iGEM 2025 — Team xmu-china

{% extends "layout.html" %}
{% block title %}Notebook{% endblock %}
{% block lead %}Document the dates you worked on your project. This should be a detailed account of the work done each
day for your project.{% endblock %}
{% block page\_content %}

![](https://static.igem.wiki/teams/5549/icon/loading.webp)

# NoteBook

Welcome to explore our iGEM 2025 Notebook, where every page bears our endless curiosity and exploration in science. Within these pages, you will discover not only experimental data and research progress, which document our scientific journey, but also the laughter of teamwork, the firm belief in overcoming challenges, and much more. This book is like a vibrant collection of stories, showcasing the spirit of XMU-China. We invite you to witness our growth and breakthroughs.

**Read Me →**

November, 2024-January, 2025

The XMU-China 2025 team officially kicked off recruitment! Over a hundred students
from different schools signed up to join us, diving into training on project design and exploring the wonders
of synthetic biology together. A vibrant and energetic team is gradually taking shape!

January-February, 2025

As the new year fireworks lit up the sky, we finalized
the formation of our team. Members got to know one another and made heartfelt wishes for the success of our
project this year!

![](https://static.igem.wiki/teams/5549/notebook/nb-fig1.webp)

February 23, 2025

Our very first offline brainstorming session! Although it was
the first time many of us met in person, everyone quickly grew comfortable.

![](https://static.igem.wiki/teams/5549/notebook/nb-fig3.webp)

We shared our project dreams over dinner, and later, the
leader and advisors led us in singing our hearts out at KTV. A night to remember!

![](https://static.igem.wiki/teams/5549/notebook/nb-fig4.webp)

March 23, 2025

Gathering again at Siming campus, Advisor Liuqin
Wu guided us on Human Practices— covering how to conduct interviews, design questionnaires,
and prepare as professional investigators. Everyone walked away with valuable skills.

![](https://static.igem.wiki/teams/5549/notebook/nb-fig5.webp)

March 30, 2025

Being a team from a coastal city, XMU-China
has always had a bond with the ocean. This year, we turned our eyes toward fish — a staple on our
dining tables — which often suffers from spoilage. We proposed developing an eco-friendly preservative.
Inspired by the sea itself, we started designing a project to extract a high-quality preservative from shrimp
and crab shells: "sourced from aquatic products, used for aquatic products."

![](https://static.igem.wiki/teams/5549/notebook/nb-fig2.webp)

April 4, 2025

Students from Siming and Xiang'an campuses gathered
in the chemistry building for lab training! Guided by our leader and advisors, we systematically
learned the basics of molecular cloning. One teammate exclaimed, "I finally became a synthetic
biology master — I can't wait to start my own experiments!"

![](https://static.igem.wiki/teams/5549/notebook/nb-fig6.webp)

April 5, 2025

A teammate Ruichen Shen designed a creative gene
circuit nicknamed the "Pop Quiz Suicide Switch". While worrying about their English test, the
team was also training hard to prepare for the upcoming Grand Jamboree.

![](https://static.igem.wiki/teams/5549/notebook/nb-fig7.webp)

April 6, 2025

XMU-China 2024 received an official commendation from
Xiamen University!

![](https://static.igem.wiki/teams/5549/notebook/nb-fig8-1.webp)

April 15, 2025

Today is our first pre experiment after the experimental
training, measuring the growth curve of the strain. Unfortunately, the first experiment did not go as
smoothly as we expected. Our culture was contaminated with other bacteria and had an unusual odor.

April 20, 2025

This is our second attempt,
and the growth of the strain is relatively normal, but the parallelism of the
experiment is not very good, and we still have room for improvement.

April 25, 2025

Teammate Yunxin Wang wrote that, in the morning,
four of us from Xiang'an filmed our team introduction video by Lake Furong. After some debate over
filming locations, we spotted black swans gliding across the water and decided to shoot with them
— the final footage turned out beautifully!

![](https://static.igem.wiki/teams/5549/notebook/nb-fig9.svg)

April 26, 2025

Can you believe it —
three teammates' birthdays all in the same week? After wrapping up our
project discussions at Xiang'an, we took a group photo and celebrated the birthdays
of Jiayue Qu, Jialin Chen, and Yanlinxin Guo with three custom cakes. What an
atmosphere! The birthday stars said: "We're so happy to be part of the XMU-China
family, may we always stay the best of friends!"

