iGEM 2025 — Team subcat-peking

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NOTEBOOK

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* [Record of Main Experimental Content](#RecordofMainExperimentalContent "Record of Main Experimental Content")

Record of Main Experimental Content

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Pre-experiment safety training：
The main content is as follows：
Personal safety:
⦁  Wear laboratory coats.
⦁  Tie long hair up and secure loose clothing or accessories.
⦁  Always wear your mask and gloves before entering the laboratory.
⦁  Remember to wash hands before and after experiments.
Laboratory safety:
⦁  No eating or drinking.
⦁  Beware of chemical reactions and toxic substances.
⦁  In order to keep the laboratory tidy, avoid placing irrelevant items, such as notebooks, or waste materials.
⦁  After completing the experiment, turn off the electricity and close the doors and windows.
Biological safety:
⦁  Conduct all sterile procedures inside a laminar flow hood.
⦁  Laboratory waste must be disinfected.
Part 1: Amplification of target gene fragment by PCR
Purpose: Amplify DNA fragments by implement PCR Procedure.
Reagent	                    Volume
PrimeSTAR Max Premix (2×)	 10 μL
Forward Primer (10–15 pmol)	 ~0.2–0.3 μM
Reverse Primer (10–15 pmol)	     ~0.2–0.3 μM
Template                      1 μL
ddH2O	                     To 50 μL total
1.Prepare 20 μL PCR mixture.
1.95°C for 3 min
2.95°C for 30 s
3.53°C for 15 s
4.72°C for 1 min
5.72°C for 10 min
（Repeat step 2,3,4 for 33 times）
2.Run PCR as described above.
3.Analyze by agarose gel electrophoresis.
4.Visualize bands under UV light.
Participants: Lawrence Zeng, Dolores Yang, Jessica Shang, Noah Sun, Yuki Zhang, Iiya Wang, He Wang, James You, Lydia Zhu, Zhenze Xie, Dong Liang, Muhuan Guo
Part 2: Plasmid construction
Purpose:
Plasmids containing antimicrobial peptides and lysozyme were constructed by agarose gel electrophoresis, ligation after digestion, plasmid transformation and plasmid extraction.
Procedure:
1. Agarose Gel Preparation and Electrophoresis
1.	Dissolve 0.1 g agarose in 100 mL TAE buffer by microwaving for 3 min.
2.	Add 1 μL GelRed dye. Pour into mold with comb;
3.	Place gel in electrophoresis chamber with TAE buffer.
4.	Mix DNA samples with loading buffer, load into wells.
5.	Run gel at 150 V for ~20 min.
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![figure1](https://static.igem.wiki/teams/5742/images/notebook/figure1.webp)

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6.	Visualize bands under UV light.
2. DNA Gel Extraction and Purification
1.	Check whether Washing Solution is adding anhydrous ethanol,
2.	Check whether Buffer B2 appear precipitate,Set the water bath pot to 50℃
3.	Cut out the gel block containing the target fragment from the agarose gel and weigh it.
4.	Add Buffer B2 into the gel block and metal bath 55 ℃
5.	Transfer sol solution into the adsorption column and 8000Xg centrifugation for 30 seconds
6.	Add Wash Buffer and 9000Xg centrifugation for 30 seconds, pour the solution in the tube.
7.	Repeat the previous step
8.	Let empty adsorption column in the centrifuge for 1 minute
9.	Put adsorption column into a clean 1.5mL centrifuge tube and add 30μl Elution Buffer into the center of the adsorption film. Stewing for 1 minute and centrifugation for 1 minute.
3. Transformation into Competent Cells
1.	Preparation for antibiotic plates (LB+1:1000 bleomycin)
2.	Thaw competent E. coli DH5α cells on the ice.
3.	Add 10μL recombinant product (plasmid) into the competent cells, gently flick vessel wall, gently mix it by using the pipette tip. Incubate on ice for 30 minutes, stationary.
4.	42℃ hot water bath heat shocks for 45s in order to create channels and pores in the cell membrane that connect the cell interior to the external environment.
5.	Remove immediately, incubate on ice for 2 minutes to reduce the temperature.
6.	Add 900μL LB culture medium. Incubate at 37℃ with shaking at 200rmp for one hour.
7.	Centrifuge and discard 800μL of the supernatant.
8.	Separate the thallus in the remaining 200μL of LB medium.
9.	Evenly spread the sample on antibiotic plates by using a sterile cell spreader.
4.Colony PCR
	Reagent	                    Volume
	PrimeSTAR Max Premix (2×)	    10 μL
	Forward Primer (10–15 pmol)	    ~0.2–0.3 μM
	Reverse Primer (10–15 pmol)	    ~0.2–0.3 μM
	Template (colonies)	              1 μL
	ddH2O	                    To 50 μL total
	1.Prepare 20 μL PCR mixture.
	1.95°C for 3 min
	2.95°C for 30 s
	3.53°C for 15 s
	4.72°C for 1 min
	5.72°C for 10 min
	（Repeat step 2,3,4 for 33 times）
	2.Run PCR as described above.
	3.Analyze by agarose gel electrophoresis.
```

![figure2](https://static.igem.wiki/teams/5742/images/notebook/figure2.webp)

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	4.Visualize bands under UV light.
Part 3 SDS-PAGE was used to verify whether the protein was expressed
Purpose:
Verify whether antimicrobial peptides and lysozyme are successfully expressed.
Procedures:
1. Protein Extraction
    ⦁	Add lysozyme and lysis buffer to bacterial pellet. Mix until fully resuspended.
    ⦁	Place tube in an insulated container with ice. Sonicate at 50% power (200–300 W) with 5-second intervals, total of 6 minutes.
    ⦁	Centrifuge lysate at 1000 × g for 10 seconds at 4°C. Collect supernatant (crude protein extract).
    ⦁	Prepare column in parallel by resuspending nickel affinity gel and equilibrating it with lysis buffer at 4°C.
2. Protein Purification
    ⦁	Mix crude extract with equilibrated resin. Shake at 4°C for 1.5 hours.
    ⦁	Load mixture into gravity column. Let drain slowly and repeat 3 times. Backup samples.
    ⦁	Wash with washing buffer and collect flow-through (wash fractions).
    ⦁	Elute protein with elution buffer. Collect elution fractions and label clearly.
3. SDS-PAGE Validation
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![figure3](https://static.igem.wiki/teams/5742/images/notebook/figure3.webp)

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Part 4 The growth curve of Pichia pastoris
Purpose:
The growth curve measurement was used to verify whether the growth ability of yeast was different when different genes were introduced.
Procedures:
Inoculate the transformants into 3 mL of YPD liquid medium and add bleomycin
Zecoin, then incubate at 30°C (OD600=0.02), The growth level of the strain was determined at a fixed time and the growth curve was drawn with the same
absorbance..
Part 5 Determination of Minimum Inhibitory Concentrations of antimicrobial peptides and lysozyme
Purpose:
Verify the bactericidal activity of antimicrobial peptides and lysozyme.
Procedures:
Prepare 1.2ml solutions of antimicrobial peptides in a centrifuge tube1(128 μg/mL)
Transfer 600μl solutions of antimicrobial peptides from tube1 into the centrifuge tube2, then add 600μl sterile water.
Transfer 600μl solutions of antimicrobial peptides from tube 2 into the centrifuge tube3, then add 600μl sterile water.
Transfer 600μl solutions of antimicrobial peptides from tube 3 into the centrifuge tube4, then add 600μl sterile water.
Transfer 600μl solutions of antimicrobial peptides from tube 4 into the centrifuge tube5, then add 600μl sterile water.
Transfer 600μl solutions of antimicrobial peptides from tube 5 into the centrifuge tube6, then add 600μl sterile water.
Transfer 600μl solutions of antimicrobial peptides from tube 6 into the centrifuge tube7, then add 600μl sterile water.
Transfer 600μl solutions of antimicrobial peptides from tube 7 into the centrifuge tube8, then add 600μl sterile water.
All antimicrobial peptides and lysozyme were diluted in gradient according to the above scheme.
Application of each sample into 96-well bacterial culture plate in order
Application of bacterial into the first line of 96-well bacterial culture plate
Application of ampicillin into the second line of 96-well bacterial culture plate
Application of LB culture medium into the third line of 96-well bacterial culture plate
Cultivate the bacterial culture until the OD value reaches 0.9–1.0 (with a CFU count of 10⁹ CFU/mL).
Perform a 1000-fold dilution to adjust the CFU count to 10⁶ CFU/mL.
Application diluted bacterial into 96-well bacterial culture plate.
Part 6 Determination of Minimum Bactericidal Concentration of antimicrobial peptides and lysozyme
Purpose:
Verify the bactericidal activity of antimicrobial peptides and lysozyme.
Procedure:
  We measured this by applying the targeted proteins and the bacteria
  (E.coli or Bacillus subtilis) onto petri dishes with LB culture medium.
  Use the protein solutions we made, dilute them to two separate centrifugal tubes
  (128 μg/ml and 64 μg/ml).
  For each type of targeted protein, we created 4 petri dishes LB culture medium.
  Each for:
  a.	128μg/ml concentration of protein + E. coli
  b.	64μg/ml concentration of protein + E. coli
  c.	128μg/ml concentration of protein + Bacillus subtilis
  d.	64μg/ml concentration of protein + Bacillus subtilis
  Add 100μl of protein to each petri dish, then use disposal cell
  Use spreader to spread the solution evenly across the culture medium.
  Use parallel pipette to add 100μl of bacteria solution to each corresponding petri dish,
  then use disposal cell spreader to spread the solution evenly across the culturemedium.
  Put the petri dishes into the constant-temperature incubator for 24 hours.
  Use the microplate reader to visualize the results.
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![figure4](https://static.igem.wiki/teams/5742/images/notebook/figure4.webp)

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Part 7 Time-Kill curve of antimicrobial peptides and lysozyme
Purpose:
Verify the bactericidal activity of antimicrobial peptides and lysozyme.
  1.Add the Bacillus subtilis and Escherichia coli separately into our antibacterial
    peptides and lysozymes, into a EP tube, using 500 μL of each component.
  2.	Add the sample into the 96 cell culture plate, starting from hour 0.
  3.	Samples were collected every 2 hours and OD600 was determined.
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![figure5](https://static.igem.wiki/teams/5742/images/notebook/figure5.webp)

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Part8 Laser Scanning Confocal Microscope observation
  Purpose: The bactericidal effect of antimicrobial peptides and lysozyme was determined by living dead cell staining.
