iGEM 2025 — Team hust-ueve-upsaclay

{% include 'menu.html' %}

# NOTEBOOK

The Records of Our Experiment Process

{% include 'footer.html' %}

➤

* [Engineering on *Escherichia coli (E. coli)*](#sec_1)
* [Engineering on *Bacillus subtilis*(the bacteria we adopt is called BS168)](#sec_6)
* [Engineering on regulation part](#sec_11)
* [Engineering on *Caenorhabditis elegans (C. elegans)*](#sec_15)

{% include 'menu.html' %}

# ENGINEERING

Progress and improvement of our project

## Engineering on *Escherichia coli (E. coli)*

## Introduction

We hope to verify the pathway from L-Tyr to naringenin in *E. coli*, so we designed three kinds of plasmids respectively contain matB-matC genes, TAL-4CL genes and CHS-CHI genes in order to synthesis naringenin using L-Tyr as ingredients. And simultaneously we want to verify the efficiency of *cre-loxp* principle in *E. coli*.In addition, we use *B. subtilis* to verify the pathway from naringenin to luteolin.

## Period 1: Electrotransformation of *pACYCDuet-matB-matC*+*pETDuet-CHS-CHI* into *E. coli*

Objective: To prepare for the transformation of *pCDFDuet-TAL-4CL* into *E. coli* and the expression of naringenin.

Practice: *pACYCDuet-matB-matC*+*pETDuet-CHS-CHI* were transformed into *E. coli*. And then *E. coli* with these two plasmids was then made into chemically competent state.

Conclusion: Before transforming the two targeted plasmids into *E. coli*, we transformed *pACYCDuet* and *pETDuet* empty plasmids into *E. coli* and coated it on plates with homologous antibiotics. After a few times of failure,we saw bacterial colony on the plates, indicating the success of the transformation process. This showed the low probability of successfully transforming two or more plasmids into one bacteria simultaneously, while it is known to all that transform one plasmid at a time costs a longer time. Therefore, we chose to firstly electrotransform two plasmids into *E. coli* and secondly chemically transform the one plasmid left into the same *E. coli*.

## Period 2: Chemically transformation of *pCDFDuet-TAL-4CL* into *E. coli*

Objective: To construct a engineering bacteria to achieve the goal of synthesizing naringenin. Thus we can verify the pathway from L-Tyr to naringenin.

Practice: *pCDFDuet-TAL-4CL* was successfully transformed into *E. coli* with the former two plasmids.

Conclusion: We can achieve the goal of simultaneously transforming numerous plasmids into the same *E. coli* even when we use two different methods of transformation.And eventually the sequencing analysis proofed that the transformation was a success.

## Period 3: Measurement of existence of enzymes by SDS-PAGE

Objective: To check whether there is expected enzymes existing or not.

Practice: We used wild-type BL21 strain (under different temperatures of 16℃, 25℃, and 37℃) as the Control group and BL21 strain with three plasmids under different temperatures (16℃, 25℃, and 37℃) as the Experiment group to do SDS-PAGE measurement.

Conclusion: We successfully obtained the enzymes that we want for producing naringenin no matter under what temperature conditions because there exist stripes in all Experiment groups and there do not exist any stripes in all Control groups. These results showed that the transformation and expression processes were successful.

## Period 4: Measurement of naringenin using HPLC

Objective: To check whether there is naringenin produced or not, which is to check whether the first half of the pathway is successful or not.

Practice: We put the engineering bacteria under different temperature conditions, which are 16℃, 25℃, and 37℃ (37℃ is the temperature at which the bacteria are cultured, 25℃ is the room temperature, and 16℃ is the temperature we calculated according to the principle of similar span) for reaction and used HPLC to measure the concentrations of the naringenin under different conditions and compared them. By the way, we used wild-type BL21 strain as the Control group.

Conclusion: No matter what temperature is used for the reaction, there always exists naringenin in the Experiment group, while there is not any naringenin in the Control group, indicating that the first half of the pathway is feasible.

## Engineering on *B. subtilis*(the bacteria we adopt is called *BS168*)

## Period 1: Construction of *pBE2R-FNS-F3’H*

Objective: To construct a plasmid that can achieve the pathway from naringenin to luteolin.

Design plasmid: According to the pathway from naringenin to luteolin, we use gene FNS to turn naringenin into apigenin, a kind of intermediate product, and then we use gene F3'H to turn apigenin into luteolin. We chose the *pBE2R* vector to hold these gene fragments, and apart from the gene fragments, we added a terminator onto the *pBE2R* vector.

Practice: We used the principle of PCR to duplicate fragments of RBS1-FNS1, RBS2-F3'H-TT, vector-pBE2R, and a terminator. And then we used the method of homologous recombination to connect these fragments and the vector into a whole plasmid.

Conclusion: We initially tried to construct a plasmid whose structure is p43promoter-FNS-terminator-p43promoter-F3'H-terminator, but because there exist many similar sequences which cause the homology to be poor, we could not use the method of homologous recombination to successfully connect these fragments. Therefore, we deleted a promoter and a terminator that are located between genes FNS and F3'H, and then the homologous recombination succeeded.

## Period 2: Transformation of *pBE2R-FNS-F3’H*

Objective: To obtain the engineering bacteria that can produce luteolin.

Practice: We made *BS168* strain into chemically competent state. And used the method of fast transformation to transform *pBE2R-FNS-F3’H* into *BS168* strain.

## Period 3: Measurement of existence of enzymes by SDS-PAGE

Objective: To see whether there are targeted enzymes in engineering bacteria.

Practice: We got the protein samples from engineering bacteria with different plasmids. The one with *pBE2R-FNS-F3'H* is the Experiment group, while the wild-type is the Control group. We set temperature ladders of 16℃, 25℃, and 37℃ (37℃ is the temperature at which the bacteria are cultured, 25℃ is the room temperature, and 16℃ is the temperature we calculated according to the principle of similar span) for both the Experiment and Control groups. Then we conducted SDS-PAGE measurement to see if we got our targeted protein.

Conclusion: Our results from the SDS-PAGE measurement showed that temperatures don't have an evident influence on the expression process. And more importantly, we successfully obtained our targeted enzymes.

## Period 4: Further Verification of Enzyme Presence by Western Blot

Objective: To further verify the presence of target enzymes in engineered bacteria. (Bands obtained from SDS-PAGE may be miscellaneous; antibody specificity is used to confirm that the obtained bands are target bands.)

Experimental Procedure: Take the protein gel samples obtained after SDS-PAGE: engineered bacteria containing the *pBE2R-FNS-F3'H* plasmid serve as the experimental group, and wild-type *BS168* strain as the control group. Perform membrane transfer on the protein samples, add primary antibody and secondary antibody after blocking, then capture images for observation.

Conclusion: For the engineered bacteria with the *pBE2R-FNS-F3'H* plasmid, clear target bands are visible in the images; for the wild-type *BS168* strain, no bands are observed. This confirms that the bands obtained from SDS-PAGE are the target bands.

## Period 5: Measurement of luteolin using HPLC

Objective: To check whether there is luteolin in the reaction system so that we can know whether the second half of the pathway is feasible or not.

Practice: We put the engineering bacteria under different temperature conditions, which are 16℃, 25℃, and 37℃ (37℃ is the temperature at which the bacteria are cultured, 25℃ is the room temperature, and 16℃ is the temperature we calculated according to the principle of similar span) for reaction and used HPLC to measure the concentrations of luteolin under different conditions and compared them. By the way, we used wild-type *BS168* bacteria as the Control group.

Conclusion: In our first attempt, we did not see any sign of luteolin’s occurrence. We reckon that the probable reasons may be as follows:

1. The plasmid we constructed was wrong, so the enzymes cannot perform their expected function.
2. It is hard for the ingredient naringenin to be transported into the engineering bacteria, which means that there lacks naringenin in the bacteria cell, so the enzyme cannot function well.

Because our sequencing result was very successful, we can rule out the influence of the first problem. To figure out whether it is the second reason influencing, we decided to construct an extracorporal reaction system. We explored different concentration conditions of naringenin, which are 0.1mM and 1mM. What’s more, we explored different reaction time conditions, which are 1h, 2h, 3h, 4h, 6h, and 9h. Moreover, we used wild-type *B. subtilis* directly reacting for 9h as Control group1 and transformed *B. subtilis* without naringrnin directly reacting for 9h as Control group2. For every single group, we set two different conditions---fracturing fluid and cracked liquid supernatant. Apart from the cofactors enzymes need to perform their functions, we also added Tris-HCl to control pH condition the same. And simultaneously, we simply repeated our first attempt to see whether it is operation caused errors.

## Engineering on regulation part

## Introduction

We screened phages targeting the *B. subtilis BS168* strain from the environment to achieve regulation of engineered bacteria. Further more, we hope to achieve dual regulation of the engineered bacteria, so we designed a component whose structure can be described as “lsl”, which stands for “loxp-terminator-loxp”. The two *loxp* component is in the same direction and the enzyme expressed by cre gene can combine to this component and cut off the stop,which is a terminator, so that perform the function of starting the gene expression process.

## Period 1: Screening of phages from the environment and their enrichment and purification

Objective: To sceen the phage targeting *E. coli* BL21 and *B. subtilis BS168* strains from the environment.

Practice: We collected soil and water samples from the flower beds and lakes on the campus of Huazhong University of Science and Technology. These samples were co-incubated with the bacterial suspensions of *E. coli* BL21 and *B. subtilis BS168* strains respectively for amplification. The phages were sceened using the **Double Agar Overlay Assay** and purified by the **phage streaking method**.

![Fail to load picture](https://static.igem.wiki/teams/5730/engineering/e-5.webp)

Figure 1. The process of phage sceening

Conclusion: We successfully sceened phages targeting the *E. coli* BL21 strain and the *B. subtilis BS168* strain from the soil, and obtained clear plaques.

## Period 2: Characterization of environmental phages and verification of their bactericidal properties

Objective: To confirm the types of the phages we have selected and verify their antibacterial efficacy.

Practice: The concentration of phages was quantified by the **Spot titer assay**. The types of phages and their bactericidal effects were determined through **Planktonic Bacteria Bactericidal experiment** and **Transmission Electron Microscopy(TEM)**.