![](https://static.igem.wiki/teams/5549/notebook/nb-fig10.svg)

April 27, 2025

Today, we tested the bactericidal effects of
chitooligosaccharides with different degrees of polymerization on *E. coli*. The experiment required us to monitor
bacterial growth continuously for 24 hours, and students from the Siming campus took shifts to complete the task.

April 28, 2025

Teammate Jiaxuan Peng wrote that: First time sleeping in the laboratory! The final exams
are approaching and we are all very anxious, but now the gene circuit I designed urgently needs data to prove its feasibility, so I am staying up
late in the laboratory monitoring the growth status of the strains. Very exhausted, but finally obtained some valuable data!

April 30, 2025

Happily, the toxicity of the chitooligosaccharides we tested was similar to what
has been reported in the literature, but we need to conduct more experiments for further verification.

May 5, 2025

We attempted to verify the toxicity of chitooligosaccharides using LB agar plates containing
the oligosaccharides, but the results were not ideal, with significant variations between different parallel groups.

![](https://static.igem.wiki/teams/5549/notebook/nb-fig11.svg)

May 5, 2025

Students from the Xiang'an campus re-evaluated the bactericidal effect of chitooligosaccharides using
the spotting method on LB plates. This has provided an experimental foundation for our use of self-produced chitooligosaccharides in sterilization.

![](https://static.igem.wiki/teams/5549/notebook/nb-fig12.svg)

June 8, 2025

We had been struggling with the low solubility of chitosan, which made it difficult to
prepare high-concentration stock solutions for degradation experiments. Today, we attempted to pretreat chitosan with hydrogen peroxide and
successfully dissolved it!

![](https://static.igem.wiki/teams/5549/notebook/nb-fig13.svg)

June 12, 2025

After weeks of stubbornly refusing to assemble, our secreted-peptide LMT mutant finally ligated,
transformed, and sequenced perfectly. Cue the silent lab-wide happy dance (so we wouldn't wake the *E. coli*).

![](https://static.igem.wiki/teams/5549/notebook/nb-fig14.webp)

June 15, 2025

Based on our preliminary research, we selected six enzymes for degrading chitosan. Today, we
successfully expressed the first one!

![](https://static.igem.wiki/teams/5549/notebook/nb-fig15.svg)

June 20, 2025

To prepare for preservative testing, we made countless PCA agar plates — filling every corner
of the biosafety cabinet and cramming the fridge. Though exhausting, it felt so rewarding.

![](https://static.igem.wiki/teams/5549/notebook/nb-fig16.svg)

June 23, 2025

Today we used fish for our preservative experiments. The lab smelled faintly of the sea, but
the parallelism of our results improved significantly. It finally feels like our chitooligosaccharides is shining!

![](https://static.igem.wiki/teams/5549/notebook/nb-fig17.svg)

June 24, 2025

Today, we tested the degradation effect of the CsnMY002 enzyme,
but the results were unsatisfactory. We need to try more enzymes.

June 26, 2025

We warmly welcomed high school iGEMers from MammothEdu-South, exchanging project ideas and
sparking shared excitement for synthetic biology.

![](https://static.igem.wiki/teams/5549/notebook/nb-fig18.svg)

July 2, 2025

Our team member Yunxin Wang wrote: Since there were not enough beakers in the Xiang'an campus
laboratory to soak the fish, I improvised by soaking it directly in a sterile bag. With this adjustment, our preservation experiment achieved
full aseptic operation throughout the process. Hopefully, the results will be more accurate now.

![](https://static.igem.wiki/teams/5549/notebook/nb-fig19.svg)

July 5, 2025

The bacterial strains in the lab are having a competition to see which one
is greener. We are screening for suitable secretion peptide mutants based on their ability to secrete green fluorescent protein.

![](https://static.igem.wiki/teams/5549/notebook/nb-fig20.svg)

July 10, 2025

After a period of discussion and design, we decided to start attempting
random mutagenesis on SaCsn46A. However, we did not obtain the correct bands, so we need to adjust the random mutagenesis conditions.

July 11, 2025

Our team member Cairui Chen wrote: Doing TLC experiments is indeed a skilled task. Using
potassium permanganate method from organic laboratory for experiments will result in too large sample dot size of the product. We need to find
a suitable method specifically for chitooligosaccharides.

![](https://static.igem.wiki/teams/5549/notebook/nb-fig21.svg)

July 13, 2025

We celebrated teammate Keshuo Zheng's birthday!

