL](#protocol)
* [PARTS](#parts)

# Protocol

## 1. Plasmid Extraction (Using a Trial Kit)

### 1.1 Introduction

Extract plasmid DNA from the bacteria.

### 1.2 Materials

TIANprep Mini Plasmid Kit

### 1.3 Procedure

* 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.
* Add 500 μL of colorless Solution RB (containing RNase A) and vortex to resuspend the bacterial pellet;
  no small bacterial clumps should remain.
* 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).
* 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.
* 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.
* Add 650 μL of Solution WB, centrifuge at 12,000×g for 1 minute, and discard the flow-through.
* Centrifuge at 12,000×g for 1–2 minutes to completely remove residual WB.
* 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).
* Centrifuge at 10,000×g for 1 minute to elute DNA. Store the eluted DNA at -20°C.

## 2. PCR

### 2.1 Introduction

A nucleic acid synthesis technique that utilizes the principle of DNA double-strand replication
to replicate specific DNA fragments outside of living organisms.

### 2.2 Materials

Table 1 General PCR Reaction System

![table1](https://static.igem.wiki/teams/5972/uploads/page/experiments/table1.webp)

### 2.3 Program

Table 2 General PCR Reaction Program

![table2](https://static.igem.wiki/teams/5972/uploads/page/experiments/table2.webp)

## 3. DNA purification

### 3.1 Introduction

A crucial step in molecular biology experiments in order to remove impurities such as primers,
dNTPs, and nonspecific amplification products in the PCR system, so as to avoid interference with subsequent
applications such as DNA sequencing, cloning, and restriction enzyme digestion.

### 3.2 Materials

TIANgel Purification Kit

### 3.3 Procedure

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.

## 4. Agarose Gel Electrophoresis

### 4.1 Introduction

An electrophoresis method that uses agar or agarose as the supporting medium. It's the standard
lab procedure for separating DNA by size.

### 4.2 Materials

#### 4.2.1 equipment

electrophoresis tank, voltage source, well combs, microwave.

#### 4.2.2 reagent

running buffer (1×TAE), agarose, 6×gel loading buffer, gelred gel dye.

### 4.3 Procedure

1. Weigh the appropriate amount of agarose powder according to the desired concentration, mix the agarose
   with the running buffer, and place in the microwave oven until the agarose is completely dissolved.
2. After cooling to about 50°C, add an appropriate amount of DNA dye and mix gently. Pour the solution into
   the gel casting mold, insert the comb, and allow it to solidify (about 30 min).
3. Remove the gel, remove the comb, and place the gel in the electrophoresis tank, adding the running
   buffer to ensure that the gel is completely submerged.Use a micropipette to carefully load the sample into
   the wells of the gel.
4. Connect the electrophoresis apparatus, set the appropriate voltage to 140 volts, and start the
   electrophoresis. The electrophoresis time varies depending on the size of the DNA fragment and gel
   concentration, typically ranging from 30 minutes to 2 hours.

## 5. Gel Extraction

### 5.1 Introduction

A standard step used to recover DNA fragments separated by electrophoresis in agarose gel.

### 5.2 Materials

TIANgel Purification Kit.

### 5.3 Procedure

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 solution 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.

## 6. Gibson Assembly

### 6.1 Introduction

A molecular biology technique used for the seamless joining of multiple DNA fragments in a
single reaction without the need for restriction enzymes or DNA ligases.

### 6.2 Materials

DNA fragments to be assembled, 2×MultiF Seamless Assembly Mix, Sterile and nuclease-free water.

### 6.3 Procedure

* Mix the purified target fragment and plasmid backbone (0.03 pmol) at a molar ratio of 3:1.
* Add ddH2O up to 20 μL.
* Ligate at 50°C for 15 minutes to obtain the ligation product.

## 7. Calcium transformation

### 7.1 Introduction

A widely used method for introducing foreign DNA into bacterial cells. This technique exploits
the natural competence of bacteria to take up DNA from their environment, facilitated by calcium ions.

### 7.2 Materials

Gibson assembly product, BL21 competent cells, sterilized LB medium, antibiotics.