![Fail to load picture](https://static.igem.wiki/teams/5730/engineering/e-6.webp)

Figure 2. The process of **Spot titer assay**

Conclusion:

(1) The **TEM electron-microscopy** results indicated that the phage targeting the *E. coli* BL21 strain was mainly filamentous phage, namely lysogenic phage; while the phage targeting the *B. subtilis BS168* strain was a combination of lysogenic and virulent phages.

(2) The **Spot titer assay** results showed that the Phage concentration (number of plaque forming units per milliliter, PFU/mL) of phage concentrated solution reached 10^9 PFU/mL.

(3) The **Planktonic Bacteria Bactericidal experiment** results proved that the phage had a good sterilization effect and could be used as a chassis organism for further modification.

## Period 3: Transformation of *pETDuet-CHS-lsl-CHI*+*pCFD-cre* and *pETDuet-CHS-lsl-CHI*+*pCDF* into *E. coli*

Objective: To design a molecular switch that can make the whole production pathway controllable.

Practice: We used the method of chemical transformation to transform *pETDuet-CHS-lsl-CHI*+*pCDF-cre* into BL21 in chemically competent state. And we used BL21 transformed with *pETDuet-CHS-lsl-CHI*+*pCDF* as the Control group.

## Period 4: Verification of the function of gene cre using SDS-PAGE

Objective: To see whether there is targeted protein to verify whether we obtained an effective *cre-loxp* structure.

Practice: We got the protein samples from engineering bacteria with different plasmids. The one with *pCDF-cre* is the Experiment group, while the one with *pCDF* is the Control group. Then we conducted SDS-PAGE measurement to see if we got our targeted protein.

Conclusion: The results of the SDS-PAGE measurement showed that both the Experiment and the Control group have targeted protein, which means that the terminator was cut off so that the targeted gene can be expressed. However, there should not be any targeted protein in the Control group. Two reasons can cause this phenomenon. Firstly, the terminator did not work out well.Secondly, the expression capacity of the target fragment is excessively strong, causing the repressor protein bound to the operon to lose its function. As a result, expression can occur without lactose induction.Therefore, in the future, we redesigned a plasmid by adding a rrNB terminator to solve the problem.

## Period 5: Western Blot

Objective: To verify whether the *cre-loxp* mechanism functions.

Experimental Procedure: Take the protein gel samples obtained after SDS-PAGE: engineered bacteria containing the *pCDF-cre* plasmid serve as the experimental group, and engineered bacteria containing the *pCDF* plasmid serve as the control group. Perform membrane transfer on the protein samples, add primary antibody and secondary antibody after blocking, then capture images for observation.

Conclusion:

(1) The TEM electron-microscopy results indicated that the phage targeting the *Escherichia coli* BL21 strain was mainly filamentous phage, namely lysogenic bacteriophage; while the phage targeting the *B. subtilis BS168* strain was a combination of lysogenic and virulent phages.

(2) The Spot titer assay results showed that the Phage concentration (number of plaque forming units per milliliter, PFU/mL) of phage concentrated solution reached 10^9 PFU/mL.

(3) The Planktonic Bacteria Bactericidal experiment results proved that the phage had a good sterilization effect and could be used as a chassis organism for further modification.

## Engineering on *Caenorhabditis elegans (C. elegans)*

## Construction of Cry Protein Expression Plasmid

We designed a plasmid to express Cry5Ba and Cry6Aa proteins in the *C. elegans* intestine. First, since the Cry proteins we used originated from Bacillus thuringiensis, heterologous expression in *B. subtilis* and *E. coli* might pose challenges. Therefore, we initially completed codon optimization. Subsequently, to enable high-level expression and purification for toxicity validation, we selected the *pET28a* vector from the pET series. The detailed design of our plasmid is outlined

Using homologous recombination technology, we designed homologous arms on the primers to recombine the gene with the vector.

The vector was linearized and subjected to PCR to obtain insertion fragments with homologous arms. Following gel verification, we performed gel recovery, followed by homologous recombination and transformation of competent *DH5α* cells. The resulting colonies were spread plate, picked individual bacterial colony, and individual bacterial colony PCR. PCR validation confirmed the presence of the desired band, which was subsequently sent for sequencing.

![Fail to load picture](https://static.igem.wiki/teams/5730/engineering/20251009184738-31-385.avif)
![Fail to load picture](https://static.igem.wiki/teams/5730/engineering/20251009184738-30-385.avif)

Figure 3. The Expression Plasmid of Cry5Ba & Cry6Aa Protein

## Engineering of the Cry5Ba&Cry6Aa Fusion Protein

During preliminary background research, we discovered that Cry5Ba and Cry6Aa proteins exhibit non-overlapping mechanisms of action and demonstrate synergistic effects when treating wild-type nematodes. Therefore, we plan to design a fusion protein combining both to achieve enhanced insecticidal efficacy. To preserve their critical domains without disrupting the secondary structure of protein folding, we employed the flexible peptide GGGGS to link the two proteins. In our project, we designed two fusion proteins: one linked by (GGGGS)1 and another by (GGGGS)2.

Using overlap PCR technology, we designed primers with complementary overlapping sequences to link the two genes. Simultaneously, through homologous recombination, the fusion gene was recombined with the vector. Below are the primers we designed with complementary overlapping sequences.

![Fail to load picture](https://static.igem.wiki/teams/5730/engineering/e-3.webp)
![Fail to load picture](https://static.igem.wiki/teams/5730/engineering/e-4.webp)

Figure 4. **Primer Design for Overlap PCR to Fuse Cry5 and Cry6 Genes**,the linker in the upper figure is (GGGGS)₂, and the linker in the lower figure is (GGGGS)₁.

{% include 'footer.html' %}

{% include 'menu.html' %}

# PROTOCOL

The specific procedures of our experiments

## Molecular construction experiments

[ ]

PCR

1. PCR system

   | Material | dosage |
   | --- | --- |
   | Primer F | 2µL |
   | Primer R | 2µL |
   | Template | 1µL |
   | ddH2O | 20µL |
   | 2× Phanta Mix | 25µL |
2. Put the sample into PCR machine, set the parameters:

   | Temperature | Time | Number of times |
   | --- | --- | --- |
   | 95℃ | 3min | ×1 |
   | 95℃ | 15min | ×n |
   | 67℃ | 15min |
   | 72℃ | 1Kb/10s |
   | 72℃ | 5min | ×1 |
   | 4℃ | infinite | ×1 |
3. n=34 for vector-PBE2R and RBS2-F3’H;n=30 for RBS1-FNS1

[ ]

Preparation of competent cells

### E.coli

1. The recipient bacterium (BL21 (DE3)) was inoculated the night before, and a single colony was selected and cultured in LB liquid medium in a shaking bed at 37℃ overnight (about 16 hours).
2. Transfer 1ml of overnight culture to 100ml LB (1:100) medium, and violently shake culture on a shaking table at 37℃ for about 2.5-3 hours (250-300 RPM) until OD600 is 0.4-0.6.
3. When the OD600 value of the cultured bacteria reaches 0.4-0.6, collect the bacteria by centrifugation at 2000×g and 4°C for 5 minutes(When the bacterial volume before centrifugation and precipitation is 50 mL, proceed with the subsequent operations as instructed; for other volumes, perform the subsequent operations after proportional conversion).
4. Resuspend the bacterial pellet with 5 mL of pre-chilled LB medium, add 5 mL of competent cell preparation reagent that has been thawed in an ice bath or ice-water bath, mix well, and incubate on ice for 5 minutes.
5. Mix gently, then aliquot appropriately into 1.5 mL or 0.5 mL centrifuge tubes as needed for the experiment, and store at -70°C.

### Preparation of E. coli Electrocompetent Cells

1. Pick a single colony of *Escherichia coli* and culture it overnight in SOC recovery medium.
2. Inoculate the overnight culture into LB medium at a ratio of 1:200 and incubate with shaking until the OD₆₀₀ reaches approximately 0.6.
3. Incubate on ice for 10 minutes, then centrifuge at 4000 rpm at 4 °C for 10 minutes and discard the supernatant.
4. Resuspend the pellet in 10% glycerol, centrifuge at 4000 rpm at 4 °C for 10 minutes and discard the supernatant.
5. Repeat step 4 (the resuspension volume can be reduced).
6. Resuspend the pellet in 10% glycerol at a ratio of 1:400, aliquot 80 μL per tube, and store at −80 °C.

### Preparation of Super Competent Bacteria

1. When the OD₆₀₀ of the cultured bacteria reaches 0.6–0.7, place the bacterial culture in an ice bath and cool for 15 minutes. **Note:** All subsequent operations must be performed at 4 °C or in an ice bath.
2. Centrifuge at 4000 g at 4 °C for 5 minutes to collect the bacteria, and discard the supernatant. **Note:** The centrifuge must be pre-cooled.
3. If the bacterial volume before centrifugal precipitation is 50 mL, proceed with the subsequent operations. If it is another volume, convert it proportionally before proceeding with the subsequent operations.
4. Gently resuspend the bacterial pellet with 20 mL of pre-cooled *Super Competent Cell Preparation Reagent A*. Be sure to resuspend very gently and slowly; otherwise, the effect will be affected.
5. Ice bath for 15 minutes.
6. Centrifuge at 4000 g at 4 °C for 5 minutes to collect the bacteria, and discard the supernatant. **Note:** The centrifuge must be pre-cooled.
7. Gently resuspend the bacterial pellet with 2 mL of pre-cooled Super Competent Cell Preparation Reagent B. Be sure to resuspend very gently and slowly; otherwise, the effect will be affected.
8. Ice bath for 15 minutes.
9. Aliquot on ice, and you can appropriately aliquot into 50–200 μL per tube as needed.
10. Use immediately or quick-freeze in liquid nitrogen or an ethanol–dry ice bath and store at −70 °C. Usually, the efficiency will not change significantly within 6 months.