  Escherichia coli and Bacillus subtilis were treated with antimicrobial peptides and lysozyme at 37 for 24 hours, respectively.
  1.The cell precipitate obtained by centrifugation was resuspended with 1XAssay Bufer, so that the cell count of the resuspended cell suspension was 1X105-6 cells /ml.
  2.Add 1-2ul of Calcein-AM (stock solution) per 1ml of cell volume, blow and mix evenly, and incubate at 37℃ in the dark for 20-25 min.
  3.Add 3-5ul of PI stock solution from the kit into the stained cells, and dye it in the dark at room temperature for 5min.
  4.Incubate the fluorescent cells for 5min minutes with 450g, and remove the staining solution by centrifugation.
  Note: It is suggested that cells should be kept away from light during fluorescent staining.
  5.Wash 450g cells with 1x PBS for 5min, resuspend the cells with 1x PBS after centrifugation, take 3-5ul drops on a clean glass slide, and after tabletting with a clean cover glass, please examine them under fluorescence microscope in time. (6) Using 490±10 nm excitation filter to simultaneously detect living cells (yellow-green fluorescence) and dead cells (red fluorescence) under fluorescence microscope. In addition, only dead cells can be observed by using 545nm emission filter. It can also be detected directly by using a suitable filter under a fluorescent enzyme-labeled instrument.
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![figure6](https://static.igem.wiki/teams/5742/images/notebook/figure6-1.webp)

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        E.coli

Part9 Morphological observation of scanning electron microscope
Purpose: The morphology of bacteria treated with antimicrobial peptides and lysozyme was observed by scanning electron microscope.
  1. Sampling: Generally, the sample size is not more than 10x10 x 5mm. Determine the target of observing the sample to avoid pulling and squeezing the sample during sampling.
  2. Cleaning: remove impurities from the observation surface and fully expose the observation surface without damaging it.
  3. Fixation: Generally, it is better to fix glutaraldehyde and acid at 4℃.
  Glutaraldehyde and acid can be fixed separately or double-fixed with "glutaraldehyde-osmium acid". After fixing for 1-3 hours, the fixed solution is sucked out, 0,1M of cool acid buffer wave (PH=7.2) is added, and the solution is rinsed for 3-4 times for 1 hour.
  4. Dehydration: Suck out the buffer wave from the vial, add ethanol to dehydrate step by step (the ethanol concentration is 30%-50%-70%6-80%-90%-100% at a time), and the dehydration time depends on the sample size, generally 15-30 minutes. Add the 1:1 mixture of isoamyl acetate and ethanol, soak for 10-20min and shake properly, discard the mixture, add pure isoamyl acetate and soak for 10-20min and shake properly.
  5. Drying: Drying is a key link in the biological preparation of scanning electron microscope. It is necessary to reduce the distortion of surface morphology caused by water evaporation surface as much as possible during the drying process, and it is necessary to ensure thorough drying. Commonly used methods include natural drying, dry drying, critical point drying, freeze drying and vacuum drying. Different drying methods will be selected for different biological samples.
  6. Sample loading: the surface is plated with gold and observed by scanning electron microscope.
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![figure8](https://static.igem.wiki/teams/5742/images/notebook/figure8.webp)

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ENGINEERING

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* [Overview](#Overview "Overview")
* [Cycle 1: Construction and verification of plasmid containing lysozyme](#Cycle1 "Cycle 1: Construction and verification of plasmid containing lysozyme")
* [Cycle 2: Construction and Verification of Plasmids Containing Antimicrobial Peptides](#Cycle2 "Cycle 2: Construction and Verification of Plasmids Containing Antimicrobial Peptides")
* [Test](#Test "Test")
* [Learn](#Learn "Learn")
* [References](#References "References")

Overview

Global population growth has made food safety a priority, yet traditional chemical preservatives raise allergy,
resistance, and carcinogenicity concerns[1,2]. Lysozyme and antimicrobial peptides are natural, safe
alternatives whose combination can reduce chemical use while enhancing shelf life and freshness[3–5].
We
screened two lysozymes and 74 antimicrobial peptides, retaining one lysozyme and nine peptides devoid of
hemolytic activity, toxicity, or long half-life. The lysozyme and antimicrobial peptide genes were then cloned
into expression vectors and transformed into Escherichia coli DH5α and Pichia pastoris GS115 respectively.
Recombinant protein expression was carried out in Pichia pastoris to construct the genetic system of lysozyme
and antimicrobial peptides. Finally, the antibacterial effect of lysozyme and antimicrobial peptides was
verified by qualitative characterization and judgment, as well as quantitative characterization and quantitative
analysis.

**Our design consists of two main cycles:**

**Cycle 1: Construction and verification of plasmid containing lysozyme**

**Cycle 2: Construction and Verification of Plasmids Containing Antimicrobial Peptides**

**Test and learn**

Generally speaking, our research is mainly divided into two cycles, the first cycle is mainly to construct
lysozyme protein needed for the experiment, and the second cycle is mainly to construct antibacterial peptide
protein needed for the experiment. After the plasmid was successfully constructed, we detected the protein
expression of antimicrobial peptides and lysozyme by SDS-PAGE. MIC, MBC, Time-Kill curve, Microscopic
observation and other methods were used to determine the antibacterial effects of antibacterial peptides and
lysozyme.

Cycle 1: Construction and verification of plasmid containing lysozyme

**Design:** Lysozyme is a natural protein with low toxicity, and egg white lysozyme has been approved to be
used in
food industry. Easy to produce, lysozyme gene has been widely studied and applied, with mature expression and
purification process, especially suitable for large-scale production by engineering microorganisms. In this
experiment, we chose lysozyme from human. We chose this gene because HLYZ has broad-spectrum antibacterial
activity. HLYZ can effectively inhibit many kinds of bacteria, especially gram-positive bacteria, by hydrolyzing
peptidoglycan in bacterial cell wall. Therefore, HLYZ is likely to play an important role in food preservation.

![figure1](https://static.igem.wiki/teams/5742/images/engineering/figure1-1.webp)

Figure 1. The plasmid maps. pPICZα A/hLYZ.

**Build:**

After confirming the successful amplification of DNA sequence(Figure 2A), we further connected the gene into
plasmid. The ligated plasmid was transformed into *Escherichia col*i by chemical transformation method and
was
preliminarily screened by antibiotics (Figure 2B). Subsequently, the single colony which resistant to
antibiotics was selected, and the plasmid construction was confirmed by DNA sequencing (Figure 2C). Furthermore,
we extracted the plasmid from *E. coli* and transformed it into *Pichia pastoris*(Figure 2D), and the
construction
of the strain was verified by colony PCR(Figure 2E).

![figure2](https://static.igem.wiki/teams/5742/images/engineering/figure2-1.webp)

Figure 2. Verification of strain construction. A: The result of target gene amplification; B: Transformation
into DH5α after ligation.C: DNA sequencing after plasmid transformation into *Escherichia coli*. D:
Successful
transformation of correct plasmid into *Pichia pastoris* GS115.E: Colony PCR results.

Cycle 2: Construction and Verification of Plasmids Containing Antimicrobial Peptides

**Design:**

Antimicrobial peptides, as natural preservatives, have strong antibacterial activity, low toxicity and
innocuity, showing great application potential and market prospects in prolonging the shelf life of food,
reducing the use of chemical preservatives and ensuring food safety. With the continuous development of
technology, the application of antimicrobial peptides in food preservation will be further expanded, providing
more green and healthy food preservation schemes. Compared with lysozyme, the reported antimicrobial peptides
are richer and more diverse. Therefore, in order to find effective and safe antimicrobial peptides, we found 74
possible antimicrobial peptide molecules by consulting the literature, and eliminated the antimicrobial peptides
with hemolysis risk and long intestinal half-life by online tools (HemoPI2 and ToxinPred).Finally,8 candidates
were selected: Cecropin A, Melittin, Buforin II, LL-37, Plectasin, Hepcidin, Piscidin-1, and Lactoferricin. We
also design a antisepsis mix protein, which connecting lysosome and 9 AMPs by flexible linker (GGGGS).

![figure3](https://static.igem.wiki/teams/5742/images/engineering/figure3-1.webp)

Figure 3. Construction results of plasmid pPICZα A/antisepsis mix, pPICZα A/Hepcidin, pPICZα A/Buforin II,
pPICZα A/Piscidin-1, pPICZα A/LL-37, pPICZα A/Cecropin A, pPICZα A/Melittin, pPICZα A/Plectasin,
pPICZα A/Lactoferricin.

**Build:**

After confirming the successful amplification of DNA sequence(Figure 2A), we further connected the gene into
plasmid. The ligated plasmid was transformed into *Escherichia coli* by chemical transformation method and
was
preliminarily screened by antibiotics (Figure 4A). Subsequently, the single colony which resistant to
antibiotics was selected, and the plasmid construction was confirmed by DNA sequencing (Figure 4B). Furthermore,
we extracted the plasmid from *E. coli* and transformed it into *Pichia pastoris*(Figure 4C), and the
construction
of the strain was verified by colony PCR(Figure 4D).

![figure4](https://static.igem.wiki/teams/5742/images/engineering/figure4-1.webp)

Figure 4 Verification of strain construction. A: Transformation into DH5α after ligation.B: DNA sequencing after
plasmid transformation into *Escherichia coli*.C: Successful transformation of correct plasmid into
*Pichia*
*pastoris* GS115.D: Colony PCR results.

Test

**1. Protein expression**

**1.1 SDS-PAGE**

*Pichia pastoris* GS115 strains carrying the correct plasmid were cultured individually at 30℃ and induced
protein
expression with 10 g/L methanol, and the proteins were purified using His-Tag affinity chromatography. SDS-PAGE
was used to verify the protein expression of the target gene. As shown in Figure 5, different bands
corresponding to the expected molecular weight were clearly observed in the purified components, confirming the
accurate expression of each protein.

![figure5](https://static.igem.wiki/teams/5742/images/engineering/figure5-2.webp)

Figure 5. The SDS-PAGE results.

**1.2 Growth curve**

The expression of protein may affect the growth level of the strain. Therefore, in order to determine whether
the protein expression of antimicrobial peptides and lysozyme affects the normal growth of the strain, we tested
the growth curve of the strain with different antimicrobial peptide protein expressions. As shown in Figure 6,
all engineered strains exhibited good growth kinetics under the test conditions. This result shows that yeast
strains can grow normally and express the required antimicrobial peptides and lysozyme proteins under this
culture condition.