### 7.3 Procedure

1. Take E. coli BL21 competent cells out of the -80°C refrigerator, and thaw on ice for 5–10 minutes.
2. Add 10 μL of Gibson assembly product 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. Colony PCR

### 8.1 Introduction

A commom way to verify if the transformation was successful, through specifically amplifying a
sequence which can only exist in recombinant plasmids. In this case, after electrophoresis, those colonies
with correct bands will turn out to have been successfully transformed, thus can be sequenced later.

### 8.2 Materials

2×Magic Green Taq SuperMix, primers, sterilized EP tubes, LB medium (containing the
corresponding antibiotic).

### 8.3 Procedure

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:

Table 3 Colony PCR Reaction System

![table3](https://static.igem.wiki/teams/5972/uploads/page/experiments/table3.webp)

4. Perform colony PCR according to the following program:

Table 4 Colony PCR Reaction Program

![table4](https://static.igem.wiki/teams/5972/uploads/page/experiments/table4.webp)

5. After PCR, perform 1% agarose gel electrophoresis.

## 9. IPTG induction expression

### 9.1 Introduction

Express the protein controlled by Lac operon.

### 9.2 Materials

1 mol/L IPTG (Isopropyl β-D-1-thiogalactopyranoside), sterilized LB medium, antibiotics.

### 9.3 Procedure

1. Inoculate 50 μL of the overnight-cultured seed solution into a vial containing 7 mL of LB medium (with
   corresponding antibiotics), 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.

## 10. Light induction expression

### 10.1 Introduction

Express the protein controlled by special photosensitive promoters.

### 10.2 Materials

Light of a speific wavelength (specialized flashlight), Petri dishes, Erlenmeyer flasks,
sterilized LB medium, corresponding antibiotics, spectrophotometer, aluminum foil.

### 10.3 Procedure

#### 10.3.1 Green Light Induction

1. Aspirate 100 μL of the corresponding bacterial solution and inoculate it into 10 mL of LB medium
   (supplemented with corresponding 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.
4. For shaking culture occasion, the remaining bacterial solution was distributed into two Erlenmeyer
   flasks (or disposable shaking flasks), also, with one set as the light group and the other as the
   non-light group. The former was illuminated by a green flashlight and shaking cultured at a 37°C shaker,
   while the latter was placed in another dark 37°C shaker.

#### 10.3.2 Blue Light Induction

The same as above, only changing the flashlight into blue.

## 11. Culture collection

### 11.1 Introduction

The technique of maintaining the vitality and genetic characteristics of microbial strains.

### 11.2 Materials

Glycerin, saline water, bacterial solution.

### 11.3 Procedure

1. Mix glycerin and saline in a 1:1 ratio to form a glycerin solution with a final concentration of 50%.
2. When used, mix according to the ratio of 50% glycerin: bacterial liquid =1:1, so that the final glycerin
   concentration is 20-30%.
3. Transfer the glycerin strains to the refrigerator at -20℃ and store for a long time.

## 12. Bacterial Harvesting and WB Sample Preparation

### 12.1 Materials

1×PBS (pH 7.4), 5×protein loading buffer.

### 12.2 Procedure

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).
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.

## 13. SDS-PAGE & Western blotting

### 13.1 Introduction

Western immunoblotting is a method of transferring a protein to a membrane and then using
antibodies for detection. Western Blot uses polyacrylamide gel electrophoresis. The subject is a protein,
the probe is an antibody, and the color rendering uses a labeled secondary antibody. The protein sample
separated by PAGE is transferred to a solid-phase carrier (such as nitrocellulose film-pvdf). The
solid-phase carrier adsorbs the protein in the form of a non-covalent bond, and can keep the type of
polypeptide separated by electrophoresis and its biological activity unchanged. The protein or polypeptide
on the solid-phase carrier is used as the antigen, and the corresponding antibody is immunologically
reacted, and then the enzyme or isotope-labeled second antibody is reacted, and the substrate is
color-developed or radioautoextended to detect the protein component of the specific target gene expression
isolated by electrophoresis. This technology is also widely used to detect the expression of protein levels.

### 13.2 Procedure

1. Prepare WB samples from the induced bacterial solutions.
2. Take out the precast 12% SDS-PAGE gel cassette (or self-made gels, with detailed preparation method in
   13.3), 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.
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.