### Bacillus subtilis

1. Targeted training overnight.
2. Pick monoclonal colonies from the plate with gun muzzle and drive it in 3mL LB cultural medium. And then culture it in the environment of 37℃、200rpm overnight.
3. Prepare cultural medium one:

   | Reagent | Volume |
   | --- | --- |
   | Competent cell preparation reagent A | 5mL |
   | Competent cell preparation reagent B | 0.625mL |
   | Competent cell preparation reagent C | 0.5mL |
   | Competent cell preparation reagent D | 0.5mL |
   | Sterile ultrapure water | 43.375mL |

   Prepare cultural medium two:

   | Reagent | Volume |
   | --- | --- |
   ||  |  |
   | --- | --- |
   | Cultural medium one | 39.6mL |
   | Competent cell preparation reagent E | 0.4mL |
4. 1. Get 80uL bacterial solution and put it into 4mL cultural medium one and culture it in the environment of 37℃、220rpm till the number of OD600 reaches 0.6 and is lower than 0.8.(about 4 to 5h)
   2. Put the above solution into 36mL cultural medium two and culture it in the environment of 37℃、100rpm for 90 min.
   3. Add 8uL Competent cell preparation reagent F and accurately culture it in the environment of 37℃、100rpm for 10min.
   4. Centrifuge under the condition 4℃、4000g for 3min and reserve 4mL the above solution. And then re-suspend.
   5. Package at 200uL/tube.

[ ]

Agarose gel electrophoresis

1. We use 1% agarose gel so we add 2g agarose per 100mL 1×TAE.
2. After adding agarose we need, heat with microwave until the solution become apparently.
3. When the temperature of the solution get lower, add 1/10000 nucleic dye.
4. Pour into the plate with fixed comb and when the gel coagulate, unplug the comb.
5. Add the samples to the well.
6. Set the voltage to 120V and run the electrophoresis for 25-35 minutes.
7. To checking the results, the location of the stripe is determined by shining a UV torch(don’t shine for long time) on the gel.

[ ]

Chemical transformation

1. Add the 10 µL plasmid into 50-100 µL competent bacterium.(Note that don't touch the beneath part of the tube by hand)
2. Put the bacterium at ice for 30 min.
3. Bathe the tube in 42℃ water for 90s(for reconstituted product is 60s), and transfer it to the ice immediately to bathe for 2 min(for reconstituted product is 5 min) .
4. Add 500-750µL non-antibiotics liquid LB medium.
5. Place the bacteria in a 37℃ shaker for 45-50 min.
6. Centrifuge in 4000 rpm for 1-2 min.
7. Pour the supernatant liquid into the remaining 50-100uL and resuspend precipitation with remaining supernate.
8. Inoculated the remaining liquid onto solid medium containing certain antibiotics and incubated at 37℃ in an incubator.

[ ]

Electrical transformation

1. Put the electrospinning cup on the ice and SOC cultural medium at the environment of 37℃ before the electrical transformation starts.
2. Add 10ng plasmids into 80µL electrically competent cells. Pay attention not to make any bubble.
3. Open the electrical transformation equipment, and set the parameters for 1.8kv, 200Ω, 0.5ms, 1mm electroporation cuvette.
4. If there does not occur any electric spark, then add the transformed cells to SOC cultural medium for 1h for revival.

[ ]

Plasmid extraction

1. Harvest 1 - 5 ml overnight cultures of bacteria cells in LB (Luria-Bertani) medium by centrifuging at 12,000 rpm for 30s, discard the medium. Fully suspend pelleted bacterial cells in 250 µL Buffer I with addition of RNase A.
2. Add 250 µL Buffer I and mix thoroughly by gently inverting the tube 4 - 6 times.
3. Add 350 µL Buffer N8, mix gently and thoroughly by inverting the tube until the color of precipitate changes from blue to light yellow completely.
4. Centrifuge at full speed 12,000 rpm for 2 min.
5. Place a spin column in a 2 ml collection tube (provided), transfer the supernatant into the spin column, close the lid and centrifuge at 12,000 rpm for 30 s.
6. Discard the filtrate. Place the spin column back to the collection tube. Add 800 µL Buffer W2 to the spin column. Close the lid and centrifuge at 12,000 rpm for 30 s.
7. Discard the filtrate. Place the spin column back to the collection tube. Centrifuge at full speed 12,000 rpm for 1 min.
8. Discard the collection tube and place the spin column in a new 1.5 ml microcentrifuge tube. To elute DNA, add 60~100 µL Buffer E to the center of the column, close the lid, let stand for 1 min, and centrifuge at 12,000 rpm for 30 s.
9. Discard the spin column.

[ ]

Homologous recombination

1. Prepare the following reaction system in an ice-water bath:

   | Component | Reaction system | Negative control c | Positive control d (if necessary) |
   | --- | --- | --- | --- |
   | DNA Assembly Mix | 5 μl | 5 μl | 5 μl |
   | Linearized carrier a | 50~200 ng | 50~100 ng | pUC19 Control Plasmid, Linearized, 1 µl |
   | Inserted fragment b | 10~200 ng |  | 500 bp Control, Fragment, 1 µl |
   | ddH2O | To 10 µl | | |
2. Place the reaction system at 50℃ and incubate for 5–15 minutes.
3. Place the centrifuge tube containing the reaction solution on ice to cool it down, followed by either performing transformation or storing it at -20℃

[ ]

Gel extraction

1. Cut the gel as thin as possible and put it in 1.5mL tubes, add 500 µL of Buffer GL in kit, and place the gel in a 65 ℃ water bath to dissolve for 4-6 min.
2. Transfer the liquid to the Spin column and then centrifuge at 12,000 rpm for 1 min.
3. Discard the solution that is in the collection tube.
4. Add 700 µL Buffer W2 (in kit), and centrifuge at 12,000 rpm for 1 min, then discard the solution that is in the collection tube.
5. Repeat step 4.
6. Discard liquid, centrifuge the empty column at 12,000 rpm for 2 min.
7. Discard the collection tube and put the column into a new 1.5 mL tube, then open the lid for 2-3 min to remove ethanol in the adsorption membrane.
8. Add 35 µL eluent solution (in kit) preheated at 65 ℃ to the center of the membrane, and stand it for 2-3 min.
9. Centrifuge at 12,000 rpm for 2 min.
10. Pipette the liquid back to the column, centrifuge at 12,000 rpm for 2 min.
11. Discard the column, measure the concentration and purity.

[ ]

Liquid recovery

1. Briefly centrifuge the PCR product, enzymatic reaction solution, or crude DNA product (including genomic DNA). Measure its volume with a pipette and transfer it to a sterile 1.5 ml or 2 ml centrifuge tube. If the sample volume is less than 100 µL, make it up to 100 µL with sterile water. For high-concentration genomic DNA, it is better to dilute it to 300 µL with sterile water to improve the recovery efficiency.
2. Add 5 times the volume of Buffer GDP, invert or vortex to mix well. If it is necessary to recover DNA fragments less than 100 bp, add an additional 1.5 times the volume (of the sample + Buffer GDP volume) of anhydrous ethanol.
3. Place the adsorption column into the collection tube. Transfer ≤700 µL of the solution to the adsorption column. Centrifuge at 12,000 rpm (13,800 × g) for 30 - 60 seconds. If the volume of the mixture is >700 µL, place the adsorption column back into the collection tube, transfer the remaining solution to the adsorption column, and centrifuge at 12,000 rpm (13,800 × g) for 30 - 60 seconds.
4. Discard the filtrate and place the adsorption column into the collection tube. Add 700 µL of Buffer GW (to which anhydrous ethanol has been added) to the adsorption column. Centrifuge at 12,000 rpm (13,800 × g) for 30 - 60 seconds.
5. Please add Buffer GW along the periphery of the adsorption column wall, or cap the tube and invert it 2 - 3 times after adding Buffer GW to help completely wash off the salts adhering to the tube wall.
6. Repeat step 5.
7. Washing with Buffer GW twice ensures that salts are completely removed, eliminating their impact on subsequent experiments.
8. Discard the filtrate and place the adsorption column into the recovery tube. Centrifuge at 12,000 rpm (13,800 × g) for 2 minutes.
9. Place the adsorption column into a sterile 1.5 ml centrifuge tube, add 20 - 30 µL of Elution Buffer to the center of the adsorption column, and let it stand for 2 minutes. Centrifuge at 12,000 rpm (13,800 × g) for 1 minute. Discard the adsorption column and store the DNA at -20℃.

## Molecular characterization experiment

[ ]

SDS-PAGE analysis

1. Installation of the glue maker and template tank
   * Take one long glass plate and one short glass plate, align the left, right and bottom ends, and insert the short glass plate into the template groove with the short glass plate facing inward.
   * Insert a slanting plate on the outside of the long glass plate to fix it.
   * Place the above template slot on the gel maker and clamp it tightly, check the leakage with water.
2. Preparation of the gel
   * Prepare the lower layer of gel: 4mL of lower layer of gel solution + 4mL of lower layer of gel buffer + 80µL of improved coagulant promoter, mix well, take appropriate amount of the lower layer of gel and inject it into the slit of the glass plate, with the liquid level in the vicinity of the white crossbar.
   * Prepare upper layer gel: 1mL of upper layer glue solution + 1mL of upper layer glue buffer + 20µL of improved coagulant promoter, mix well, take the appropriate amount of upper layer glue and inject it into the slit of the glass plate, with the liquid level equal to the upper part of the short glass plate.
   * Insert a comb, try to avoid air bubbles at the upper sample hole, remove the comb after the gel is solidified, remove the template tank from the glue maker and put it into the electrophoresis tank.
3. Mix 400mL UP water with 100mL 5× SDS-PAGE solution to get the electrode buffer, add the electrode buffer to the template tank, and the liquid level inside and outside the template tank should be equal.
4. Protein Sampling
   * Centrifuge the sample at 8,000 rpm for 5 minutes.
   * Discard the supernatant.
   * Add 500 μL of PBS to the centrifuge tube and resuspend the sample.
   * Pipette 10 μL of the sample into an EP tube, and place the tube on an ice box.
   * Add 40 μL of instant protein loading buffer (5×).
   * Place the tube in a metal bath heater and heat at 100°C for 10 min.
   * Centrifuge the heated sample at 12,000 rpm for 2 minutes.
5. Turn on the electrophoresis machine, the mode is constant voltage, 120V for 65min, the current is 1200mA.
6. After electrophoresis is completed, place the gel in a box containing Coomassie Blue Staining Solution, shake in the shaker and stain for 30min.
7. The stained gel was rinsed several times with tap water until the bottom color of the gel became transparent.