![](https://static.igem.wiki/teams/5549/notebook/nb-fig22.svg)

July 14, 2025

The HPLC column pressure has been consistently high over the past two days. Although everyone
suspects macromolecules might be affecting the column, no clear improvement was observed regardless of the mobile phase used. With the help of her
senior, Meihan tried various methods, but the pressure remained stubbornly high. Eventually, even her senior concluded that the column might simply be
broken. Meihan was upset because this could delay the entire experiment. As a last resort, she decided to flush the column with the mobile phase once
more. In the early hours of the morning, when no one held much hope, the pressure finally began to drop!!! Meihan loaded her samples overnight, hoping
for good results the next day.

July 16, 2025

We attempted to use the aniline blue plate method to identify different chitosan
enzymatic hydrolysis products, but found little difference in the diffusion of chitooligosaccharides with different degrees of polymerization.
We need to find a new identification method.

![](https://static.igem.wiki/teams/5549/notebook/nb-fig23.svg)

July 20, 2025

After days of mutagenesis trials, using freshly prepared manganese chloride finally yielded
correct bands — a breakthrough, though mutation rates remain to be determined.

![](https://static.igem.wiki/teams/5549/notebook/nb-fig24.svg)

July 23, 2025

Today, we tried using the DNS method for the quantitative detection of chitooligosaccharides.
The solution turned a beautiful reddish-brown after the reaction—we succeeded!

![](https://static.igem.wiki/teams/5549/notebook/nb-fig25.svg)

July 25, 2025

We finally found the optimal developing solvent formulation for the TLC
experiment. Today, we used it to identify chitooligosaccharides with different degrees of polymerization, and the results met our expectations!

![](https://static.igem.wiki/teams/5549/notebook/nb-fig26.svg)

July 26, 2025

After a period of exploration, we have finally defined the experimental conditions for producing
chitosanase and degrading chitosan. We can now start testing the degradation capabilities of different enzymes!

![](https://static.igem.wiki/teams/5549/notebook/nb-fig27.svg)

July 27, 2025

Our teammate Nanqin Lin wrote We'd been stuck for days: co-electroporating our knockout fragment with
the editing plasmid just wouldn't take.

Our instructor Junhong suggested a two-step rescue—first
get both plasmids in, then add the fragment later.

I blurted out, *"But I've never pulled off a
double-antibiotic transformation…"*

He looked me dead in the eye and said, *"This time you will."*

Next morning—boom—double-resistant colonies on the plate.

Sometimes all it takes is a senior's faith and a fresh set of cuvettes.

July 31, 2025

Do you remember the "Pop Quiz Suicide Switch"? The creator of our
earlier gene circuit unveiled an upgraded version featuring: the "Wish Fulfilled" promoter, "Clear Mind" RBS, "Successful Experiment"
factor, and "Never-ending" terminator. With such a circuit, success feels guaranteed!

![](https://static.igem.wiki/teams/5549/notebook/nb-fig28.svg)

August 1, 2025

Our lab member Ruichen Shen wrote: Picked 88 mutants of CsnCA for colony PCR. Although
very few were successful, we still obtained over ten valuable mutants.

![](https://static.igem.wiki/teams/5549/notebook/nb-fig29.svg)

Our team member Cairui Chen wrote: Sometimes certain bacterial strains can be unpredictable
in experiments. They perform well in the first screening but fail in the second. Yes, we're looking at you, LMT variant M13L.

August 3, 2025

For the first time since summer started, one teammate finished experiments
before 10 pm! Of course, we celebrated with delicious food afterwards.

August 4, 2025

Our team member Yunxin Wang wrote: Today we successfully constructed the plasmid
containing the SaCsn46A mutants sequence. Thanks to Ruichen Shen for assisting with the transformation! We had several failed transformation
attempts before, but Ruichen Shen succeeded effortlessly. We truly appreciate his skilled hands in transformation!

![](https://static.igem.wiki/teams/5549/notebook/nb-fig30.svg)

August 5, 2025

Our team member Cairui Chen wrote: This is our first time conducting a preservation
experiment on fruit! The chilled blueberries were delicious, but not very suitable for the experiment, as the properties varied too much between
individual blueberries. We will select other fruits.

And our lab member Ruichen Shen wrote: The lab ran out of TAE buffer, and no one could run gels.
I tried making the stock solution myself, and it worked exceptionally well.

August 6-8, 2025-CCiC

Off to the China Conference on iGEM Competition we went!
Swapped stickers, pins, and stories with teams from ZZU, XJTLU, ZJU, and more.
Our poster has received praise and likes.
Fellow iGE*M*ers loved our seafood-preservation angle and tossed us fresh ideas
We soaked up keynote talks from professors and biotech CEOs, then raced
back to the hotel to scribble down every last insight.
Three days, a suitcase full of new friends, and brains bursting with inspiration—mission accomplished!