### 13.3 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.

## 14. Configuration of culture medium

### 14.1 Introduction

Medium refers to a nutrient matrix formulated from a combination of different nutrients that
supplies microorganisms, plants, or animals (or tissues) for growth and reproduction. Generally, it contains
several types of substances such as carbohydrates, nitrogen-containing substances, inorganic salts
(including trace elements), vitamins, and water. Media vary in its function, configuration and application,
where the use of a medium is generally achieved by changing its configuration.

### 14.2 Materials

Tryptone, yeast extract, NaCl, deionized water, agar.

### 14.3 Procedure

#### 14.3.1 LB liquid medium

1. Weigh 10 g tryptone, 5 g yeast extract, and 10 g sodium chloride.
2. Add about 800 mL of deionized water, stir thoroughly to dissolve.
3. Add deionized water to bulk the medium to 1 L.
4. Sterilize at 115°C for 25 minutes (or 121°C for 20 minutes).

#### 14.3.2 LB solid medium

The same as liquid medium, with 2% (w/v) agar powder added as a coagulant before sterilization.

#### 14.3.3 LB semi-solid medium

The same as liquid medium, with 0.25% (w/v) agar powder added as a coagulant before
sterilization.

## 15. Bacteria Motility Verification

### 15.1 Introduction

Visualize the diffusion ability of an engineered bacteria through semi-solid medium.

### 15.2 Materials

LB semi-solid medium, corresponding antibiotics.

### 15.3 Procedure

#### 15.3.1 Original method

1. Heat and melt the LB semi-solid medium, cool to approximately 55°C, add corresponding antibiotic, 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.

#### 15.3.2 Optimized approach

Basically the same in operation details as above, with the order of induction and inoculation
changed. The bacterial solution (before induction) was spot on the center of the prepared semi-solid LB
medium plate first. Then the plate was induced using a flashlight with a specific wavelength.

## 16. Multi-Mode Microplate Reader Detection

### 16.1 Introduction

To detect the flurorescent intensity of the engineered bacteria after being induced in contrast
to that without induction.

### 16.2 Procedure

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.

## 17. Microscopic Observation

### 17.1 Introduction

See and record the movement and motion situation of the engineered bacteria based on ordinary
optics microscopy through 100×oil immersion lens, with a total magnification ratio of 1000×.

### 17.2 Materials

Glass slides, coverslips, ordinary optics microscope.

### 17.3 Procedure

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.

# Parts

### 1. Introduction

For us, the Parts in iGEM are the "cornerstone" of our project—from building the genetic circuit for light-controlled bacterial movement to ensuring the stable function of vaccine strains, all rely on standardized and highly active parts. We not only strictly screen and verify parts to ensure experimental efficiency, but also optimize and submit new parts to the Registry, which not only lays a solid foundation for our own project, but also contributes to the shared collaboration of global synthetic biology.

### 2. Basic parts

Table 1 Previously Published Basic Parts Used in This Study

![table5](https://static.igem.wiki/teams/5972/uploads/page/experiments/table5.webp)
![table6](https://static.igem.wiki/teams/5972/uploads/page/experiments/table6.webp)

Table 2 Basic Parts Uploaded in This Study

![table7](https://static.igem.wiki/teams/5972/uploads/page/experiments/table7.webp)
![table8](https://static.igem.wiki/teams/5972/uploads/page/experiments/table8.webp)

Composite parts

Table 3 Composite Parts Uploaded in This Study

![table9](https://static.igem.wiki/teams/5972/uploads/page/experiments/table9.webp)
![table10](https://static.igem.wiki/teams/5972/uploads/page/experiments/table14.webp)
![table11](https://static.igem.wiki/teams/5972/uploads/page/experiments/table12.webp)
![table11](https://static.igem.wiki/teams/5972/uploads/page/experiments/table13.webp)

{% endblock %}

{% extends "layout.html" %}
{% block page\_content %}

### LAB

# RESULTS

### OVERVIEW

* [INTRODUCTION](#introduction)
* [SECTION 1](#section1)
* [SECTION 2](#section2)
* [SECTION 3](#section3)
* [SECTION 4](#section4)
* [SECTION 5](#section5)

# Introduction

## 1.1 Research Background

In this project, we plan to use Escherichia coli as a substitute for Edwardsiella piscicida to
construct an engineered strain that exhibits linear movement under blue light and tumbling movement under
green light. We will further combine this engineered strain with a light irradiation device to achieve
precise control over the movement state of Escherichia coli, thereby enhancing the invasive ability of the
vaccine strain and improving the efficacy of immersion immunization.