[ ]

Western Blot

1. Make SDS-PAGE gel for detected protein.
2. Membrane transfer
   * Prepare membrane transfer: 5× Transfer Buffer 200 mL + 200 mL Methanol + 600 mL UP water.
   * Pour the membrane transfer buffer onto a white porcelain plate and a flat dish, and prepare filter paper and cotton in the same size as the size of the clamp. Prepare three pieces of filter paper on each side of the clamp and soak them in the buffer together.
   * Cotton is placed on the black clamp surface, above the three layers of filter paper in turn, there can be no air bubbles between the filter paper, use the roller to roll a few circles to discharge the air bubbles.
   * After electrophoresis is completed, remove the template tank, pry up the glass plate with a spatula, and cut away the upper layer of glue. Put the glue into the buffer and transfer it to the filter paper.
   * Take the NC membrane and soak it in methanol for 30 seconds.
   * Carefully put the membrane into UPH2O and soak for 2 minutes.
   * Carefully put the membrane into transfer buffer and equilibrate for at least 5 minutes.
   * Put the membrane on the adhesive (if the membrane is smaller than the adhesive, just put the membrane on the target strip position), the membrane is near the white clip part, the adhesive is against the black color.
   * Put three layers of filter paper on the membrane to drive out air bubbles.
   * Put another layer of filter paper, clip in buffer, put into the electrophoresis tank (pay attention to the direction of the electrode, black to black).
   * Put in the WB exclusive ice pack, pour in the membrane transfer buffer.
   * Let the electrophoresis tank let that as in the ice box, surrounded by ice packs, and fill the remaining area with water, but do not go over the electrophoresis tank.
   * Electrophoresis instrument set constant current 300mA, transfer membrane for 100min.
3. Closure
   * Prepare closure solution: 0.1 g BSA + 20 mL TBST, shake well.
   * After electrophoresis, take out the membrane and soak it in the sealing solution, shake for 1 hour.
4. Primary antibody incubation
   * Discard the containment solution and rinse the membrane and box with 1× TBST.
   * Add 1× TBST to wash the membrane and shake for 10 minutes.
   * Discard the TBST, add the primary antibody. Incubate at 4°C on a shaker overnight.
5. Secondary antibody incubation
   * Recover primary antibody. Wash with 1× TBST, shake for 10 minutes and wash three times.
   * Wrap the box with tin foil.
   * Add the secondary antibody and shake for 2 hours away from light.
   * Recover secondary antibody. Wash with 1× TBST, shake for 10 minutes, and wash three times, taking care to avoid light.
6. Developing
   * Use the PerkinElmer EnVision 2103 Multilabel Reader for scanning. First, spray alcohol and wipe the instrument. Then, hold the NC membrane with tweezers and place it on the microplate.
   * Mix the ELISA Femto Luminol and ELISA Femto Stabilizer at a ratio of 1:1 to prepare 400 μL of developing solution. Then, add the developing solution dropwise onto the NC membrane to make it completely cover the membrane.
   * Observe and capture images via the software.

[ ]

HPLC

| HPLC Conditions | |
| --- | --- |
| Component | Condition |
| Equipment | Agilent 1200LC |
| HPLC Column | TC-C18(2), 4.6 x 250 mm, 5 µm, 400 bar |
| Mobile Phase A | 100% Methanol |
| Mobile Phase B | 0.1% Formic Acid in Water |
| Mobile Phase Composition | A: 60%, B: 40% |
| Flow Rate | 1 mL/min |
| Detection Wavelength (Naringenin) | 285 nm |
| Detection Wavelength (Luteolin) | 350 nm |

1. **Culture & Harvest:** The engineered BS168 and wild-type BS168 strains were cultured overnight, and the cells were harvested by centrifugation.
2. **Lysate Preparation:** A portion of the cell pellet was resuspended and disrupted (e.g., by sonication or bead-beating) to create a crude lysate.
3. **Clarification:** The lysate was centrifuged (12,000 × g, 10 min) to remove cell debris, and the resulting supernatant was collected as the enzyme source.
4. **Reaction Setup:** The reaction mixture was assembled by combining the supernatant with the substrate and co-factors as detailed in Table X. The final volume was adjusted with an appropriate buffer.
5. **Incubation & Termination:** The reaction was incubated at 37°C for 9 hours and then terminated by heat inactivation at 98°C for 10 minutes.
6. **Sample Workup:** The terminated reaction was centrifuged (12,000 × g, 10 min) to precipitate denatured proteins.
7. **Analysis:** The clarified supernatant was analyzed by HPLC to quantify the reaction products.

[ ]

Phage Sampling and processing

* Preparation of host bacteria
  1. All frozen bacterial stocks were stored at −80 °C in 25% v/v glycerol.
  2. Inoculate 3 mL of bacterial media with glycerol stock in LB media, the inoculated media was incubated at 37 °C and 180 rpm for 16–18 h to promote bacterial growth.
  3. Overnight cultures were subsequently diluted 1:100 in 50 mL of fresh bacterial cell media and incubated for 2–3 h to allow bacterial cells to reach the mid-exponential growth phase.
* Preparation of phage stocks solution
  PB solution:

  | Component | Concentration |
  | --- | --- |
  | NaCl | 68 mM |
  | MgSO4 | 10 mM |
  | Tris HCl | 10 mM |
  | pH | 7.5 |

  1. Take 20 mL soil and water samples in 50 mL centrifuge tubes. For the soil sample, add 20 mL phage buffer PB, mix well, and let it stand for a certain period of time.
  2. Centrifuge both the soil and water samples at 5000g for 10 minutes.
  3. Filter through a 0.22μm filter to obtain the phage stocks solution.
* Preparation of phage concentrated solution

  LB powder: Mix well in advance for rapid preparation of culture medium containing phage, with the ratio of peptone: yeast extract: NaCl = 2:1:2.
  1. Pour 20 mL of the phage stocks solution into a conical flask (usually a 50 mL conical flask), add 0.5 g of LB powder and 20 μl of 2M CaCl2 solution. Filter through a 0.22 μm filter. Inoculate the host bacteria solution at a ratio of 1:200.
  2. Cultivate at 37℃ and 220 rpm for overnight.
  3. Centrifuge at 5000g for 10 minutes to obtain the phage concentrated solution.

[ ]

Double-layer flat plate screening

1. Spread 5 ml of solid medium with a 1.5% agar concentration onto the plate.
2. Mix 5, 50, and 500 μL of phage concentrated solution with 500 μL of host bacteria that are in the logarithmic growth phase, and then combine it with 4.5 mL of solid medium with a 0.7% agar, immediately pour it onto the top layer of the agar plate.
3. Place the plate in a 37°C incubator for overnight cultivation, and then count the number of plaques at the end.

[ ]

Spot titer assay

1. Mix 500 μL of the host bacteria that are in the logarithmic growth phase with 4.5 mL of 0.7% agar medium, and then pour it onto the 1.5% agar plate.
2. Centrifuge the phage concentrated solution at 5000g for 10 minutes.
3. Take the supernatant and perform a 10-fold gradient dilution.
4. Take 5 μl of the diluted solution and drop it onto the double-layer plate, marking it accordingly.
5. When the droplets have completely dried, incubate overnight at 37 ℃ in room air, and count the plaques.

Phage concentration (number of plaque forming units per milliliter, PFU/mL) was determined using this technique. Phage stock was serially diluted in PBS and plated on bacteriallawns to enumerate the number of infectious virions present inthe stock.

[ ]

Transmission electron microscopy

1. Dilute the overnight culture of the host bacteria at a ratio of 1:40, add it to the LB medium, and shake for 2 hours to achieve an OD600 value of 0.6 in the bacterial solution.
2. Take 4 ml of the bacteria, centrifuge at 3000g for 2 minutes, discard the supernatant, and resuspend the bacteria in 1 ml of ddH2O.
3. Repeat Step 2 three times.
4. Centrifuge the phage concentrated solution at 5000g for 10 minutes.
5. Add phage (10^11 pfu/ml) to the bacteria solution, incubate for 30 minutes, and then aspirate 20 μl and drop it onto the copper mesh (dark side up) for Transmission electron microscopy observation.

[ ]

Appendix (about phage)

1. Phage purification
   * Pick a clear phage spot and draw lines on the 1.5% agar plate.
   * Mix 500 μL of the host bacteria that are in the logarithmic growth phase with 4.5 mL of 0.7% agar medium, and then pour it onto the streaked plate.
   * Place the plate in a 37°C incubator for overnight cultivation, pick out clear plaques and repeat the process once more to isolate a single phage.
2. Phage enlarged cultivation
   * Pick out clear plaque and elute it in the host bacteria, place the plate in a 37°C incubator for overnight cultivation.
   * Centrifuge at 5000g for 10 minutes to obtain phage stock solution, 4℃ for preservation.
3. Planktonic activity of phage against host bacteria
   * Centrifuge the phage concentrated solution at 5000g for 10 minutes.
   * Based on the concentration of virus particles in the phage enrichment solution obtained from Spot titer assay, an appropriate amount of supernatant was mixed with the host bacteria with an OD600 value of 1.0, so that the concentration of virus particles in the mixture was 10 times that of the bacteria.
   * Every two hours, the bacterial cell concentration in the mixture was determined using spectrophotometry, with the detection wavelength set at 600 nm.