![figure6](https://static.igem.wiki/teams/5742/images/engineering/figure6-1.webp)

Figure 6. Growth curves of 10 recombinant strains.

**2. Functional Test**

**2.1 Minimum Inhibitory Concentration (MIC) determination**

The minimum antibacterial concentration (MIC) of biological preservatives was determined by Broth microdilution
method to evaluate their antibacterial activity. Single colonies of the test strains *E. coli* and
*Bacillus*
*subtilis* were inoculated into LB medium for overnight culture, and the resulting suspension was adjusted
to 106
CFU mL-1 in fresh medium. Dispense 100 μL of the purified protein, from 128 to 2 μg·mL-1,
into a 96-well
microplate. Subsequently, 100 μL of standardized bacterial suspension was added to each well and the plates were
incubated at 37℃ for 24 h. Wells containing only sterile LB medium can serve as negative controls, while wells
containing bacteria serve as positive controls to confirm bacterial viability.

The MIC50 of the purified protein is shown in Table 1. From MIC results, we can see that Plectasin
has the
strongest antibacterial effect on both *Escherichia coli* and *Bacillus subtilis*. When the
concentration of
Plectasin is 4 μg/ml, 50% bacteria can be killed. The MIC50 values of all antimicrobial peptides are
below 16
μg/ml, which indicates that all antimicrobial peptides involved in the experiment have certain antibacterial
activity. However, the MIC50 value of lysozyme is higher, this means the bacteriostatic effect of
lysozyme is
slightly weak, but the MIC50 value is also below 32 μg/ml.Thus lysozyme also has certain
bacteriostatic effect.

**Table 1. The Inhibitory concentration curve results.**

|  |  |  |
| --- | --- | --- |
| Drug | MIC50 | |
| *E. coli* | *Bacillus subtilis* |
| Cecropin A | 8 μg/ml | 16 μg/ml |
| Melittin | 8 μg/ml | 16 μg/ml |
| LL-37 | 8 μg/ml | 4 μg/ml |
| Plectasin | 4 μg/ml | 4 μg/ml |
| Piscidin-1 | 8 μg/ml | 4 μg/ml |
| Hepcidin | 8 μg/ml | 16 μg/ml |
| Lactoferricin | 8 μg/ml | 16 μg/ml |
| Buforin II | 8 μg/ml | 16 μg/ml |
| HLYZ | 16 μg/ml | 32 μg/ml |
| Antisepsis mix | 16 μg/ml | 16 μg/ml |

**2.2 Time-Kill curve assay**

The growth of bacteria at different time points was monitored by optical density method (OD600). By
drawing the
growth curve of bacteria treated with antimicrobial peptides, we can know the inhibitory effect of antimicrobial
peptides on bacteria and how long it takes for bacteria to recover their growth ability after antimicrobial
peptides treatment. Equal volumes of biopreservatives and bacterial suspensions were mixed and incubated at 30℃
for 24 h, and absorbance was measured at 0, 2, 4, 6, 8 and 24 h to determine the growth curve of bacteria under
the condition of adding different biopreservatives.

As shown in Figure 7, the addition of biological preservatives inhibited the growth of bacteria to varying
degrees. From the curve results, the inhibitory effect of some antibacterial peptides will gradually weaken with
the increase of time. In the experiment of inhibiting *Escherichia coli*, four antimicrobial
peptides(Cecropin A,
Lactoferricin, LL-37 and Plectasin) showed strong activity. With the increase of culture time, the growth of
bacteria was still inhibited to a great extent. In order to explore whether there is a combined effect between
antibacterial peptides and lysozyme, we mixed all antibacterial peptides with lysozyme and determined the
antibacterial activity of the mixed samples. The results show that the mixed samples have certain antibacterial
activity, but the combined effect is not significant, which may be because we mixed all antibacterial peptides,
some of which have poor antibacterial activity or antagonistic effect, resulting in poor mixed effect. Later, we
will consider the combination of two, so as to find the best combination scheme with antibacterial effect.
Compared with *Escherichia coli*, most antimicrobial peptides have better antibacterial effect on
*Bacillus*
*subtilis*. Notably, Plectasin showed the most significant inhibitory effects against *E. coli* and
*Bacillus*
*subtilis*.

![figure7](https://static.igem.wiki/teams/5742/images/engineering/figure7-1.webp)

Figure 7. Time-Kill curve. A: *E. coli*; B: *Bacillus subtilis*.

**2.3 Laser Scanning Confocal Microscope observation**

The bacterial suspension was incubated with different biopreservatives for a total of 10 h at 37℃, followed by
double staining of live and dead cells using Calcein-AM/PI. Calcein-AM is hydrolyzed by intracellular esterase
after entering the cell to produce a strong anionic green fluorescence product, thereby labeling the living
cells with green fluorescence. Propidium iodide (PI) cannot cross the cell membrane of living cells and only
enters when the membrane is damaged; It inserts into the DNA, producing a red fluorescence that labels dead
cells. This differential staining protocol labels both live and dead cells in all samples, and their
visualization under confocal microscopy provides a rapid quantitative assessment of the antimicrobial efficacy
of bio preservatives.

As shown in Figure 8, all tested bio preservatives exerted bactericidal activity against both Escherichia coli
and Bacillus subtilis, although the extent of killing varied markedly among treatments. Notably, exposure to
Plectasin or the LL-37 resulted in a pronounced reduction in B. subtilis viability, indicating potent
bactericidal efficacy against this species. Conversely, Cecropin A left a substantially higher residual viable
count of B. subtilis, revealing a comparatively weak activity against this bacterium. In contrast, Plectasin was
less effective against E. coli than against B. subtilis, whereas LL-37 displayed superior bactericidal
performance against E. coli relative to its effect on B. subtilis. Through the observation under confocal
microscope, we confirmed that our antimicrobial peptide protein has certain killing ability to bacteria.
Moreover, our lysozyme also has a certain bactericidal effect on Bacillus subtilis. Through the screening of the
above experiments, we determined that Plectasin and LL-37 are the best antibacterial peptides we have obtained
at present, and further we want to observe the morphology of the treated bacteria by scanning electron
microscope.

![figure8](https://static.igem.wiki/teams/5742/images/engineering/figure8-1.webp)

Figure 8. Laser Scanning Confocal Microscope results. Laser Scanning Confocal Microscope results. A: Bacillus
subtilis; B: E. coli; C: Bacterial mortality.

**2.4 Morphological observation of scanning electron microscope**

Further, we want to observe the morphology of bacteria treated with antibacterial peptides by scanning electron
microscope. We chose two antibacterial peptides (Plectasin and LL-37) which had the most remarkable effect in
the previous antibacterial experiment and chose AMP as the positive control. By scanning electron microscope, we
can see that compared with the bacteria not treated with antimicrobial peptides, the surfaces of
*Escherichia*
*coli* and *Bacillus subtilis* were damaged to varying degrees after treated with antimicrobial
peptides, which
means that our antimicrobial peptides may damage the cell wall or cell membrane structure of bacteria, thus
causing bacterial death. The specific bactericidal mechanism of antimicrobial peptides needs further analysis.

![figure9](https://static.igem.wiki/teams/5742/images/engineering/figure9-1.webp)

Figure 9. Scanning electron microscope observation results. A: *E. coli*; B: *Bacillus subtilis*.

Learn

1. In this study, lysozyme and antimicrobial peptides were successfully expressed, but yields remained modest,
most likely due to suboptimal promoter selection and inadequate codon optimization. Therefore, we intend to
implement codon-promoter joint coordination, co-express chaperones, and employ a high-density fermentation
strategy to significantly improve production titers and biological activity.

2. Functional assays indicate limited antimicrobial potency of the resulting bio preservatives, suggesting that
the expression environment may impair the conformation or stability of the fusion protein. Therefore, in the
future, we will use multiple expression systems in combination: try to compare different hosts to determine the
system that best preserves the natural fold and maximizes antimicrobial efficacy.

3. We also need to obtain more proteins, so as to repeat the experiment many times to obtain more accurate data.
Therefore, increasing protein production is the most important thing that we need to optimize and improve in the
next step. At present, our experiment only found that our antibacterial peptide can inhibit the growth of
bacteria, and the specific mechanism has not been resolved. Further, we may analyze the bactericidal mechanism
of this antibacterial peptide.

References

```
1)	World Health Organization (WHO). Foodborne diseases: emerging threats to public health. World Health Organization, https://www.who.int/data/gho/data/themes/who-estimates-of-the-global-burden-of-foodborne-diseases (2022).
2)	Gallo, M., Ferrara, L., Calogero, A., Montesano, D. & Naviglio, D. Relationships between food and diseases: What to know to ensure food safety. Food Res Int 130, 109414, doi:10.1016/j.foodres.2020.109414 (2020).
3)	Singh, S. et al. Recent approaches in the application of antimicrobial peptides in food preservation. World J Microbiol Biotechnol 40, 315, doi:10.1007/s11274-024-04126-4 (2024).
4)	Nawaz, N. et al. Lysozyme and Its Application as Antibacterial Agent in Food Industry. Molecules 27, doi:10.3390/molecules27196305 (2022).
5)	Abdou, A. M. & Awad, D. A. B. Lysozyme Peptides as a Novel Nutra-Preservative to Control Some Food Poisoning and Food Spoilage Microorganisms. Probiotics Antimicrob Proteins, doi:10.1007/s12602-024-10226-2 (2024).
```

{% endblock %}

{% extends "layout.html" %}
{% block title %}Experiments{% endblock %}
{% block page\_header %}

EXPERIMENTS

{% endblock %}
{% block page\_content %}

* [Preparation of culture medium](#Preparationofculturemedium "Preparation of culture medium")
* [The Construction of Plasmid](#TheConstructionofPlasmid "The Construction of Plasmid")
* [Functional Test](#FunctionalTest "Functional Test")

Preparation of culture medium

```
Goal:Prepare a culture environment with rich nutrients for bacterial growth
Materials:
Tryptone
Yeast Extract
ddH2O
Pipette and tips
PCR tube
EP tube
Peptone
K2HPO4
KH2PO4
Gloves
Lab coat
Procedure:
LB Medium: Weigh 2.5 g tryptone, 1.25 g yeast extract, 2.5 g NaCl. Add distilled water to 250 mL. Sterilize at 121°C for 30 min. For solid medium, add 2% agar powder before sterilization.