![](https://static.igem.wiki/teams/5549/notebook/nb-fig31.webp)

August 10, 2025

With the help of students from the Xiang'an campus, our progress has accelerated
significantly. The enzymes SaCsn46A and CsnCA tested today showed pr*o*mising degradation effects.

August 11, 2025

Our team member Keshuo Zheng wrote: Today was a long day! One member
of our team simultaneously conducted molecular cloning experiments and monitored cell growth status. Even though things got a bit hectic,
we still achieved successful outcomes in the end!

![](https://static.igem.wiki/teams/5549/notebook/nb-fig32.svg)

August 11, 2025

Today, we tried using the Hammer Bacterial Lysate to lyse the bacterial strains.
It was expensive, and we had high h*o*pes for it, but its performance was disappointing. We still need to use the ultrasonic instrument.

![](https://static.igem.wiki/teams/5549/notebook/nb-fig33.svg)

August 14, 2025

The typhoon is about to hit, and Jiaxuan and Meihan, who stayed up
late working on their experiment, decided to bunk overnight in the break room—494, zzzzz...

Luckily, their lab buddies left some snacks behind—munch, munch, munch... zzz

Who's to say this isn't a modern twist o*n* "Willow-lined shore, dawn breeze, fading moon"?

("Willows"—by the shore, howling typhoon, not a *moon* in sight...)

August 16, 2025

Today's experimental results were somewhat disheartening. We found that constructing enzyme mutants in the pSB1C3
vector rarely produced the correct plasmids, indicating we might need to change the vector.

August 20, 2025

The pET-28a(+) vector came to our rescue! Today, several McChoA
enzyme mutants we constructed showed a high rate of correct bands.

Team member Keshuo Zheng wrote: Today we wrapped up the LMT improvement experiments.
We've successfully validated the mutation effect of C14A and obtained promising data!

![](https://static.igem.wiki/teams/5549/notebook/nb-fig34.svg)

August 22, 2025

After several days of effort, we finally obtained over 100 bacterial strains
containing SaCsn46A enzyme mutants. We have decided to start the "Great Enzyme Screening" experiment soon.

![](https://static.igem.wiki/teams/5549/notebook/nb-fig35.svg)

August 24, 2025

Today was a big day for our XMU-China iGEM team! We held a science outreach event at the Xiamen
Science and Technology Museum, themed "sourced from aquatic products, used for aquatic products" Our goal was twofold: to spark curiosity in kids and teens about the hidden
value in shrimp and crab shells, teaching them to appreciate these marine resources and understand the importance of seafood freshness and safety. On
the other hand, we were thrilled to share our team's innovative solution for recycling crustacean waste, offering the public a glimpse into the amazing
potential of synthetic biology to make a difference.

![](https://static.igem.wiki/teams/5549/notebook/nb-fig36.svg)

August 26, 2025

Our "Great Enzyme Screening" experiment started today. We carried out screening of 104 variants
of the randomly mutated chitosanase. The workload was tremendous, yet everyone worked together seamlessly, passing the tasks along like a relay.
Step by step, we pushed through — and finally, we were rewarded with solid results.

![](https://static.igem.wiki/teams/5549/notebook/nb-fig37.svg)

September 1, 2025

Our lab member Ruichen Shen wrote: The new semester has begun! We got a new ice box and drew
little caricatures of our team members on it.

![](https://static.igem.wiki/teams/5549/notebook/nb-fig38.svg)

September 2, 2025

Today, while preparing to purify proteins, Meihan discovered that
the AKTA protein purification system she usually uses was out of order. As a result, she had to seek help from others.
Fortunately, Advisor Yanhao Chen provided extensive assistance. He guided Meihan in using a new protein purification system
and successfully helped her purify the proteins. Grateful for such a supportive team!

September 3, 2025

Our team member Nanqin Lin, who is exploring the construction of a
suicide switch, wrote: The issue that had been troubling us for almost two weeks—where the Donor for genomic insertion
consistently showed 2000 bp in PCR—has finally been resolved! It turned out that the first and fourth segments were
preferentially connecting. We succeeded by performing the connections in batches!

September 7, 2025

Our teammate Cairui Chen wrote: Today, under the guidance of our advisor Ziyan Han,
I successfully learned how to use our lab's new AKTA system to purify the protein SaCsn46A mutants. It was incredibly rewarding to see
the solution turn blue during the Bradford assay test—a clear sign of successful purification!