## *Caenorhabditis elegans (C. elegans)* experiment

[ ]

Molecular Construction of Cry5Ba and Cry6Aa

### 1. Amplification of Target Gene - PCR

1. PCR Reaction System

   | Component | Volume Added |
   | --- | --- |
   | Template DNA (Cry5Ba, Cry6Aa) | 2µL |
   | F Primer | 2µL |
   | R Primer | 2µL |
   | ddH2O | 19µL |
   | Mix | 25µL |
   | Total | 50µL |
2. PCR Program Settings

   *Fig. 1 PCR Parameters (for Cry5Ba)*

   ![Fail to load picture](https://static.igem.wiki/teams/5730/protocol/c/protocol-c-1.webp)

### 2. Electrophoresis

1. Gel Preparation

   | Component | Amount |
   | --- | --- |
   | agarose | 0.25g |
   | 10X TAE buffer | 2.5ml |
   | ddH2O | Top up to 25mL |
2. Heat the mixture in a microwave, add 2.5μL of nucleic acid dye, stir, pour the hot mixture into a mold, and wait for solidification.
3. Load the sample into the well and perform gel electrophoresis.
4. After electrophoresis, observe under UV light, then cut and recover the target band.

### 3. Gel Extraction and Purification

1. Add an equal volume of Buffer GDP, and heat in a metal bath until the gel melts.
2. Place the adsorption column into a collection tube, transfer the solution to the adsorption column, and centrifuge at 12,000 rpm for 1 minute.
3. Discard the lower solution, place the adsorption column back into the collection tube, add 300μL of Buffer GDP to the adsorption column, and centrifuge.
4. Discard the lower solution, place the adsorption column back into the collection tube, add 700μL of Buffer GW (with anhydrous ethanol added) to the adsorption column, and centrifuge.
5. Repeat Step 4 (add 700μL of Buffer GW with anhydrous ethanol again and centrifuge).
6. Dry the adsorption column and centrifuge to remove residual ethanol.
7. Add 30μL of ddH₂O for elution, centrifuge, discard the upper solution, and the collected DNA is in the lower collection tube.

### 4. Homologous Recombination

1. Measure the concentration of the gene recovered in the previous step, calculate the volumes of the linearized vector and target gene to be added (total volume: 5μL).
2. Add 5μL of 2× Seamless Cloning Mix.
3. Incubate in a PCR instrument at 50°C for 30 minutes, then at 12°C for 5 minutes.
   (Note: There are two tubes of Cry5B gene: one has a concentration of over 50 ng/μL (1.6μL of gene + 3.4μL of vector), and the other has a concentration of 41.65 ng/μL. However, the linearized vector used for the first experiment is suspected to be exhausted, so the ratio must be recalculated in subsequent experiments.)

### 5. Transformation

1. Add 10μL of the reaction sample (all contents in the PCR tube) to a tube of competent cells (DH5α or BL21), mix gently, and stand on ice for 30 minutes (in a clean bench).
2. Perform heat shock in a 42°C water bath for 90 seconds, quickly transfer back to the ice bath, and stand for 3 minutes (to restore the cell membrane).
3. Add 500μL of LB medium without antibiotics, mix well, and incubate with shaking at 37°C for 45 minutes.
4. Centrifuge at 4,000 rpm for 1 minute to precipitate bacterial cells (skip centrifugation if the concentration is too high). Aspirate the medium, leaving 100μL of medium, and mix well. Take 30μL (both undiluted and diluted as controls) and spread evenly on LB plates containing kanamycin (kana), then incubate at 37°C.

[ ]

Protein Expression of Cry5Ba and Cry6Aa

### 1. Induction of Protein Expression

1. Inoculation of Culture Medium: Inoculate 1 mL of overnight engineered bacterial culture (1% inoculum) into 100 mL LB liquid medium. Add 1‰ kanamycin (50 mg/mL stock solution).
2. Initial Culture: Incubate conical flasks in a 37°C constant-temperature shaking incubator at 220 rpm for approximately 2 hours until the culture OD600 reaches 0.3–0.7 (mid-log phase).
3. Expression induction: Add 0.5‰ IPTG (1M stock solution) to the conical flask, mix thoroughly, and place in a 16°C constant-temperature shaking incubator. Incubate at 160 rpm for 16–24 hours.

### 2. Bacterial Lysis and Soluble Expression Validation

1. Bacterial Density Measurement: Transfer 100 μL of induced bacterial culture into a 1.5 mL EP tube. Dilute 10-fold with 900 μL of ultrapure water and vortex to mix. Use ultrapure water as a blank control. Measure the OD600 of the diluted bacterial suspension using a spectrophotometer and record the actual OD value. Assuming a target OD value of 30, calculate the required volume of Buffer A for resuspension using the following formula.

   ![Expert Interview](https://static.igem.wiki/teams/5730/protocol/c/protocol-c-2.webp)
2. Cell Centrifugation and Resuspension: Transfer bacterial suspensions to pre-chilled centrifuge tubes. Top up with ultrapure water to achieve equal weights (weight tolerance ≤0.1 g). Centrifuge at 4°C (6000 rpm for 10 min). Discard the supernatant and retain the bacterial pellet. Resuspend each tube of bacterial pellet with pre-chilled Buffer A at the calculated resuspension volume. Vortex gently until bacteria are uniformly distributed throughout the solution.
3. Ultrasonic disruption: Perform ultrasonic disruption in an ice bath (parameters: 5 seconds on, 3 seconds off, for 30 minutes at 4°C). Transfer the disrupted sample to a pre-cooled centrifuge tube and store at 4°C for later use.

### 3. Soluble Expression Validation

1. SDS-PAGE Electrophoresis: Assemble the vertical electrophoresis chamber, insert the prepared gel plates, and pour 300 mL of Tris electrophoresis buffer into the middle compartment of the chamber. Remove the comb and load samples into the loading wells (loading buffer – marker – each sample – loading buffer). Cover the top lid and connect the leads. Start at 80V. Once samples enter the separating gel, increase the voltage to 120V. Stop electrophoresis when the bromophenol blue band reaches the bottom of the gel.
2. Staining and Decolorization: Remove the gel blocks after electrophoresis and immerse them in Coomassie Brilliant Blue staining solution. Seal and place in a 60°C oven for staining for 40 minutes. Rinse the gel blocks under running water, then immerse them in decolorizing solution and shake overnight until the gel background becomes transparent. Observe the band distribution under a white light transilluminator and analyze the results.

[ ]

Appendix

### 1. Preparation of LB Solid Medium (100mL)

| Component | Volume Added |
| --- | --- |
| LB Broth | 2.5g |
| Agar | 1.5g |
| ddH2O | Top up to 100mL |

1. Dissolve the components by sonication, and wrap the container with parafilm.
2. Sterilize in an autoclave at 121°C for 15-20 minutes.

### 2. Plasmid Extraction

1. Column Equilibration: Add 500μL of Equilibration Buffer BL to Adsorption Column CP4 (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 put the adsorption column back.
2. Add 5-15mL of overnight-cultured bacterial solution to a centrifuge tube, centrifuge at 12,000 rpm for 1 minute, and aspirate the supernatant as much as possible.
3. Add 500μL of Solution P1 to the tube with bacterial pellets, and thoroughly resuspend the pellets using a pipette or vortex oscillator.
4. Add 500μL of Solution P2, gently invert the tube up and down 6-8 times to fully lyse the bacteria. Do not use a vortex oscillator; lysis time should not exceed 5 minutes.
5. Add 700μL of Solution P3, immediately invert up and down gently 6-8 times (mix quickly to avoid local precipitation) – white flocculent precipitate will appear. Centrifuge at 12,000 rpm for 10 minutes, and a precipitate will form at the bottom of the tube. (Do not use a vortex oscillator.)
6. Add the supernatant from Step 5 to Filter Column CS (placed in a collection tube) in batches, centrifuge at 12,000 rpm for 2 minutes. Carefully transfer the filtrate (from the collection tube) to Adsorption Column CP4 (placed in a new collection tube) in batches. If there is residual liquid in the filter column, extend the centrifugation time of Step 5; if there is a small amount of precipitate at the bottom of the collection tube, avoid aspirating it.
7. Centrifuge Adsorption Column CP4 at 12,000 rpm for 1 minute, discard the waste liquid, and put the column back into the collection tube.
8. Add 600μL of Washing Buffer PW to the column, centrifuge at 12,000 rpm for 1 minute, discard the waste liquid, and put the column back.
9. Repeat Step 9 (add 600μL of Washing Buffer PW again and centrifuge), then let the column stand to volatilize residual ethanol.
10. Put Adsorption Column CP4 back into the collection tube and centrifuge at 12,000 rpm for 2 minutes to remove residual washing buffer.
11. Transfer the column to a clean centrifuge tube, add 100-300μL of Elution Buffer TB to the center of the adsorption membrane (avoid touching the membrane with the pipette tip), stand at room temperature for 2 minutes, and centrifuge at 12,000 rpm (~13,400×g) for 1 minute to collect the plasmid solution.