At present, we have completed the construction of four groups of plasmids, which have been
introduced into Escherichia coli BL21 to construct engineered strains. We have verified that the target
proteins can be successfully expressed and initially tested their effects on the movement state of bacteria.

# Section 1: Plasmid Amplification and Reconstruction

In this project, pBBR1MCS-5, pDawn, and pSR58.6 were used as vectors. The main target genes
included CheA, CheY, CheZ, and sfGFP. The electrophoresis results of the main fragments and vector backbones
used in this study are shown in the figure below.

![results1](https://static.igem.wiki/teams/5972/uploads/page/results/results1.webp)

Figure 1 Electrophoresis Image of the CheA, CheY and CheZ Fragments

![results2](https://static.igem.wiki/teams/5972/uploads/page/results/results2.webp)

Figure 2 Electrophoresis Image of the sfGFP Fragment

![results3](https://static.igem.wiki/teams/5972/uploads/page/results/results3.webp)

Figure 3 Electrophoresis Image of the pBBR1MCS-5 Vector Backbone

![results4](https://static.igem.wiki/teams/5972/uploads/page/results/results4.webp)

Figure 4 Electrophoresis Image of the pDawn Vector Backbone

![results5](https://static.igem.wiki/teams/5972/uploads/page/results/results5.webp)

Figure 5 Electrophoresis Image of the pSR58.6 Vector Backbone

The aforementioned fragments and vector backbones were successfully assembled into the
recombinant plasmids listed in the table below using a seamless cloning method.

Table 1 Main Plasmids Used in This Experiment

![table1](https://static.igem.wiki/teams/5972/uploads/page/results/table1.webp)

# Section 2: Verification of Light-responsive Pathways

Centered on constructing light-controlled E. coli BL21 engineered strains that move straight
under blue light and tumble under green light, this project relies on CheA, CheY, and CheZ proteins to
control bacterial movement. Western Blot and fluorescence detection are key experiments to verify protein
expression and regulation efficiency, laying the foundation for subsequent movement capability studies.

Western blot directly confirms whether target proteins are expressed: Using Flag-tagged
plasmids (e.g., BL21-pDawn-CheZ-Flag), it detects protein expression differences under light induction.
Results show blue light significantly increases CheZ expression, confirming the "light signal → target
protein expression" pathway works; weak bands in dark groups indicate minor basal expression, guiding
subsequent optimizations.

![results6](https://static.igem.wiki/teams/5972/uploads/page/results/results6.webp)

Figure 6 WB Development Result Image of BL21-pDawn-CheZ-Flag

Under green light induction conditions, similar results were also observed. Taking
BL21-pSR43.6r&pSR58.6-CheA-Flag as an example:

![results7](https://static.igem.wiki/teams/5972/uploads/page/results/results7.webp)

Figure 7 WB Development Result Image of BL21-pDawn-CheZ-Flag

Fluorescence detection (with sfGFP as reporter gene) quantifies expression efficiency:
Experiments on BL21-pDawn-sfGFP and BL21-pSR43.6r&pSR58.6 show light boosts sfGFP expression in a
time-dependent manner, confirming inducible and quantifiable light-responsive pathways.

![results8](https://static.igem.wiki/teams/5972/uploads/page/results/results8.webp)

Figure 8 Relative Fluorescence Intensity of Bacterial Culture under Blue Light
Induction

![results9](https://static.igem.wiki/teams/5972/uploads/page/results/results9.webp)

Figure 9 Together, they verify the light-controlled expression system is functional,
ensuring
reliable attribution of subsequent movement experiment results to protein function rather than expression
errors.

# Section 3:

## Verification 1: Macroscopic Verification via Semi-Solid Medium Culture Assay

To preliminarily verify the motility of the engineered strains, we designed a semi-solid medium
culture assay. Owing to its semi-solid nature, this medium exhibits both a certain degree of fluidity and
stability. Utilizing semi-solid medium to assess bacterial diffusive motility (i.e., motility test) enables
more intuitive observation of phenomena, making it a fundamental and crucial identification technique in
microbiology laboratories.