YPD Medium: Weigh 2.5 g yeast extract, 5 g tryptone. Prepare glucose stock separately, sterilize separately. Add water to 250 mL and autoclave at 121°C for 20 min. Add glucose before use. Add 2% agar powder for solid medium.
BMGY Medium:, Weigh 0.5 g yeast extract, 1g peptone,0.15g K2HPO4
0.59g KH2PO4, Dissolve with 50ml of water. Sterilize at 121°C for 20 min. (5 mL 10×YNB, 100 μ l of 50× biotin and 500μL of glycerol were added during fermentation.)
BMMY Medium:Weigh 0.5 g yeast extract, 1g peptone,0.15g K2HPO4,0.59g KH2PO4, Dissolve with 50ml of water. Sterilize at 121°C for 20 min. (During fermentation, 5ml of 10× YNB, 100μ l of 50× biotin and 0.5 g of methanol were added.)
```

The Construction of Plasmid

![figure1](https://static.igem.wiki/teams/5742/images/experiments/figure1.webp)

```
⦁  Construction of pPICZα A/hLYZ
    Goal: Construction of plasmid containing lysozyme hLYZ
    A: Obtain the target gene
    Materials:
    Template DNA
    PrimeSTAR Max Premix
    Forward Primer (10 μM)
    Reverse Primer (10 μM)
    ddH2O
    Pipette and tips
    PCR tube
    EP tube
    Agarose
    TAE Buffer
    GelRed
    DNA Loading Buffer
    DNA marker
    Procedure:
    1.Dispense template DNA into tubes.
    2.Add corresponding forward and reverse primers.
    3.Add PrimeSTAR Max Premix and ddH2O to each tube.
    4.Mix gently and run PCR using:
    95˚ 3 minutes
    one cycle:
    95˚for 30 seconds
    55˚for 30 seconds
    72˚ for 50 seconds
    This cycle repeats for 33 times
    72˚ for 10 minutes
    B: Agarose Gel Electrophoresis
    Goal: Verify whether the target gene is constructed correctly
    Materials:
    Agarose
    TAE Buffer
    GelRed
    DNA Loading Buffer
    DNA marker
    Procedure:
    1.	Dissolve 0.1 g agarose in 100 mL TAE buffer by microwaving for 3 min.
    2.	Add 1 μL GelRed dye. Pour into mold with comb;
    3.	Place gel in electrophoresis chamber with TAE buffer.
    4.	Mix DNA samples with loading buffer, load into wells.
    5.	Run gel at 150 V for ~20 min.
    6.	Visualize bands under UV light.
    C: DNA Gel Extraction
    Goal: Obtaining DNA fragments with correct construction.
    Materials:
    Binding Buffer
    Wash Buffer
    Elution Buffer
    Procedure:
    ⦁	Check whether Washing Solution is adding anhydrous ethanol,
    ⦁	Check whether Buffer B2 appear precipitate,Set the water bath pot to 50℃
    ⦁	Cut out the gel block containing the target fragment from the agarose gel and weigh it.
    ⦁	Add Buffer B2 into the gel block and metal bath 55 ℃
    ⦁	Transfer sol solution into the adsorption column and 8000Xg centrifugation for 30 seconds
    ⦁	Add Wash Buffer and 9000Xg centrifugation for 30 seconds, pour the solution in the tube.
    ⦁	Repeat the previous step
    ⦁	Let empty adsorption column in the centrifuge for 1 minute
    ⦁	Put adsorption column into a clean 1.5mL centrifuge tube and add 30μl Elution Buffer into the center of the adsorption film. Stewing for 1 minute and centrifugation for 1 minute.
    D: Restriction enzyme digestion and ligation
    Goal: Obtaining DNA fragments with restriction sites.
    Materials:
    Plasmid DNA
    Restriction enzyme 1
    Restriction enzyme 2
    10× Reaction Buffer
    Nuclease-free Water
    Ligation buffer
    T4 ligase
    Insert DNA fragment
    Digested vector
    ddH2O
    Procedure:
    1.Mix all components in a 30 μL total reaction volume.
    2.Incubate at 37°C for 3 hours.
    3.Mix insert fragmen(step 2)t with ligation buffer, T4 ligase, digested vector,
    And ddH2O.
    4.Incubate at room temp for 1 hour.
    E: Transformation into E.coli Competent Cells
    Goal: Expression of plasmid in Escherichia coli.
    Materials:
    Competent E. coli cells
    LB culture medium
    Peptone, yeast extract, NaCl (for making LB agar)
    Kanamycin
    Petri dishes
    Sterile cell spreader
    Transformed bacterial colony plates
    Procedure:
    ⦁	Preparation for antibiotic plates (LB+1:1000 bleomycin)
    ⦁	Thaw competent E. coli DH5α cells on the ice.
    ⦁	Add 10μL recombinant product (plasmid) into the competent cells, gently flick vessel wall, gently mix it by using the pipette tip. Incubate on ice for 30 minutes, stationary.
    ⦁	42℃ hot water bath heat shocks for 45s in order to create channels and pores in the cell membrane that connect the cell interior to the external environment.
    ⦁	Remove immediately, incubate on ice for 2 minutes to reduce the temperature.
    ⦁	Add 900μL LB culture medium. Incubate at 37℃ with shaking at 200rmp for one hour.
    ⦁	Centrifuge and discard 800μL of the supernatant.
    ⦁	Separate the thallus in the remaining 200μL of LB medium.
    ⦁	Evenly spread the sample on antibiotic plates by using a sterile cell spreader.
    F: Colony PCR
    Goal: Verification whether the strain is constructed correctly.
    Materials:
    PrimeSTAR Max Premix (2×)
    Forward Primer (10–15 pmol)
    Reverse Primer (10–15 pmol)
    ddH2O
    Template (colonies)
    Procedure:
    Reagent	                    Volume
    PrimeSTAR Max Premix (2×)	 10 μL
    Forward Primer (10–15 pmol)	 ~0.2–0.3 μM
    Reverse Primer (10–15 pmol)	 ~0.2–0.3 μM
    Template (colonies)	         1 μL
    ddH2O	                    To 50 μL total
    1.Prepare 20 μL PCR mixture.
    1.95°C for 3 min
    2.95°C for 30 s
    3.53°C for 15 s
    4.72°C for 1 min
    5.72°C for 10 min
    （Repeat step 2,3,4 for 33 times）
    2.Run PCR as described above.
    3.Analyze by agarose gel electrophoresis.
    4.Visualize bands under UV light.
    G: Plasmid Extraction
    Goal: Obtain the successfully constructed plasmid.
    Materials: Plasmid extraction kit
    Procedure:
    Cell Lysis:
    1.Harvest 1.5–5 mL overnight bacterial culture; centrifuge at 8,000 × g for 2 min; discard supernatant.
    2.Resuspend pellet in 250 μL Buffer SP1; mix thoroughly.
    3.Add 250 μL Buffer SP2; mix gently by inversion 5–10 times; incubate 2–4 min at
    room temperature.
    4.Add 350 μL Buffer SP3; mix gently by inversion 5–10 times
    Binding and Washing:
    1.Centrifuge at 12,000 × g for 5–10 min; transfer lysate to spin column; centrifuge at 8,000 × g for 30 sec; discard flow-through.
    2.(Optional) Add 500 μL Buffer DW1; centrifuge at 9,000 × g for 30 sec; discard flow-through.
    3.Add 500 μL Wash Solution; centrifuge at 9,000 × g for 30 sec; discard flow-through.
    4.Repeat wash step once.
    5.Dry spin column at 9,000 × g for 1 min.
    Elution:
    1.Place spin column in a clean 1.5 mL tube.
    2.Add 50–100 μL Elution Buffer to membrane center; incubate 1 min; centrifuge for 1 min to elute DNA.
    H: Transforming plasmid into Pichia pastoris.
    Goal: Make lysozyme protein express in Pichia pastoris.
    Materials:
    GS115 Chemically Competent Cell
    Yeast transformation promoting reagent
    Pichia pastoris transformation solution
    Procedure:
    1. Take 5μg of plasmid to be transformed, cut by enzyme and recover;
    2. Take a sterile 1.5 ml EP tube, add 1-5ug of precooled linear plasmid (the volume is not higher than 15 μL), 5 μL of yeast transformation promoter, 100 μL of GS115 competent cells melted on ice and 500 ml of Pichia pastoris transformation solution in turn, gently turn and mix evenly for 6-8 times.
    3.Water bath at 30°C for 30min, gently turn over and mix evenly for 6-8 times every 15min.
    4. Add 20 mL of dimethyl sulfoxide to each branch (to improve the conversion efficiency, it is not necessary).
    5.Take a water bath at 42°C for 15min, gently turn over and mix evenly for 6-8 times every 7.5min.
    6.The supernatant was instantly centrifuged at 12000rpm, and each YPG Plus (or YPG) with lml was resuscitated at 30℃ for 1 hour (to improve the transformation efficiency, it was not necessary).
    7.The supernatant was instantly centrifuged at 12000rpm, resuspended with 100μ 0.9% NaCl, smeared and cultured at 30℃ for 3-7 days.
    8. Verify whether the strain is constructed correctly by colony PCR.
⦁  Construction of pPICZα A/Cecropin A
    A: Obtain the target gene
    Goal: Obtaining a gene fragment for constructing antibacterial peptide Cecropin A
    Materials: The specific primers of antimicrobial peptide A were designed,
    and the other materials were the same as those in 2.1-A.
    Procedure: The procedure is basically the same as 2.1-A, and the
      PCR extension time is adjusted according to the fragment length.
    B: Agarose Gel Electrophoresis
    Goal: Verify whether the target gene is constructed correctly
    Materials: Materials were the same as those in 2.1-B.
    Procedure: The procedure is basically the same as 2.1-B.
    C: DNA Gel Extraction
    Goal: Obtaining DNA fragments with correct construction.
    Materials: Materials were the same as those in 2.1-C.
    Procedure: The procedure is basically the same as 2.1-C.
    D: Restriction enzyme digestion and ligation
    Goal: Obtaining DNA fragments with restriction sites.
    Materials: Materials were the same as those in 2.1-D.
    Procedure: The procedure is basically the same as 2.1-D.
    E: Transformation into E.coli Competent Cells
    Goal: Expression of plasmid in Escherichia coli.
    Materials: Materials were the same as those in 2.1-E.
    Procedure: The procedure is basically the same as 2.1-E.
    F: Colony PCR
    Goal: Verification whether the strain is constructed correctly.
    Materials: Materials were the same as those in 2.1-F.
    Procedure: The procedure is basically the same as 2.1-F.