### 3. Toxicity Testing of Obtained Cry5B, Cry6A, and a Series of Fusion Proteins

1. Survival Analysis
   * Day 1 (D1)
     1. Prepare L4-stage synchronized nematodes and NGM plates (including medium-sized and small-sized plates).
     2. Inoculate a single colony of *OP50 E. coli* into antibiotic-free LB medium. Culture with shaking at 200 rpm and 37°C for 16 hours. Prepare a fresh culture for each use.
   * Day 2 (D2)
     1. Prepare FUDR-containing OP50 bacterial solution and seed NGM plates:
        1. (1) Dispense 50 μL of 50 mg/mL FUDR into PCR tubes for storage (ensure sterility).
        2. (2) Take 3 mL of freshly cultured OP50 bacterial solution and mix it with 48 μL of 50 mg/mL FUDR (calculate the volume proportion to avoid concentration errors).
        3. (3) Seed 100 μL of the FUDR-OP50 mixture onto each medium-sized NGM plate.
        4. (4) Use a sterilized and cooled spreader to spread the bacterial solution evenly across the plate surface.
        5. (5) Seal the prepared plates with parafilm (to prevent contamination and moisture loss) and incubate at 37°C for 24 hours.
     2. Synchronize nematodes for L4-stage preparation:
        1. (1) For each nematode strain (labeled D1–D2), select 15–25 adult worms (adjust the number based on their egg-laying capacity).
        2. (2) Perform synchronization for 3–4 hours to collect eggs, and remove the parent worms after synchronization (ensure no adult worms remain in the egg culture).
        3. Recommendation: The development from nematode egg to L4 stage takes approximately 56 hours. To avoid delayed picking of L4-stage worms, it is recommended to start synchronization between 10:00 and 14:00 (local time).
   * Day 4 (D4)
     1. Pre-treat L4-stage nematodes with FUDR:
        1. (1) Transfer more than 100 L4-stage synchronized nematodes to the FUDR-seeded NGM plates (prepared on D2).
        2. (2) Incubate the plates at 20°C for 24 hours (this step inhibits nematode egg hatching and ensures only adult worms are used in subsequent toxicity tests).
        3. (3) Adjust the number of nematodes per plate as needed: For small-sized NGM plates, use 30 worms per plate, with 3 replicate plates per protein treatment group (total of n protein solution groups, depending on the number of fusion proteins tested).
     2. Prepare Cry protein-containing OP50 bacterial solution and seed small NGM plates:
        1. (1) Prepare a mixed solution of Cry protein (Cry5B, Cry6A, or fusion protein) and OP50, ensuring the final concentration of the Cry protein is 15 mg/L (verify the concentration with a spectrophotometer if necessary).
        2. (2) Seed 100 μL of the Cry-OP50 mixture onto each small-sized NGM plate.
        3. (3) Spread the solution evenly with a sterilized and cooled spreader.
        4. (4) Seal the plates and incubate at 25°C for 12 hours (to allow the bacterial lawn to form).
        5. Note: Prepare control plates (seeded with OP50 alone, without Cry protein) simultaneously to eliminate the influence of OP50 on nematode survival.
   * Day 5 (D5)* Conduct toxicity treatment and survival counting:
       1. (1) Transfer the FUDR-pretreated nematodes (from D4) to the small NGM plates containing Cry protein (prepared on D4): 30 worms per plate, 3 replicate plates per Cry protein solution.
       2. (2) Incubate all plates at 25°C.
       3. (3) Perform survival counting every 12 hours: Record the number of live nematodes per plate. Continue counting until the survival rate of any group drops below 10% or all nematodes in the group are dead.
       4. Operation Note: Remove dead nematodes from the plates immediately after counting and incinerate them (to prevent cross-contamination between plates).
   * Example of Survival Counting Table

     |  | strain | | | | date | | | |  | strain | | | | date | | | |
     | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
     | Date | C/D | | 1 | | 2 | | 3 | | Date | C/D | | 1 | | 2 | | 3 | |
     |  | / | | / | | / | | / | |  | / | | / | | / | | / | |
     | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
     |  | / | | / | | / | | / | |  | / | | / | | / | | / | |
     |  | / | | / | | / | | / | |  | / | | / | | / | | / | |
     |  | / | | / | | / | | / | |  | / | | / | | / | | / | |
     |  | / | | / | | / | | / | |  | / | | / | | / | | / | |
     |  | strain | | | | date | | | |  | strain | | | | date | | | |
     | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
     | Date | C/D | | 1 | | 2 | | 3 | | Date | C/D | | 1 | | 2 | | 3 | |
     |  | / | | / | | / | | / | |  | / | | / | | / | | / | |
     | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
     |  | / | | / | | / | | / | |  | / | | / | | / | | / | |
     |  | / | | / | | / | | / | |  | / | | / | | / | | / | |
     |  | / | | / | | / | | / | |  | / | | / | | / | | / | |
     |  | / | | / | | / | | / | |  | / | | / | | / | | / | |
     |  | strain | | | | date | | | |  | strain | | | | date | | | |
     | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
     | Date | C/D | | 1 | | 2 | | 3 | | Date | C/D | | 1 | | 2 | | 3 | |
     |  | / | | / | | / | | / | |  | / | | / | | / | | / | |
     | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
     |  | / | | / | | / | | / | |  | / | | / | | / | | / | |
     |  | / | | / | | / | | / | |  | / | | / | | / | | / | |
     |  | / | | / | | / | | / | |  | / | | / | | / | | / | |
     |  | / | | / | | / | | / | |  | / | | / | | / | | / | |

     * *Note: "C/D" refers to "Censor/Death"; fill in the number of live nematodes in the "/ " blanks.*
2. Median Lethal Concentration (LC₅₀) Determination- *Note: The preparation method for nematode synchronization is consistent with that described in "1) Survival Analysis" (ensure L4-stage synchronization to maintain experimental consistency).*
   1. Nematode Culture: Prepare L4-stage larvae of the Caenorhabditis elegans N2 wild-type strain.
   2. The Cry protein was diluted in HEPES buffer to create a concentration gradient (e.g., 10 µM, 5 µM, 2.5 µM, 1.25 µM). For each concentration, a minimum of three replicates were prepared.
   3. Prepare 48-well microtiter plates for toxicity testing: Seed each well of a 48-well microtiter plate with 140 µL of S medium and 40 µL of an E. coli OP50 suspension (OD600 = 0.3), prepared in S medium.
   4. Add FUDR to inhibit nematode reproduction: Add 5 μL of 8×10⁻³ mol/L FUDR to each well
   5. Each well was treated with 10 µL of either the toxin solution or HEPES buffer (which served as the control).
   6. Add 5 µL of L4-stage larvae (approximately 20 larvae resuspended in S medium) to each well.
   7. Wrap the culture plate with moist paper towels, place it in a sealed container to maintain humidity, and incubate at 25°C for 5 days.
   8. Score nematode survival in each well under a microscope. Motile nematodes are scored as alive. For non-motile nematodes, gently prod with a pick; if no response is observed, score them as dead. Record the survival status of at least 100 nematodes per concentration group.
   9. LC50 calculation: Plot the survival rate against the toxin concentration. Determine the LC50 (the toxin concentration that kills 50% of the nematodes) through curve fitting.

{% include 'footer.html' %}

➤

* [Results on synthetic biology experiment on *Caenorhabditis elegans (C. elegans)*](#sec_1)
* [Results on synthetic biology experiment on *Escherichia coli(E.coli)*](#sec_4)
* [Results on synthetic biology experiment on *Bacillus subtilis*](#sec_9)
* [Results on synthetic biology experiment on regulation part Introduction](#sec_12)

{% include 'menu.html' %}

# RESULTS

What we have achieved

## Results on synthetic biology experiment on *C. elegans*

## 1.1 Construction of the expression plasmid of Cry5Ba , Cry6Aa and Cry5Ba-Cry6Aa Fusion Protein

We constructed plasmids capable of expressing Cry5Ba and Cry6Aa as well as their fusion proteins, and verified the successful construction and transformation of the plasmids.

![Figure 26 r101.avif](https://static.igem.wiki/teams/5730/result/nematode/r101.avif)

Figure 1. The agarose gel electrophoresis of protein fragments M-MF024 marker; 1-Cry5Ba; 2-Cry6Aa; 3 Fusion; 4 pET28a\_linear vector

Analysis: The PCR amplification bands of Cry5BA (approximately 1800 bp), Cry6Aa (approximately 1200 bp), the fusion protein (approximately 3000 bp), and the pET-28a vector (approximately 5000 bp) align with the theoretical lengths estimated based on the designed primer positions, demonstrating that these target genes have been successfully obtained.

![Figure 27 r102.avif](https://static.igem.wiki/teams/5730/result/nematode/r102.avif)

Figure 2. Plates of the different transformed Cry6Aa(a), Cry5Ba(b) and fusion(c) *E. coli*

Analysis: From the result of plate coating of LB solid medium with antibiotic Kan, the medium successfully grew dispersed single colonies, which indicated that the bacterium was successfully screened and transformed.

![Figure 28 r103.avif](https://static.igem.wiki/teams/5730/result/nematode/r103.avif)

Figure 3. Horizontal sequencing maps comparison for Cry5Ba, Cry6Aa and fusion

Analysis: The sequencing result can show that the target element and the plasmid vector were correctly connected; there were no heterogeneous peaks in the sequencing result, and the sequence of the actual plasmid is identical with the designed plasmid.

## 1.2 Verification of proteins by Western blot

![Figure 29 r104.avif](https://static.igem.wiki/teams/5730/result/nematode/r104.avif)

Figure 4. The western blot of Cry5Ba, Cry6Aa and fusion protein

Analysis: The protein bands on the western blot gel for Cry5Ba (approximately 65.45 kDa), Cry6Aa (approximately 44.33 kDa), and the fusion protein (approximately 110 kDa) corresponded to their theoretical lengths based on their molecular weights, indicating that these target proteins were successfully expressed in the engineered bacteria.

## 1.3 Nematode survival Analysis Experiment

![Figure 30 r105.avif](https://static.igem.wiki/teams/5730/result/nematode/r105.avif)

Figure 5. The Cry protein survival curve (round 1)

Analysis: According to the survival curve, Cry6Aa has already begun to cause the death of nematodes, while the killing effect of Cry5Ba is not obvious. At 96 hours, due to the poor effect of the FUDR we used, a large number of larvae appeared, so we terminated this experiment.

![Figure 31 r106.avif](https://static.igem.wiki/teams/5730/result/nematode/r106.avif)

Figure 6. The Cry protein survival curve (round 2)

Analysis: To accelerate the insecticidal rate, in the second set of survival analysis experiments, we modified the original experimental conditions by directly dropping protein solutions onto the culture medium. The results showed that 24 hours after protein application, the survival rates of the Cry5Ba group, Cry6Aa group, and Fusion protein group were all lower than 6%, whereas the survival rate of the control group remained higher than 75%. These findings confirm that both Cry5Ba protein and Cry6Aa protein, as well as their fusion protein, exhibit excellent insecticidal efficacy, which is consistent with our initial hypothesis.

## Results on synthetic biology experiment on *E. coli*

## Introduction

We aim to design the *E. coli* to be a factory that can turn L-Tyr and malonate into naringenin. We transformed three plasmids containing six genes into the same *E. coli*. Then we conducted verification experiments on *E.coli* to ensure the enzymes were correctly expressed and functioning well.

## Part 1: Plasmids construction

The three plasmids containing the target genes were synthesized by company. Then we transformed the plasmids into the *E. coli*.