In our hypothesis, overexpression of the CheZ protein would dephosphorylate CheY-A, thereby
inhibiting the clockwise rotation of Escherichia coli (E. coli) flagella, weakening their tumbling motility,
and simultaneously promoting counterclockwise rotation to enhance their straight-line motility. The weakened
tumbling ability and strengthened straight-line motility of E. coli would manifest as enhanced diffusive
motility on semi-solid medium, possibly with an accelerated diffusion rate—specifically, forming larger
colony areas within the same time period.

To validate whether the effect of CheZ overexpression aligns with our hypothesized outcomes
under IPTG induction, we constructed the pBBR1-MCS-5-CheZ system.

![results10](https://static.igem.wiki/teams/5972/uploads/page/results/results10.webp)

Figure 10 Results of semi-solid medium culture for the pBBR1-MCS-5-CheZ system(From
left to right: Plates 1 and 3 are IPTG-induced groups; Plate 2 is the uninduced control group)

From the semi-solid culture results, the colonies in the two experimental groups (IPTG-induced)
were indeed larger than those in the middle control group (uninduced). This confirms that overexpression of
the CheZ protein can effectively promote the straight-line motility of E. coli.

Conversely, overexpression of the CheY protein or CheA protein (a phosphokinase) would increase
the production of CheY-A, thereby promoting the counterclockwise rotation of E. coli flagella, enhancing
their tumbling motility, and inhibiting clockwise rotation to weaken their straight-line motility. The
strengthened tumbling ability and weakened straight-line motility of E. coli would manifest as reduced
diffusive motility on semi-solid medium, possibly with a slower diffusion rate—specifically, forming smaller
bacterial lawn areas within the same time period. To verify whether the effects of CheY and CheA
overexpression match our hypothesized outcomes under IPTG induction, we further constructed the
pBBR1-MCS-5-CheY and pBBR1-MCS-5-CheA systems.

![results11](https://static.igem.wiki/teams/5972/uploads/page/results/results11.webp)

Figure 11 Results of semi-solid medium culture for the pBBR1-MCS-5-CheY system(From
left to right: Plates 1 and 2 are IPTG-induced groups; Plate 3 is the uninduced control group)

![results12](https://static.igem.wiki/teams/5972/uploads/page/results/results12.webp)

Figure 12 Results of semi-solid medium culture for the pBBR1-MCS-5-CheZ system(From
left to right: Plates 1 and 2 are IPTG-induced groups; Plate 3 is the uninduced control group)

From the semi-solid culture results, the colonies in the IPTG-induced groups were either the
same size as or smaller than those in the uninduced control group. This partially aligns with our expected
outcomes. We hypothesize that this discrepancy may stem from human operational errors during sample
spotting, which caused slight variations in the volume of bacterial culture added—leading to differences in
colony size. To a certain extent, this result supports the validity of our initial hypothesis, but further
verification is required to confirm the exact scenario.

In our design, under green light induction, the pSR43.6-pSR58.6-CheA and pSR43.6-pSR58.6-CheY
systems would overexpress the CheA and CheY proteins, respectively. This would increase CheY-A production,
enhance tumbling motility, and weaken straight-line motility of the bacteria. On semi-solid medium, this
would manifest as reduced diffusive motility and a slower diffusion rate—specifically, forming smaller
bacterial lawn areas within the same time period.

![results13](https://static.igem.wiki/teams/5972/uploads/page/results/results13.webp)

Figure 13 Results of semi-solid medium culture for the pSR43.6-pSR58.6-CheY system

![results14](https://static.igem.wiki/teams/5972/uploads/page/results/results14.webp)

Figure 14 Results of semi-solid medium culture for the pSR43.6-pSR58.6-CheA system

From the results in Figures 13 and 14, there was no significant difference in colony size
between the green light-induced groups and the non-induced groups. This confirms that the variations in
colony size observed in the pBBR1-MCS-5 system were likely caused by experimental errors. However, this does
not sufficiently prove that overexpression of CheA or CheY has no effect on flagellar tumbling motility.
Similarly, while the colony results from CheZ overexpression in semi-solid medium do not fully confirm
enhanced straight-line motility, they provide preliminary macroscopic evidence of a potential effect.
Further experiments are needed to clarify the exact impact of these protein overexpressions.