    G: Plasmid Extraction
    Goal: Obtain the successfully constructed plasmid.
    Materials: Materials were the same as those in 2.1-G.
    Procedure: The procedure is basically the same as 2.1-G.
    H: Transforming plasmid into Pichia pastoris.
    Goal: Make lysozyme protein express in Pichia pastoris.
    Materials: Materials were the same as those in 2.1-H.
    Procedure: The procedure is basically the same as 2.1-H.
⦁  Construction of pPICZα A/Melittin
    A: Obtain the target gene
    Goal: Obtaining a gene fragment for constructing antibacterial peptide Melittin
    Materials: The specific primers of antimicrobial peptide A were designed,
    and the other materials were the same as those in 2.1-A.
    Procedure: The procedure is basically the same as 2.1-A, and the
      PCR extension time is adjusted according to the fragment length.
    B: Agarose Gel Electrophoresis
    Goal: Verify whether the target gene is constructed correctly
    Materials: Materials were the same as those in 2.1-B.
    Procedure: The procedure is basically the same as 2.1-B.
    C: DNA Gel Extraction
    Goal: Obtaining DNA fragments with correct construction.
    Materials: Materials were the same as those in 2.1-C.
    Procedure: The procedure is basically the same as 2.1-C.
    D: Restriction enzyme digestion and ligation
    Goal: Obtaining DNA fragments with restriction sites.
    Materials: Materials were the same as those in 2.1-D.
    Procedure: The procedure is basically the same as 2.1-D.
    E: Transformation into E.coli Competent Cells
    Goal: Expression of plasmid in Escherichia coli.
    Materials: Materials were the same as those in 2.1-E.
    Procedure: The procedure is basically the same as 2.1-E.
    F: Colony PCR
    Goal: Verification whether the strain is constructed correctly.
    Materials: Materials were the same as those in 2.1-F.
    Procedure: The procedure is basically the same as 2.1-F.
    G: Plasmid Extraction
    Goal: Obtain the successfully constructed plasmid.
    Materials: Materials were the same as those in 2.1-G.
    Procedure: The procedure is basically the same as 2.1-G.
    H: Transforming plasmid into Pichia pastoris.
    Goal: Make lysozyme protein express in Pichia pastoris.
    Materials: Materials were the same as those in 2.1-H.
    Procedure: The procedure is basically the same as 2.1-H.
⦁  Construction of pPICZα A/ pPICZα A/Buforin II
    A: Obtain the target gene
    Goal: Obtaining a gene fragment for constructing antibacterial peptide Buforin II.
    Materials: The specific primers of antimicrobial peptide A were designed,
    and the other materials were the same as those in 2.1-A.
    Procedure: The procedure is basically the same as 2.1-A, and the
      PCR extension time is adjusted according to the fragment length.
    B: Agarose Gel Electrophoresis
    Goal: Verify whether the target gene is constructed correctly
    Materials: Materials were the same as those in 2.1-B.
    Procedure: The procedure is basically the same as 2.1-B.
    C: DNA Gel Extraction
    Goal: Obtaining DNA fragments with correct construction.
    Materials: Materials were the same as those in 2.1-C.
    Procedure: The procedure is basically the same as 2.1-C.
    D: Restriction enzyme digestion and ligation
    Goal: Obtaining DNA fragments with restriction sites.
    Materials: Materials were the same as those in 2.1-D.
    Procedure: The procedure is basically the same as 2.1-D.
    E: Transformation into E.coli Competent Cells
    Goal: Expression of plasmid in Escherichia coli.
    Materials: Materials were the same as those in 2.1-E.
    Procedure: The procedure is basically the same as 2.1-E.
    F: Colony PCR
    Goal: Verification whether the strain is constructed correctly.
    Materials: Materials were the same as those in 2.1-F.
    Procedure: The procedure is basically the same as 2.1-F.
    G: Plasmid Extraction
    Goal: Obtain the successfully constructed plasmid.
    Materials: Materials were the same as those in 2.1-G.
    Procedure: The procedure is basically the same as 2.1-G.
    H: Transforming plasmid into Pichia pastoris.
    Goal: Make lysozyme protein express in Pichia pastoris.
    Materials: Materials were the same as those in 2.1-H.
    Procedure: The procedure is basically the same as 2.1-H.
⦁  Construction of pPICZα A/LL-30
    A: Obtain the target gene
    Goal: Obtaining a gene fragment for constructing antibacterial peptide LL-30.
    Materials: The specific primers of antimicrobial peptide A were designed,
    and the other materials were the same as those in 2.1-A.
    Procedure: The procedure is basically the same as 2.1-A, and the
      PCR extension time is adjusted according to the fragment length.
    B: Agarose Gel Electrophoresis
    Goal: Verify whether the target gene is constructed correctly
    Materials: Materials were the same as those in 2.1-B.
    Procedure: The procedure is basically the same as 2.1-B.
    C: DNA Gel Extraction
    Goal: Obtaining DNA fragments with correct construction.
    Materials: Materials were the same as those in 2.1-C.
    Procedure: The procedure is basically the same as 2.1-C.
    D: Restriction enzyme digestion and ligation
    Goal: Obtaining DNA fragments with restriction sites.
    Materials: Materials were the same as those in 2.1-D.
    Procedure: The procedure is basically the same as 2.1-D.
    E: Transformation into E.coli Competent Cells
    Goal: Expression of plasmid in Escherichia coli.
    Materials: Materials were the same as those in 2.1-E.
    Procedure: The procedure is basically the same as 2.1-E.
    F: Colony PCR
    Goal: Verification whether the strain is constructed correctly.
    Materials: Materials were the same as those in 2.1-F.
    Procedure: The procedure is basically the same as 2.1-F.
    G: Plasmid Extraction
    Goal: Obtain the successfully constructed plasmid.
    Materials: Materials were the same as those in 2.1-G.
    Procedure: The procedure is basically the same as 2.1-G.
    H: Transforming plasmid into Pichia pastoris.
    Goal: Make lysozyme protein express in Pichia pastoris.
    Materials: Materials were the same as those in 2.1-H.
    Procedure: The procedure is basically the same as 2.1-H.
⦁  Construction of pPICZα A/Plectasin
    A: Obtain the target gene
    Goal: Obtaining a gene fragment for constructing antibacterial peptide Plectasin.
    Materials: The specific primers of antimicrobial peptide A were designed,
    and the other materials were the same as those in 2.1-A.
    Procedure: The procedure is basically the same as 2.1-A, and the
      PCR extension time is adjusted according to the fragment length.
    B: Agarose Gel Electrophoresis
    Goal: Verify whether the target gene is constructed correctly
    Materials: Materials were the same as those in 2.1-B.
    Procedure: The procedure is basically the same as 2.1-B.
    C: DNA Gel Extraction
    Goal: Obtaining DNA fragments with correct construction.
    Materials: Materials were the same as those in 2.1-C.
    Procedure: The procedure is basically the same as 2.1-C.
    D: Restriction enzyme digestion and ligation
    Goal: Obtaining DNA fragments with restriction sites.
    Materials: Materials were the same as those in 2.1-D.
    Procedure: The procedure is basically the same as 2.1-D.
    E: Transformation into E.coli Competent Cells
    Goal: Expression of plasmid in Escherichia coli.
    Materials: Materials were the same as those in 2.1-E.
    Procedure: The procedure is basically the same as 2.1-E.
    F: Colony PCR
    Goal: Verification whether the strain is constructed correctly.
    Materials: Materials were the same as those in 2.1-F.
    Procedure: The procedure is basically the same as 2.1-F.
    G: Plasmid Extraction
    Goal: Obtain the successfully constructed plasmid.
    Materials: Materials were the same as those in 2.1-G.
    Procedure: The procedure is basically the same as 2.1-G.
    H: Transforming plasmid into Pichia pastoris.
    Goal: Make lysozyme protein express in Pichia pastoris.
    Materials: Materials were the same as those in 2.1-H.
    Procedure: The procedure is basically the same as 2.1-H.
⦁  Construction of pPICZα A/Hepcidin
    A: Obtain the target gene
    Goal: Obtaining a gene fragment for constructing antibacterial peptide Hepcidin.
    Materials: The specific primers of antimicrobial peptide A were designed,
    and the other materials were the same as those in 2.1-A.
    Procedure: The procedure is basically the same as 2.1-A, and the
      PCR extension time is adjusted according to the fragment length.
    B: Agarose Gel Electrophoresis
    Goal: Verify whether the target gene is constructed correctly
    Materials: Materials were the same as those in 2.1-B.
    Procedure: The procedure is basically the same as 2.1-B.
    C: DNA Gel Extraction
    Goal: Obtaining DNA fragments with correct construction.
    Materials: Materials were the same as those in 2.1-C.
    Procedure: The procedure is basically the same as 2.1-C.
    D: Restriction enzyme digestion and ligation
    Goal: Obtaining DNA fragments with restriction sites.
    Materials: Materials were the same as those in 2.1-D.
    Procedure: The procedure is basically the same as 2.1-D.
    E: Transformation into E.coli Competent Cells
    Goal: Expression of plasmid in Escherichia coli.
    Materials: Materials were the same as those in 2.1-E.
    Procedure: The procedure is basically the same as 2.1-E.
    F: Colony PCR
    Goal: Verification whether the strain is constructed correctly.
    Materials: Materials were the same as those in 2.1-F.
    Procedure: The procedure is basically the same as 2.1-F.
    G: Plasmid Extraction
    Goal: Obtain the successfully constructed plasmid.
    Materials: Materials were the same as those in 2.1-G.
    Procedure: The procedure is basically the same as 2.1-G.
    H: Transforming plasmid into Pichia pastoris.
    Goal: Make lysozyme protein express in Pichia pastoris.
    Materials: Materials were the same as those in 2.1-H.
    Procedure: The procedure is basically the same as 2.1-H.
⦁  Construction of pPICZα A/Piscidin-1
    A: Obtain the target gene
    Goal: Obtaining a gene fragment for constructing antibacterial peptide Piscidin-1.
    Materials: The specific primers of antimicrobial peptide A were designed,
    and the other materials were the same as those in 2.1-A.
    Procedure: The procedure is basically the same as 2.1-A, and the
      PCR extension time is adjusted according to the fragment length.
    B: Agarose Gel Electrophoresis
    Goal: Verify whether the target gene is constructed correctly
    Materials: Materials were the same as those in 2.1-B.
    Procedure: The procedure is basically the same as 2.1-B.
    C: DNA Gel Extraction
    Goal: Obtaining DNA fragments with correct construction.