Table 1. The information of plasmids designed for *E. coli*

| Plasmid name | Expected size |
| --- | --- |
| pCDFDuet-TAL-4CL | 7409 |
| pACYCDuet-matB-matC | 6784 |
| pETDuet-CHS-CHI | 7170 |

### 1.1 Electric transformation of *pACYCDuet-matB-matC* and pETDuet-CHS-CHI

We used the method of electric transformation to simultaneously transform two plasmids into the *E. coli*. And then we coated the transformed *E. coli* on board that has two kinds of corresponding antibiotics to see if the transformation was successful.

![Expert Interview](https://static.igem.wiki/teams/5730/result/micro/molecue-renew-1.avif)

Figure 7. The board of *E. coli* transformed with two plasmids

Analysis: The transformed *E. coli* can grow on the board that has two kinds of corresponding antibiotics---Amp and Cap. This indicates that the transformed *E. coli* has the resistance genes of these two antibiotics, which is to say that the transformation process was likely successful.

### 1.2 Chemical transformation of pCDFDuet-TAL-4CL

We made the former *E. coli* into chemically competent cells and then we used the method of chemical transformation to transform the third plasmid into the *E. coli* in order to raise the successful rate of obtaining engineering bacteria that has three plasmids. Then we coated the transformed *E. coli* on the board that has three kinds of corresponding antibiotics---Amp、Smr and Cap.

![Expert Interview](https://static.igem.wiki/teams/5730/result/micro/molecue-renew-2.avif)

Figure 8. The board of *E. coli* transformed with three plasmids

Analysis: The transformed *E. coli* can grow on the board that has three kinds of corresponding antibiotics---Amp、 Smr and Cap. This indicates that the transformed *E. coli* has the resistance genes of these three antibiotics, which is to say that the transformation process was likely successful. Then we sent the board for company to conduct bacteria PCR. The result shows that the engineering bacteria has the three constructed plasmids(the six targeted genes), which is to say that the transformation processes in parts 1.1 and 1.2 were successful.

## Part 2: Molecular expression measurement

In our experiment, it was essential to determine whether *E. coli* successfully expressed the target enzyme we needed. Therefore, we used SDS-PAGE for verification and investigated whether temperature and the addition of an inducer are factors affecting the expression of *E. coli*.

We designed the experiment as follows: the control group consisted of engineered *E. coli* cultured at 16°C, 25°C, and 37°C without the inducer, while the experimental group comprised engineered *E. coli* cultured at the same three temperatures (16°C, 25°C, 37°C) with the inducer added. After processing, the samples were loaded into the gel lanes, and the results of the protein gel were observed.

![Expert Interview](https://static.igem.wiki/teams/5730/result/micro/f5.avif)

Figure 9. The SDS-PAGE gel of *E. coli* transformed with three plasmids

Analysis:For the control group, the results showed that almost no target protein was detected in *E. coli* cultured at 37°C, whereas clear target protein bands were visible in the samples cultured at 16°C and 25°C. This indicates that even without inducing the engineered *E. coli*, we could still obtain the desired target enzyme within this specific temperature range.
For the experimental group, the protein band of *E. coli* cultured at 37°C was the faintest, while the band intensities of the samples cultured at 16°C and 25°C were nearly identical. Meanwhile, due to the distortion of the gel, the position of the matC band may not be accurate.But in combination with the sequencing results, the result can still suggest that the expression levels of *E. coli* were approximately the same within this temperature range.(16℃ and 25℃)

Although the lack of a His-tag prevented us from performing Western Blotting, we still obtained the desired results: the engineered *E. coli* strain successfully expressed the target protein we needed, and this expression could be achieved without induction within a specific temperature range (indicating that the gene we introduced has a strong expression capacity).

## Part 3: Functional measurement

The successful construction of the naringenin biosynthetic pathway in the engineered *Escherichia coli* required a reliable method to confirm the production of this target intermediate. Since naringenin contains a conjugated ring system with phenolic hydroxyl groups, it exhibits strong and characteristic ultraviolet (UV) absorption. Literature indicates a definitive absorption maximum for naringenin at 285 nm. Therefore, we established a High-Performance Liquid Chromatography (HPLC) method with UV detection specifically tuned to this wavelength. This approach was designed to unambiguously identify and quantify naringenin synthesis by the engineered E. coli strain based on its intrinsic photophysical properties.

![Expert Interview](https://static.igem.wiki/teams/5730/result/micro/hplc1.avif)

Figure 10. HPLC result of *E. coli*

Analysis: Based on the chromatographic results, the detection method was validated using standard compounds, confirming its reliability for our analytical purposes. Comparative analysis revealed that the sample containing the engineered *E. coli* strain exhibited a peak at 7.1 minutes, which aligns with the retention time of the naringenin standard. Moreover, no corresponding peak was observed in the blank control. These results indicate that the engineered *E. coli* successfully produced naringenin, demonstrating the effective construction of the intended biosynthetic pathway.

## Results on synthetic biology experiment on *B. subtilis*

## Introduction

We aim to design the *B. subtilis* to be a factory that can turn naringenin into luteolin. We transformed one plasmid containing two genes into the *B. subtilis*. Then we conducted verification experiments on *B. subtilis* to ensure the enzymes were correctly expressed and functioning well.

## Part 1: Plasmids construction

First we constructed the plasmid containing the two targeted genes. Then we transformed it into the *B. subtilis*.

Table 2. The information of plasmid designed for *B. subtilis*

| Plasmid name | Expected size |
| --- | --- |
| pBE2R-RBS1-FNS1-RBS2-F3’H-TT | 9411 |

### 1.1 Construction of pBE2R-FNS1-F3’H

We used the method of PCR to copy fragments of genes and vector, and then we used the method of homologous recombination to combine the fragments. Then we transformed the plasmid into the *B. subtilis* as Experiment group and coated it on the board that has the corresponding antibiotic---Kan to see if the transformation process was successful. Simultaneously, we transformed an empty pBE2R plasmid into the *Bacillus subtilis* as Control group. In this part, we attempted to construct pBE2R plasmid for two times. But we failed for the first time because the homology of the fragments was poor. ( Because we have many similar sequences to combine ) Then we redesigned the pBE2R plasmid and deleted one promoter and one terminator to increase homology and then we tried the second time.

![Expert Interview](https://static.igem.wiki/teams/5730/result/micro/r7.webp)

Figure 11. The board of *B. subtilis* transformed with one plasmid

Analysis: The transformed *B. subtilis* can grow on the board that has Kan. This indicates that the transformed *B. subtilis* has the resistance gene of Kan, which is to say that the transformation process was likely successful.

![Expert Interview](https://static.igem.wiki/teams/5730/result/micro/p.avif)

Figure 12. The bacteria PCR result of transformed *B. subtilis*

Analysis: This is the bacteria PCR result of pBE2R. The fragment frag1’s length is 1200. Although the length of the band shown in the result is not exactly 1200bp, but based on the sequence result and the fact that the band is a result of bacteria PCR, we can reckon that the transformation process was successful.

![Expert Interview](https://static.igem.wiki/teams/5730/result/micro/r9.webp)

Figure 13. The sequence result of transformed *B. subtilis*

Analysis: The sequencing result shows that the sequence we designed was the same as the measured sequence. This indicates that the transformation process was successful.

## Part 2: Molecular expression measurement

Whether *B. subtilis* successfully expresses the target enzyme determines our ability to convert naringenin to luteolin. Therefore, it is particularly important to confirm whether the protease expressed by the engineered *B. subtilis* is the target protein.

To obtain more accurate results during plasmid construction, we added a His-tag to the plasmid introduced into *Bacillus subtilis*. This allowed the expressed product to be specifically recognized by antibodies, enabling us to obtain clear images and accurate results via Western Blotting.

### 2.1 Verification via SDS-PAGE Gel

We conducted preliminary verification using SDS-PAGE.

To verify whether the temperature can affect the expression of the target protein, we redesigned the experiment:

Control group: *B. subtilis* transformed with the empty plasmid (i.e., pBE2R) and cultured at 16°C, 25°C, and 37°C.

Experimental group: *B. subtilis* transformed with the normal plasmid (i.e., pBE2R-FNS-F3’H) and cultured at the same three temperatures (16°C, 25°C, 37°C).

![Expert Interview](https://static.igem.wiki/teams/5730/result/micro/11.avif)

Figure 14. The SDS-PAGE gel of *B. subtilis* transformed with one plasmid

Analysis:The experimental results showed two key findings:

Temperature had little effect on the expression of the engineered *B. subtilis*—the *B. subtilis* transformed with the normal plasmid exhibited expression at all three temperatures, with bands of similar intensity.

Unexpectedly, *B. subtilis* transformed with the empty plasmid also showed a relevant band in the target molecular weight range. Under normal circumstances, transformed with an empty plasmid should not enable *Bacillus subtilis* to produce the target product. The appearance of this band might be attributed to one of the following two reasons:

1.Experimental operation error, where the control group was actually *B. subtilis* transformed with the normal plasmid.

2.The obtained expression product was not the target protein we needed.

Thus, we decided to use Western Blotting to verify whether the obtained protein was the target protein and to clarify the reason for the band appearance in the control group.

### 2.2 Verification via Western Blot

In the previous experiment, we set up groups with different temperatures, but the gel results showed that both *B. subtilis* transformed with the normal plasmid and those transformed with the empty plasmid exhibited target bands.

![Expert Interview](https://static.igem.wiki/teams/5730/result/micro/20251009220219-1984-44.avif)

Figure 15. The Western Blot result of *B. subtilis* transformed with one plasmid.1

Analysis:Subsequent Western Blotting of this protein gel further confirmed that all these bands corresponded to the target protein.

This ruled out the possibility that the obtained protein was not the desired target protein, leading us to suspect that either the *B. subtilis* in the control group was also transfected with the normal plasmid or there was an error during sample loading onto the protein gel.

To resolve this, we redesigned the experiment:

Control group: *B. subtilis* transformed with the empty plasmid (i.e., pBE2R) and cultured at 25°C.

Experimental group: *B. subtilis* transformed with the normal plasmid (i.e., pBE2R-FNS-F3’H) and cultured at 25°C.