Therefore, we conducted the following microscopic observation assay to investigate bacterial
motility at the microscopic level.

## Verification 2: Microscopic Investigation via Microscopic Field Observation

To directly identify changes in the motility of engineered strains from a microscopic
perspective, we designed a microscopic observation assay to visualize the movement trajectories of E. coli
in a liquid environment, as shown in the figures below.

![results15](https://static.igem.wiki/teams/5972/uploads/page/results/results15.webp)

Figure 15 Microscopic movement trajectories of strains from the pDawn-CheZ system

Figure 15 presents the movement trajectories of four representative E. coli cells selected for
observation. Microscopic observations revealed that the strains overexpressing CheZ exhibited a noticeable
enhancement in straight-line motility. Compared to the macroscopic observations from the semi-solid medium
assay, this microscopic evidence further confirms our expected outcomes.

![results16](https://static.igem.wiki/teams/5972/uploads/page/results/results16.webp)

Figure 16 Microscopic movement trajectories of strains from the pSR43.6-pSR58.6-CheY
system

The E. coli cells observed in Figure 16 were strains overexpressing CheY. Observations of their
motility revealed a significant decrease in movement ability. This is consistent with our previous
hypothesis that overexpression of CheY enhances tumbling motility. We propose that if E. coli exhibits
strengthened tumbling motility and weakened straight-line motility, it would appear as slow in-place
wriggling rather than rapid movement under microscopic observation. This further confirms the success of our
strain engineering. The unexpected diffusion results from the earlier semi-solid medium assay may be
attributed to the difficulty of detecting changes in E. coli flagellar motility using macroscopic
observation methods.

# Section 4: Verification Assay for the Infectivity of Edwardsiella Piscicida

![results17](https://static.igem.wiki/teams/5972/uploads/page/results/results17.webp)

Figure 17 Infection assay of Edwardsiella piscicida labeled with red fluorescent
protein

![results18](https://static.igem.wiki/teams/5972/uploads/page/results/results18.webp)

Figure 18 Infection assay of Edwardsiella piscicida labeled with green fluorescent
protein

In this set of experiments, we constructed E. piscicida strains labeled with red fluorescent
protein and green fluorescent protein, respectively, and subjected them to attenuation treatment before
conducting infection assays. Through immersion infection assays on zebrafish larvae, we finally detected the
infection efficiency using fluorescence microscopy, as shown in Figures 17 and 18. Observations revealed
that the vaccine strains primarily accumulated in the gills, digestive tract, and skin surface of the
zebrafish larvae. This is consistent with findings from previously reviewed literature, which indicate that
vaccine strains typically enter the fish body through these three pathways and then reach immune organs such
as the liver via bodily fluids to activate the immune response—thereby exerting their function as a vaccine.

# Section 5: Conclusion and Outlook

## Conclusion

Our goal was to design a light-inducible system for precise control of Edwardsiella piscicida
motility. This would allow accurate immersion vaccination in fish. As a first step, we performed
proof-of-concept studies in E. coli.

Using pBBR1-MCS-5, we showed that CheZ, CheA, and CheY can alter flagellar behavior. We then
used pDawn and pSR43.6r&pSR58.6 systems to achieve light-inducible control. Under blue light, cells were
expected to swim linearly. Under green light, cells were expected to tumble.

We successfully constructed 13 engineered strains. PCR and gel electrophoresis confirmed
correct assembly. Western blotting verified protein expression. Semi-solid assays and microscopy
demonstrated changes in motility consistent with our design. sfGFP served as a reporter to measure
light-inducible expression levels. These data supported our prediction of motility regulation.

To test translational application, we performed zebrafish immersion infection with
fluorescently labeled Edwardsiella piscicida. Bacteria localized to major entry points and immune organs.
This confirmed the feasibility of our proposed vaccination concept.

## Outlook

Time limits restricted our current work to concept validation. In the future, we will:

1. Repeat experiments to ensure reproducibility.
2. Combine blue- and green-light-inducible systems in a single strain. This will allow ratio-dependent
   control of linear versus tumbling motility.
3. Transfer the system to Edwardsiella piscicida. This should enable precise, light-controlled infection
   during immersion vaccination. The strategy may improve efficiency, dosage control, and delivery accuracy
   in aquaculture vaccines.

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