    Materials: Materials were the same as those in 2.1-C.
    Procedure: The procedure is basically the same as 2.1-C.
    D: Restriction enzyme digestion and ligation
    Goal: Obtaining DNA fragments with restriction sites.
    Materials: Materials were the same as those in 2.1-D.
    Procedure: The procedure is basically the same as 2.1-D.
    E: Transformation into E.coli Competent Cells
    Goal: Expression of plasmid in Escherichia coli.
    Materials: Materials were the same as those in 2.1-E.
    Procedure: The procedure is basically the same as 2.1-E.
    F: Colony PCR
    Goal: Verification whether the strain is constructed correctly.
    Materials: Materials were the same as those in 2.1-F.
    Procedure: The procedure is basically the same as 2.1-F.
    G: Plasmid Extraction
    Goal: Obtain the successfully constructed plasmid.
    Materials: Materials were the same as those in 2.1-G.
    Procedure: The procedure is basically the same as 2.1-G.
    H: Transforming plasmid into Pichia pastoris.
    Goal: Make lysozyme protein express in Pichia pastoris.
    Materials: Materials were the same as those in 2.1-H.
    Procedure: The procedure is basically the same as 2.1-H.
⦁  Construction of pPICZα A/Lactoferricin
    A: Obtain the target gene
    Goal: Obtaining a gene fragment for constructing antibacterial peptide Lactoferricin.
    Materials: The specific primers of antimicrobial peptide A were designed,
    and the other materials were the same as those in 2.1-A.
    Procedure: The procedure is basically the same as 2.1-A, and the
      PCR extension time is adjusted according to the fragment length.
    B: Agarose Gel Electrophoresis
    Goal: Verify whether the target gene is constructed correctly
    Materials: Materials were the same as those in 2.1-B.
    Procedure: The procedure is basically the same as 2.1-B.
    C: DNA Gel Extraction
    Goal: Obtaining DNA fragments with correct construction.
    Materials: Materials were the same as those in 2.1-C.
    Procedure: The procedure is basically the same as 2.1-C.
    D: Restriction enzyme digestion and ligation
    Goal: Obtaining DNA fragments with restriction sites.
    Materials: Materials were the same as those in 2.1-D.
    Procedure: The procedure is basically the same as 2.1-D.
    E: Transformation into E.coli Competent Cells
    Goal: Expression of plasmid in Escherichia coli.
    Materials: Materials were the same as those in 2.1-E.
    Procedure: The procedure is basically the same as 2.1-E.
    F: Colony PCR
    Goal: Verification whether the strain is constructed correctly.
    Materials: Materials were the same as those in 2.1-F.
    Procedure: The procedure is basically the same as 2.1-F.
    G: Plasmid Extraction
    Goal: Obtain the successfully constructed plasmid.
    Materials: Materials were the same as those in 2.1-G.
    Procedure: The procedure is basically the same as 2.1-G.
    H: Transforming plasmid into Pichia pastoris.
    Goal: Make lysozyme protein express in Pichia pastoris.
    Materials: Materials were the same as those in 2.1-H.
    Procedure: The procedure is basically the same as 2.1-H.
⦁  Construction of pPICZα A/antisepsis mix
    A: Obtain the target gene
    Goal: Obtaining a gene fragment for constructing antibacterial peptide antisepsis mix.
    Materials: The specific primers of antimicrobial peptide A were designed,
    and the other materials were the same as those in 2.1-A.
    Procedure: The procedure is basically the same as 2.1-A, and the
      PCR extension time is adjusted according to the fragment length.
    B: Agarose Gel Electrophoresis
    Goal: Verify whether the target gene is constructed correctly
    Materials: Materials were the same as those in 2.1-B.
    Procedure: The procedure is basically the same as 2.1-B.
    C: DNA Gel Extraction
    Goal: Obtaining DNA fragments with correct construction.
    Materials: Materials were the same as those in 2.1-C.
    Procedure: The procedure is basically the same as 2.1-C.
    D: Restriction enzyme digestion and ligation
    Goal: Obtaining DNA fragments with restriction sites.
    Materials: Materials were the same as those in 2.1-D.
    Procedure: The procedure is basically the same as 2.1-D.
    E: Transformation into E.coli Competent Cells
    Goal: Expression of plasmid in Escherichia coli.
    Materials: Materials were the same as those in 2.1-E.
    Procedure: The procedure is basically the same as 2.1-E.
    F: Colony PCR
    Goal: Verification whether the strain is constructed correctly.
    Materials: Materials were the same as those in 2.1-F.
    Procedure: The procedure is basically the same as 2.1-F.
    G: Plasmid Extraction
    Goal: Obtain the successfully constructed plasmid.
    Materials: Materials were the same as those in 2.1-G.
    Procedure: The procedure is basically the same as 2.1-G.
    H: Transforming plasmid into Pichia pastoris.
    Goal: Make lysozyme protein express in Pichia pastoris.
    Materials: Materials were the same as those in 2.1-H.
    Procedure: The procedure is basically the same as 2.1-H.
```

Functional Test

```
⦁  The growth curve of Pichia pastoris
    Goal: The growth curve measurement was used to verify whether the growth ability
    of yeast was different when different genes were introduced.
    Materials:
    LB liquid medium
    Pichia pastoris
    Bleomycin Zecoin
    Procedure:
    Inoculate the transformants into 3 mL of YPD liquid medium and add bleomycin
    Zecoin, then incubate at 30°C (OD600=0.02), The growth level of the strain was determined at a fixed time and the growth curve was drawn with the same
    absorbance..
⦁  SDS-PAGE of antimicrobial peptide protein and lysozyme protein
    Goal: Antimicrobial peptides and lysozyme proteins were obtained by protein
    induction and verified by SDS-PAGE.
    Materials:
    gloves
    lab coat
    parallel pipette
    pipette tip
    dd water
    electrophoresis apparatus
    30% Acr-Bis
    Lower gel buffer
    Upper gel buffer
    10% Gel polymerization catalyst
    TEMED Substitute
    Tris
    SDS
    Glycine
    His-tag Purification Resin
    Procedure:
    ⦁	Inoculate the strain into 3 mL of YPD liquid medium and add bleomycin Zecoin, then incubate at 30°C for 24 h to obtain the primary seed culture.
    ⦁	Inoculate the primary seed culture into 100 mL of BMGY medium at a 2% inoculum rate, incubate at 30°C until the OD600 reaches approximately 1.5, and measure the growth curve.
    ⦁	Then centrifuge at 4°C and 5000 rpm for 5 minutes, remove the supernatant, and collect the cells. Resuspend in 50 mL BMMY medium and incubate at 30°C, adding 10 g/L methanol every 24 hours during the incubation period.
    ⦁	Harvest the cells after 72 hours of protein induction.
    All following experiment below was conducted with an ice bath
    ⦁	Collect the previous bacterial colonies, centrifuge at 6000 rpm for 3 minutes
    ⦁	Collect the supernatant liquid, then store under -20°C
    Strain lysis:
    ⦁	Combine 4 μL of lysis buffer, lysozyme solution(1mg/ml) under 0°C, let it sit for 30min
    ⦁	Supersonic lysis under 300W power for 5min (the sonication process consists of a cycle, shifting between 5-second sonication and 5-second breaks).
    ⦁	Centrifuge at 6000 rpm under 4°C Celsius for ten minutes.
    ⦁	The combination of 4ml supernatant liquid and 1ml of Purification Resin (BeyoGoldTMHis-Tag), let it sit for 2 hours.
    ⦁	Transfer the formulae into an affinity layer adsorption column, collect the filtered liquid, and label the substance as liquid number 1.
    ⦁	Wash the affinity column again four times using the wash buffer and collect the liquid in four separate EP tubes.
    ⦁	Then, wash the same column four more times using the elution buffer, and collect the filtered liquid in four other EP tubes.
    ⦁	To evaluate the resulting proteins, we ran SDS-Page.
    Make SDS-Page gel
    Separation gel:
    Distilled Water: 4.8 mL(To adjust volume and provide a solvent environment)
    30% Acr - Bis (29:1): 10.0 mL(Acrylamide - Bisacrylamide mixture, main component for gel polymerization)
    Lower Gel Buffer (4X): 5.0 mL(Provide appropriate pH and ionic strength for separating gel)
    10% Gel Polymerization Catalyst:0.2 mL(Initiate gel polymerization reaction, usually contains ammonium persulfate)
    - TEMED Substitute: 0.008 mL(Accelerate the polymerization process, analogous to TEMED function)
    Staking gel:
      - Distilled Water:2.33 mL(Solvent and volume adjustment)
    - 30% Acr - Bis (29:1):0.67 mL(Acrylamide - Bisacrylamide for stacking gel structure)
    - Upper Gel Buffer (4X): 1 mL(Provide suitable pH and ionic strength for stacking gel)
    - 10% Gel Polymerization Catalyst:0.04 mL(Trigger stacking gel polymerization)
    - TEMED Substitute:0.004 mL(Speed up stacking gel formation)
    Electrophoresis Solution:Tris 3g, Glycine 14.4g, and SDS 1g. Add double-distilled water to bring the volume up to 1L. When using, dilute it 5 times to obtain the 1×working solution.
    Run electrophoresis: run at 80V for 20 minutes, then switch to 180V and run for an additional 40 minutes.
    ⦁	In order to observe the proteins visually, we dyed them using CBBS (Coomassie brilliant blue staining).

⦁  Determination of Minimum Inhibitory Concentrations of antimicrobial peptides and lysozyme
    Goal: Verify the bactericidal activity of antimicrobial peptides and lysozyme.
    Materials:
    Escherichia coli K-12
    Bacillus subtilis
    LB culture medium
    Petri dishes
    Microcentrifuge tubes
    Pipette tips
    Sterile water
    Incubator
    Ampicillin
    microplate reader
    96-well bacterial culture plate
    Procedure:
    Prepare 1.2ml solutions of antimicrobial peptides in a centrifuge tube1(128 μg/mL)
    Transfer 600μl solutions of antimicrobial peptides from tube1 into the centrifuge tube2, then add 600μl sterile water
    Transfer 600μl solutions of antimicrobial peptides from tube 2 into the centrifuge tube3, then add 600μl sterile water
    Transfer 600μl solutions of antimicrobial peptides from tube 3 into the centrifuge tube4, then add 600μl sterile water
    Transfer 600μl solutions of antimicrobial peptides from tube 4 into the centrifuge tube5, then add 600μl sterile water
    Transfer 600μl solutions of antimicrobial peptides from tube 5 into the centrifuge tube6, then add 600μl sterile water
    Transfer 600μl solutions of antimicrobial peptides from tube 6 into the centrifuge tube7, then add 600μl sterile water
    Transfer 600μl solutions of antimicrobial peptides from tube 7 into the centrifuge tube8, then add 600μl sterile water
    All antimicrobial peptides and lysozyme were diluted in gradient according to the above scheme.