![Expert Interview](https://static.igem.wiki/teams/5730/result/micro/20251009215948-1983-44.avif)

Figure 16. The Western Blot result of *B. subtilis* transformed with one plasmid.2

Analysis:After gel electrophoresis, Western Blotting was performed on the obtained protein gel. The images clearly showed that the *B. subtilis* transformed with the normal plasmid exhibited a target band, while no target band was observed in the *B. subtilis* transformed with the empty plasmid.

This successfully confirmed that the earlier appearance of a band in the *B. subtilis* transformed with the empty plasmid was due to experimental operation errors, and verified that we had obtained the desired target product.

## Part 3: Function measurement

For the engineered *B. subtilis*, which was designed to convert naringenin into the final product luteolin, the analytical method needed to detect and distinguish both compounds simultaneously. Luteolin, sharing a similar flavonoid structure but with extended conjugation, possesses a distinct UV absorption profile, with a reported maximum at 350 nm. To monitor the intended bioconversion, we developed a unified HPLC-UV method capable of detecting both naringenin (at 285 nm) and luteolin (at 350 nm) within a single run. This dual-wavelength detection strategy was essential to verify the specific activity of the *B. subtilis* strain by confirming not only the presence of luteolin but also the concomitant consumption of the naringenin precursor.

![Expert Interview](https://static.igem.wiki/teams/5730/result/micro/hplc2.avif)

Figure 17. The HPLC result of *B. subtilis*

Analysis: In contrast, the sample with the engineered *B. subtilis* strain did not show any peak at the retention time corresponding to the luteolin standard, while the blank control again remained clear of such signals. This suggests that the conversion from naringenin to luteolin was not achieved, indicating a failure in the functional assembly of the pathway in the engineered *B. subtilis*.

## Results on synthetic biology experiment on regulation part

## Introduction

We planed to screen phages targeting the *B. subtilis* BS168 strain from the environment to achieve regulation of engineered bacteria. After that, we aim to construct a plasmid containing “lsl” component and transform it into the *E. coli* to verify its function. While eventually in the application process, we will use phages as a medium to import this molecular switch.

## Part 1: Plasmid construction

We constructed and transformed a plasmid containing CHS and CHI genes and “lsl” component and a plasmid containing cre gene into the same *E. coli* as Experiment group. We tried for two times but we failed for the first time probably because the effect of the “cre” gene was too strong causing the lac operator to break. And we transformed a plasmid containing CHS and CHI genes and “lsl” component and an empty plasmid into the same *E. coli* as Control group.

Table 3. The information of plasmids designed for regulation part

| Plasmid name | Expected size |
| --- | --- |
| pETDuet-CHS-lsl-CHI | 7382 |
| pCDF-cre | 4783 |

### 1.1 Construction of pETDuet-CHS-lsl-CHI

![Expert Interview](https://static.igem.wiki/teams/5730/result/micro/.avif)

Figure 18. The board of *E. coli* of Experiment group

Analysis: The transformation result of the Control group and the Experiment group shows that the transformed *Escherichia coli(E.coli)* can grow on the board that has two corresponding antibiotics. This indicates that the transformation process was likely successful.

![Expert Interview](https://static.igem.wiki/teams/5730/result/micro/r18.webp)

Figure 19. The sequence result of *E. coli* of Experiment group

Analysis: The sequencing result shows that there is a blank in the blue rectangular area, which is the position of the “lsl” component. This indicates that the transformation process was successful and cre gene was functioning well.

### 1.2Construction of pCDF-cre

![Expert Interview](https://static.igem.wiki/teams/5730/result/micro/r19.webp)

Figure 20. The sequence result of the construction of pCDF-cre plasmid

Analysis: The sequence result shows that the sequence we designed was the same as the measured sequence. This indicates that our construction of pCDF-cre plasmid using the method of PCR was successful.

## Part 2: Molecular expression measurement

To confirm whether our Cre-loxP system functions properly, we decided to conduct verification experiments in Escherichia coli (E. coli) BL21. The experiment was designed as follows: E. coli transfected with the pCDFDuet-CHS-lsl-CHI plasmid served as the control group, while E. coli co-transfected with the pCDFDuet-CHS-lsl-CHI plasmid and pCDFDuet-Cre plasmid served as the experimental group. Our hypothesis was that if the constructed Cre-loxP system was correct, the control group would be unable to express the target protein, whereas the experimental group would successfully express it.

### 2.1 Verification via SDS-PAGE Gel

Protein samples obtained from the above experiment were analyzed using SDS-PAGE, and the gel image results were as follows:

![Expert Interview](https://static.igem.wiki/teams/5730/result/micro/lsl-cre-sds-page.avif)

Figure 21. The SDS-PAGE gel of *Escherichia coli(E.coli)* of Control&Experiment group

Analysis:First, a band corresponding to CHS was observed in both the control group and the experimental group, indicating that the plasmids were successfully transfected into E. coli and induced for protein expression. However, a band corresponding to the CHI protein was detected in both groups, suggesting that CHI was expressed in E. coli of both the control and experimental groups.

### 2.2 Verification via Western Blot

To identify the cause of the CHI band appearance, we performed Western Blot analysis on the protein gel.

![Expert Interview](https://static.igem.wiki/teams/5730/result/micro/20251009212845-1958-44.avif)

Figure 22. The Western Blot result of *Escherichia coli(E.coli)* of Control&Experiment group

Analysis: In the Western Blot image, CHI bands were still detected in both groups of E. coli (note: only CHI carries a His-tag).

Based on the above results, we proposed the following potential reasons:

1.The pCDFDuet-CHS-lsl-CHI plasmid transfected into the control group was contaminated with Cre. This contamination would lead to the excision of the terminator by Cre (a band suspected to correspond to Cre was observed at the respective position in the control group’s protein gel).

2.The terminator in the plasmid failed to exert its intended function.

In future experiments, we will further investigate the cause of the CHI band appearance in the control group to verify whether our Cre-loxP system indeed functions as expected.

## Part 3: Phage Screening

### 3.1 Screening of phages from the environment and their enrichment and purification

We collected soil and water samples from flower beds and lakes across the campus of Huazhong University of Science and Technology. Each sample was co-incubated with bacterial suspensions of either *Escherichia coli(E.coil)* BL21 strain or *Bacillus subtilis* BS168 strain for amplification. Phages were subsequently screened using the double agar overlay assay and purified via the phage streaking method.

![Expert Interview](https://static.igem.wiki/teams/5730/result/micro/r22.webp)

Figure 23. **Plaque morphology of phage targeting engineered bacteria on a double-layer agar plate**. (a)(b).Phages targeting *E. coli* BL21 strain; (c)-(f).Phages targeting *Bacillus subtilis* BS168 strain. The red arrows and boxes indicate the location of the clear bacterial colonies

Analysis: Figure 23 shows the plaque obtained for the phages targeting engineered bacteria. The plaques were characterized by a transparent center with cloudy edges. The results in Figure 23 confirm the successful isolation of phages from environmental soil samples that are specific to the two engineered host bacteria. Evidence of their bactericidal efficacy is provided by the formation of clear plaques in double-layer agar assays. At the same time, we found that even for the phage plaques targeting the same engineered bacteria, they present different sizes and different degrees of roughness at the edges (Fig 23a & Fig 23b; Fig 23c & Fig 23f), which also indicates that the phages we isolated are not a single species but a mixed group. It is proposed that this heterogeneous phage community collectively contributes to the overall antibacterial outcome.

### 3.2 Characterization of environmental phages and verification of their bactericidal properties

![Expert Interview](https://static.igem.wiki/teams/5730/result/micro/result-phage-microscope-renew.avif)

Figure 24. **TEM observation of E. coli with phage Transmission electron microscopy (TEM) observation of engineered bacteria with phage**. (a)(b). Phage was attaching themselves into *E. coli BL21* strain host cell membrane; (c)(d). Phage was attaching themselves into *B. subtilis* BS168 strain host cell membrane. The red arrows and boxes indicate the location of phages. The scale bar represents 200 nm

Analysis:
TEM analysis reveals a diversity of phage morphologies in the *E. coli* phage-enriched liquid. As shown in Figures 24a (including insets), filamentous phages are the predominant morphology observed. This is complemented by the presence of tadpole-shaped phages, as depicted in Figure 24b. These findings collectively confirm that the phage population is polyclonal, with filamentous phages constituting the major type.

Transmission electron microscopy (TEM) in Figures 24c, 24d reveals the morphological diversity of bacteriophages infecting *B. subtilis*. The population includes both filamentous and tadpole-shaped phages, indicating a similarly mixed phage community as observed with *E. coli*.

![Expert Interview](https://static.igem.wiki/teams/5730/result/micro/r24.webp)

Figure 25. **Spot titer assay of Phages**. Phages and polymer controls using modified double-layer agar method.

Analysis: Quantitative analysis of the phage enrichment solutions (Spot titer assay) revealed that the *E. col*i phage lysate reached an endpoint dilution of 10^-6. The titer of the original solution was calculated as 5.6×10^8 PFU/mL based on the plaque count from the 10^-5 dilution. In parallel, the *Bacillus subtilis* phage lysate exhibited an endpoint dilution of 10^-7, with the plaque count at the 10^-6 dilution indicating an original concentration of 8.2×10^9 PFU/mL.

These findings indicate that the phage-enriched solutions contained not only highly lytic phages but also a diversity of temperate phages, which collectively contributed to effective and persistent suppression of bacterial growth.

### 3.3 Outlook

We have successfully isolated environmental phages targeting *E. coli* and *B. subtilis*, and confirmed their lytic efficacy through planktonic bactericidal assays. These phages can serve as effective biological tools to precisely terminate the luteolin synthesis reaction.

Future work will focus on further purification of the phage isolates through continuous enrichment and screening, which is expected to enhance their lytic activity while eliminating potentially harmful contaminating phages, thereby improving overall safety. Furthermore, we plan to employ genetic engineering to augment phage functionality. Specifically, modification of phage genomes will not only enhance their bactericidal capacity for more reliable reaction termination but also, in combination with the Cre-loxP system, enable the delivery of cre recombinase genes. This integrated approach will establish a dual-control mechanism for regulating the synthetic pathway, effectively creating a programmable, **phage-based switch**.

{% include 'footer.html' %}