    Application of each sample into 96-well bacterial culture plate in order
    Application of bacterial into the first line of 96-well bacterial culture plate
    Application of ampicillin into the second line of 96-well bacterial culture plate
    Application of LB culture medium into the third line of 96-well bacterial culture plate
    Cultivate the bacterial culture until the OD value reaches 0.9–1.0 (with a CFU count of 10⁹ CFU/mL).
    Perform a 1000-fold dilution to adjust the CFU count to 10⁶ CFU/mL.
    Application diluted bacterial into 96-well bacterial culture plate.
⦁  Time-Kill curve of antimicrobial peptides and lysozyme
    Goal: Verify the bactericidal activity of antimicrobial peptides and lysozyme.
    Materials:
    Bacillus subtilis
    Escherichia coli
    Lab coat
    Gloves
    EP tube
    Lysozymes
    Antibacterial peptides
    Pipette and tips
    Cell culture plate
    Procedure:
    1.Add the Bacillus subtilis and Escherichia coli separately into our antibacterial
    peptides and lysozymes, into a EP tube, using 500 μL of each component.
    2.	Add the sample into the 96 cell culture plate, starting from hour 0.
    3.	Samples were collected every 2 hours and OD600 was determined.
⦁  Laser Scanning Confocal Microscope observation
    Goal: The bactericidal effect of antimicrobial peptides and lysozyme was
    determined by living dead cell staining.
    Materials:
    Escherichia coli K-12
    Bacillus subtilis
    LB culture medium
    Petri dishes
    Microcentrifuge tubes
    Pipette tips
    Sterile water
    Incubator
    Ampicillin
    Procedure:
    Escherichia coli and Bacillus subtilis were treated with antimicrobial peptides and lysozyme at 37 for 24 hours, respectively.
    1.The cell precipitate obtained by centrifugation was resuspended with 1XAssay Bufer, so that the cell count of the resuspended cell suspension was 1X105-6 cells /ml.
    2.Add 1-2ul of Calcein-AM (stock solution) per 1ml of cell volume, blow and mix evenly, and incubate at 37℃ in the dark for 20-25 min.
    3.Add 3-5ul of PI stock solution from the kit into the stained cells, and dye it in the dark at room temperature for 5min.
    4.Incubate the fluorescent cells for 5min minutes with 450g, and remove the staining solution by centrifugation.
    Note: It is suggested that cells should be kept away from light during fluorescent staining.
    5.Wash 450g cells with 1x PBS for 5min, resuspend the cells with 1x PBS after centrifugation, take 3-5ul drops on a clean glass slide, and after tabletting with a clean cover glass, please examine them under fluorescence microscope in time. (6) Using 490±10 nm excitation filter to simultaneously detect living cells (yellow-green fluorescence) and dead cells (red fluorescence) under fluorescence microscope. In addition, only dead cells can be observed by using 545nm emission filter. It can also be detected directly by using a suitable filter under a fluorescent enzyme-labeled instrument.
⦁  Morphological observation of scanning electron microscope
    Goal: The morphology of bacteria treated with antimicrobial peptides and lysozyme was observed by scanning electron microscope.
    Materials:
    Escherichia coli K-12
    Bacillus subtilis
    LB culture medium
    Petri dishes
    Microcentrifuge tubes
    Pipette tips
    Sterile water
    Incubator
    Ampicillin
    Procedure:
    1. Sampling: Generally, the sample size is not more than 10x10 x 5mm. Determine the target of observing the sample to avoid pulling and squeezing the sample during sampling.
    2. Cleaning: remove impurities from the observation surface and fully expose the observation surface without damaging it.
    3. Fixation: Generally, it is better to fix glutaraldehyde and acid at 4℃.
    Glutaraldehyde and acid can be fixed separately or double-fixed with "glutaraldehyde-osmium acid". After fixing for 1-3 hours, the fixed solution is sucked out, 0,1M of cool acid buffer wave (PH=7.2) is added, and the solution is rinsed for 3-4 times for 1 hour.
    4. Dehydration: Suck out the buffer wave from the vial, add ethanol to dehydrate step by step (the ethanol concentration is 30%-50%-70%6-80%-90%-100% at a time), and the dehydration time depends on the sample size, generally 15-30 minutes. Add the 1:1 mixture of isoamyl acetate and ethanol, soak for 10-20min and shake properly, discard the mixture, add pure isoamyl acetate and soak for 10-20min and shake properly.
    5. Drying: Drying is a key link in the biological preparation of scanning electron microscope. It is necessary to reduce the distortion of surface morphology caused by water evaporation surface as much as possible during the drying process, and it is necessary to ensure thorough drying. Commonly used methods include natural drying, dry drying, critical point drying, freeze drying and vacuum drying. Different drying methods will be selected for different biological samples.
    6. Sample loading: the surface is plated with gold and observed by scanning electron microscope.
```

{% endblock %}

{% extends "layout.html" %}
{% block title %}Results{% endblock %}
{% block page\_header %}

RESULTS

{% endblock %}
{% block page\_content %}

* [Overview](#Overview "Overview")
* [Construction of plasmids](#Constructionofplasmids "Construction of plasmids")
* [Protein expression](#Proteinexpression "Protein expression")
* [Functional Test](#FunctionalTest "Functional Test")
* [Future plans](#Futureplans "Future plans")
* [References](#References "References")

Overview

With the increasing global population, food safety has gradually become the focus of global
attention[1]. Food
preservation and preservative play a crucial role in the food industry. Although traditional chemical
preservatives are effective in extending the shelf life of food, their widespread use poses many health risks,
including allergic reactions, drug resistance issues, and potential carcinogenicity[2]. Therefore,
the
development of natural, green and harmless preservatives has become an important trend in the development of the
food industry.

As natural antibacterial ingredients, lysozyme and antimicrobial peptides have become hot topics in food
preservative research in recent years due to their excellent antibacterial properties and good
safety[3,4,5].
The combination of the two can not only enhance the antibacterial effect, but also reduce the dependence on
traditional chemical preservatives, further improving food safety and freshness.

The main goal of this project is to develop a novel food preservative technology to improve the shelf life and
safety of food through the combination of lysozyme and antimicrobial peptides. We first screened from 2
lysozymes[6,7] and 74 antimicrobial peptides[8-81], eliminated antimicrobial peptides with
hemolytic risk,
toxicity, and long intestinal half-life, and finally screened out 1 lysozyme and 9 antimicrobial peptides. The
lysozyme and antimicrobial peptide genes were then cloned into expression vectors and transformed into
*Escherichia coli* DH5α and *Pichia pastoris* GS115 respectively. We expressed antimicrobial peptides
and lysozyme
proteins by *Pichia pastoris* and separated and purified these proteins by affinity chromatography column.
Finally, the antibacterial effect of lysozyme and antimicrobial peptides was verified by qualitative
characterization and judgment, as well as quantitative characterization and quantitative analysis.

Construction of plasmids

**2.1 Amplification of target genes and vector fragment**

We obtained target genes for constructing plasmid pPICZα A/antisepsis mix, pPICZα A/Hepcidin, pPICZα A/Buforin
II, pPICZα A/Piscidin-1, pPICZα A/LL-37, pPICZα A/Cecropin A, pPICZα A/Melittin, pPICZα A/Plectasin, pPICZα
A/hLYZ, pPICZα A/Lactoferricin using PCR technology, as shown in Figure 1A. We obtained fragments of
approximately 1600bp, 80bp, 80bp, 80bp, 130bp, 130bp, 90bp, 130bp, 460bp, 160bp in length, which are consistent
with our expected sizes.

Subsequently, we selected EcoRI and NotI restriction sites for enzymatic digestion of the target genes and
vector fragment (as shown in Figure 1B). We obtained fragments of approximately 3600bp in length, which are
consistent with our expected sizes.

![figure1](https://static.igem.wiki/teams/5742/images/results/figure1-1.webp)

Figure 1. Gel electrophoresis validation. A: PCR results; B: vector enzyme
digestion results.

**2.2 Fragment ligation and transformation**

The digested PCR fragment and vector were ligated using T4 DNA ligase and then we transformed them into
*Escherichia coli* DH5α, as shown in Figure 2A.The results preliminarily proved that the strain
construction was
successful. In order to further confirm the result, we sequenced the DNA(Figure 2C). The results of DNA
sequencing are completely consistent with expectations, demonstrating that all ten target gene fragments were
precisely integrated into pPICZα A.

Compared with bacteria, yeast is a eukaryote, which is more closely related to human beings, and the protein
expressed by yeast may be safer for human beings. Therefore, we chose *Pichia pastoris* as the strain for
protein
expression. The verified plasmids were then extracted from *E. coli* and transformed into *Pichia
pastoris* GS115
(Figure 2D). Further, in order to verify the correctness of the strain construction, we selected Single colony
from the plate for colony PCR and observed the results by gel electrophoresis. Consistent with expectations, all
candidates are the same size as expected (Figure 2B). In order to determine whether antibacterial peptides and
lysozyme can be expressed normally, we want to induce the strain and verify whether the protein can be expressed
normally after being transformed into yeast by SDS-PAGE.

![figure2](https://static.igem.wiki/teams/5742/images/results/figure2-1.webp)

Figure 2. Construction results of plasmid pPICZα A/antisepsis mix, pPICZα
A/Hepcidin, pPICZα A/Buforin II, pPICZα A/Piscidin-1, pPICZα A/LL-37, pPICZα A/Cecropin A, pPICZα A/Melittin,
pPICZα A/Plectasin, pPICZα A/hLYZ, pPICZα A/Lactoferricin. A: Transformation into DH5α after ligation; B: Colony
PCR results; C: Sequencing results; D: Successful transformation of correct plasmid into Pichia pastoris GS115.

Protein expression

**3.1 SDS-PAGE**

*Pichia pastoris* GS115 strains carrying the correct plasmid were cultured individually at 30℃ and induced
protein
expression with 10 g/L methanol, and the proteins were purified using His-Tag affinity chromatography. SDS-PAGE
was used to verify the protein expression of the target gene. As shown in Figure 3, different bands
corresponding to the expected molecular weight were clearly observed in the purified components, confirming the
accurate expression of each protein.

![figure3](https://static.igem.wiki/teams/5742/images/results/figure3-2.webp)

Figure 3. The SDS-PAGE results.

**3.2 Growth curve**

The expression of protein may affect the growth level of the strain. Therefore, in order to determine whether
the protein expression of antimicrobial peptides and lysozyme affects the normal growth of the strain, we tested
the growth curve of the strain with different antimicrobial peptide protein expressions. As shown in Figure 4,
all engineered strains exhibited good growth kinetics under the test conditions. This result shows that yeast
strains can grow normally and express the required antimicrobial peptides and lysozyme proteins under this
culture condition.

![figure4](https://static.igem.wiki/teams/5742/images/results/figure4-1.webp)

Figure 4. Growth curves of 10 recombinant strains.

Functional Test

**4.1 Minimum Inhibitory Concentration (MIC) determination**

We hope that through our experiments, antibacterial peptides with the strongest bactericidal effect can be
screened, so as to be combined with lysozyme to achieve the purpose of food preservation. First, we tested the
minimum inhibitory concentration of each antimicrobial peptide. The minimum antibacterial concentration (MIC) of
biological preservatives was determined by Broth microdilution method to evaluate their antibacterial activity.
Single colonies of the test strains E. coli and Bacillus subtilis were inoculated into LB medium for overnight
culture, and the resulting suspension was adjusted to 106 CFU mL-1 in fresh medium.
Dispense 100 μL of the
purified protein, from 128 to 2 μg·mL-1, into a 96-well microplate. Subsequently, 100 μL of
standardized
bacterial suspension was added to each well and the plates were incubated at 37℃ for 24 h. Wells containing only
sterile LB medium can serve as negative controls, while wells containing bacteria serve as positive controls to
confirm bacterial viability.

The MIC50 of the purified protein is shown in Table 1. From MIC results, we can see that Plectasin
has the
strongest antibacterial effect on both Escherichia coli and Bacillus subtilis. When the concentration of
Plectasin is 4 μg/ml, 50% bacteria can be killed. The MIC50 values of all antimicrobial peptides are
below 16
μg/ml, which indicates that all antimicrobial peptides involved in the experiment have certain antibacterial
activity. However, the MIC50 value of lysozyme is higher, this means the bacteriostatic effect of
lysozyme is
slightly weak, but the MIC50 value is also below 32 μg/ml.Thus lysozyme also has certain
bacteriostatic effect.

**Table 1. The Inhibitory concentration curve results.**

|  |  |  |
| --- | --- | --- |
| Drug | MIC50 | |
| *E. coli* | *Bacillus subtilis* |
| Cecropin A | 8 μg/ml | 16 μg/ml |
| Melittin | 8 μg/ml | 16 μg/ml |
| LL-37 | 8 μg/ml | 4 μg/ml |
| Plectasin | 4 μg/ml | 4 μg/ml |
| Piscidin-1 | 8 μg/ml | 4 μg/ml |
| Hepcidin | 8 μg/ml | 16 μg/ml |
| Lactoferricin | 8 μg/ml | 16 μg/ml |
| Buforin II | 8 μg/ml | 16 μg/ml |
| HLYZ | 16 μg/ml | 32 μg/ml |
| Antisepsis mix | 16 μg/ml | 16 μg/ml |

**4.2 Time-Kill curve assay**

Based on MIC results, we initially found that some antimicrobial peptides have good antibacterial effects, such
as LL-37 and Piscidin-1, among which Plectasin has the best antibacterial effects. In order to further verify
this result and find the most suitable antibacterial peptide, we tested the bactericidal effect of antibacterial
peptide after treatment for different times. Based on the experimental results of MIC, we found that some
bacteria could not be killed even if the concentration of antimicrobial peptides had risen to the highest
concentration (128μg/ml). Therefore, in order to ensure that antibacterial peptides can kill most bacteria, in
the subsequent experiments, we all use antibacterial peptides with a concentration of 128μg/ml for experiments.
The growth of bacteria at different time points was monitored by optical density method (OD600). By
drawing the
growth curve of bacteria treated with antimicrobial peptides, we can know the inhibitory effect of antimicrobial
peptides on bacteria and how long it takes for bacteria to recover their growth ability after antimicrobial
peptides treatment. Equal volumes of bio preservatives and bacterial suspensions were mixed and incubated at 30℃
for 24 h, and absorbance was measured at 0, 2, 4, 6, 8 and 24 h to determine the growth curve of bacteria under
the condition of adding different bio preservatives.

As shown in Figure 5, the addition of biological preservatives inhibited the growth of bacteria to varying
degrees. From the curve results, the inhibitory effect of some antibacterial peptides will gradually weaken with
the increase of time. In the experiment of inhibiting *Escherichia coli*, four antimicrobial
peptides(Cecropin A,
Lactoferricin, LL-37 and Plectasin) showed strong activity. With the increase of culture time, the growth of
bacteria was still inhibited to a great extent. In order to explore whether there is a combined effect between
antibacterial peptides and lysozyme, we mixed all antibacterial peptides with lysozyme and determined the
antibacterial activity of the mixed samples. The results show that the mixed samples have certain antibacterial
activity, but the combined effect is not significant, which may be because we mixed all antibacterial peptides,
some of which have poor antibacterial activity or antagonistic effect, resulting in poor mixed effect. Later, we
will consider the combination of two, so as to find the best combination scheme with antibacterial effect.
Compared with *Escherichia col*i, most antimicrobial peptides have better antibacterial effect on
*Bacillus*
*subtilis*. Notably, Plectasin showed the most significant inhibitory effects against *E. coli* and
*Bacillus*
*subtilis*.

![figure5](https://static.igem.wiki/teams/5742/images/results/figure5-1.webp)

Figure 5. Time-Kill curve. A: *E. coli*; B: *Bacillus subtilis*.

**4.3 Laser Scanning Confocal Microscope observation**

The bacterial suspension was incubated with different bio preservatives for a total of 10 h at 37℃, followed by
double staining of live and dead cells using Calcein-AM/PI. Calcein-AM is hydrolyzed by intracellular esterase
after entering the cell to produce a strong anionic green fluorescence product, thereby labeling the living
cells with green fluorescence. Propidium iodide (PI) cannot cross the cell membrane of living cells and only
enters when the membrane is damaged; It inserts into the DNA, producing a red fluorescence that labels dead
cells. This differential staining protocol labels both live and dead cells in all samples, and their
visualization under confocal microscopy provides a rapid quantitative assessment of the antimicrobial efficacy
of bio preservatives.

As shown in Figure 6, all tested bio preservatives exerted bactericidal activity against both Escherichia coli
and Bacillus subtilis, although the extent of killing varied markedly among treatments. Notably, exposure to
Plectasin or the LL-37 resulted in a pronounced reduction in B. subtilis viability, indicating potent
bactericidal efficacy against this species. Conversely, Cecropin A left a substantially higher residual viable
count of B. subtilis, revealing a comparatively weak activity against this bacterium. In contrast, Plectasin was
less effective against E. coli than against B. subtilis, whereas LL-37 displayed superior bactericidal
performance against E. coli relative to its effect on B. subtilis. Through the observation under confocal
microscope, we confirmed that our antimicrobial peptide protein has certain killing ability to bacteria.
Moreover, our lysozyme also has a certain bactericidal effect on Bacillus subtilis. Through the screening of the
above experiments, we determined that Plectasin and LL-37 are the best antibacterial peptides we have obtained
at present, and further we want to observe the morphology of the treated bacteria by scanning electron
microscope.

![figure6](https://static.igem.wiki/teams/5742/images/results/figure6-1.webp)

Figure 6. Laser Scanning Confocal Microscope results. A: Bacillus subtilis; B: E. coli; C: Bacterial mortality.

**4.4 Morphological observation of scanning electron microscope**

Further, we want to observe the morphology of bacteria treated with antibacterial peptides by scanning electron
microscope. We chose two antibacterial peptides (Plectasin and LL-37) which had the most remarkable effect in
the previous antibacterial experiment and chose AMP as the positive control. By scanning electron microscope, we
can see that compared with the bacteria not treated with antimicrobial peptides, the surfaces of Escherichia
coli and Bacillus subtilis were damaged to varying degrees after treated with antimicrobial peptides, which
means that our antimicrobial peptides may damage the cell wall or cell membrane structure of bacteria, thus
causing bacterial death. The specific bactericidal mechanism of antimicrobial peptides needs further analysis.

![figure7](https://static.igem.wiki/teams/5742/images/results/figure7-1.webp)

Figure 7. Scanning electron microscope observation results. A: *E. coli*; B: *Bacillus subtilis*.

Future plans

Through the above experiments, we determined that antibacterial peptides and lysozyme played an important role
in inhibiting bacterial growth, suggesting that antibacterial peptides and lysozyme may play a key role in
preventing food spoilage, and they can be used as important components of food preservatives. At the same time,
there are still some shortcomings in our experiments. Due to time constraints, some of our experiments have not
been done perfectly. First of all, due to the limitation of our protein purification process at present, the
purity of antibacterial peptides we obtained is not enough, so the protein concentration used in our experiment
may be inconsistent with the actual antibacterial concentration. In order to obtain more accurate results, we
need to optimize the protein purification process, so as to obtain higher purity protein.Secondly,at present,
our experiment only found that our antibacterial peptide can inhibit the growth of bacteria, and the specific
mechanism has not been resolved. Further, we may analyze the bactericidal mechanism of this antibacterial
peptide. Finally, we also need to obtain more proteins, so as to repeat the experiment many times to obtain more
accurate data. In the follow-up experiments, we will try to combine our antimicrobial peptides with lysozyme to
explore whether the combination of them can have better bactericidal effects.

In the future, the work will be carried out simultaneously at three levels: improving expression efficiency,
ensuring biosecurity, and promoting industrial implementation: the yield and activity of lysozyme and
antimicrobial peptides will be significantly improved through codon and promoter collaborative optimization, the
introduction of molecular chaperones, and the optimization of fermentation processes, and the synergistic
antimicrobial spectrum of the two against foodborne pathogens will be systematically evaluated. Construct
suicide switches that can induce suicide switches, carry out environmental release risk assessment,
immunogenicity and long-term impact research on intestinal flora to ensure that GMO hosts and products comply
with food safety regulations of various countries. On this basis, a low-cost continuous purification process and
a GMP-level quality control system are established to complete scale-up verification from laboratory to
industrial scale and realize commercial application.

References

```
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