September 10, 2025

Our team member Yunxin Wang wrote: Today, we finalized the use of CFU/g as the metric to evaluate
the preservation effect in our experiment. To prepare for this, we made a large number of PCA plates. My skills in pouring and arranging plates have
now been fully mastered!

![](https://static.igem.wiki/teams/5549/notebook/nb-fig39.svg)

September 11, 2025

We've occupied almost all the instruments in the lab that can heat liquids, because
today we set up many different temperatures to test enzyme stability across various conditions.

Our team member Cairui Chen wrote: Testing enzyme activity under different pH conditions
is not easy. We've used up a lot of pH test strips just to calibrate the pH correctly.

September 12, 2025

Our team member Yunxin Wang wrote: Today, we attempted to use chitosan degradation
products for fish preservation for the first time. While buying the fish, we encountered an incredibly cute puppy at the store. From now on,
I'll always buy fish from this supermarket!

![](https://static.igem.wiki/teams/5549/notebook/nb-fig40.svg)

September 15, 2025

Our teammate Jiaxuan Peng wrote: We ran multiple enzyme kinetics experiments
today, but all failed. When chitosan was not treated with hydrogen peroxide, the reducing sugar background was very low, making it
impossible to detect any enzymatic activity. After treating chitosan with hydrogen peroxide, the reducing sugar background showed a direct
linear relationship with substrate concentration, yet we still couldn't observe any enzyme effect and have to give up this experiment. What a
disheartening day!

![](https://static.igem.wiki/teams/5549/notebook/nb-fig41.svg)

September 17, 2025

In Xiang'an Campus, team member Jiayue Qu wrote: While conducting experiments to
test the effectiveness of secreted proteins for the POC section, we obtained exceptionally clean and clear protein gel results! The outcomes
are closely aligned with our expectations — truly exciting!

![](https://static.igem.wiki/teams/5549/notebook/nb-fig42.svg)

September 18, 2025

We prepared a large amount of secreted chitosanase and used it to degrade chitosan. With these
preparations in hand, we are now ready to launch the preservative experiment to verify the preservation effect of our mutants.

![](https://static.igem.wiki/teams/5549/notebook/nb-fig43.svg)

September 19, 2025

A strange smell has appeared in the lab? Where is it coming from? Oh no, it's from the bacterial
contamination of the chitosan degradation products in the supernatant from our POC experiment. We need to repeat the experiment.

September 25, 2025

We used the protein obtained from yesterday's experiment to degrade chitosan. The TLC and HPLC
results confirmed that the protein has good degradation activity!

September 27, 2025

Our team member Nanqin Lin, who is exploring the construction of a suicide switch, wrote:
After changing the promoter, we finally detected the correct band for the genomically inserted bacteria! I'm genuinely thrilled! Although
subsequent sequencing revealed that the *ccdB* segment was still missing...

![](https://static.igem.wiki/teams/5549/notebook/nb-fig44.svg)

September 28, 2025

Our team member Cairui Chen wrote: Today, Ruichen Shen suggested that we adjust
the dilution factor for tomorrow's experiment on the preservation of chitosan degradation products. Oh, but he forgot that the last plating
was already done yesterday—by tomorrow we'll have all the data. Poor guy, his brain must be fried from staying up late writing the results
section last night.

September 29, 2025

Our lab member Ruichen Shen wrote: Completed the first colony count test
for the preservation experiment using degradation products. The preservation effect of the degradation products is remarkably good!

October 1, 2025

Today, Meihan pulled an all-nighter for LCA calculation. During a break, she went to witness
the breaking dawn: the first light piercing through the sky, the early mist rising, kites soaring high, and winds sweeping across the horizon.
She reminisced about verses of journeys, turbulence, and perseverance, and wished for a smooth wiki freeze.

![](https://static.igem.wiki/teams/5549/notebook/nb-fig45.svg)

October 8, 2025

Wiki Freeze!

{% endblock %}

{% extends "layout.html" %}
{% block title %}Engineering Success{% endblock %}
{% block lead %}Demonstrate engineering success in a technical aspect of your project by going through at least one iteration of the engineering design cycle. This achievement should be distinct from your Contribution for Bronze.{% endblock %}
{% block page\_content %}

![](https://static.igem.wiki/teams/5549/icon/loading.webp)

[Overview](#section1)

[Part 1](#section2)

[Introduction](#section2-1)

[Cycle 1](#section2-2)

[Cycle 2](#section2-3)

[Cycle 3](#section2-4)

[Cycle 4](#section2-5)

[Conclusion](#section2-6)

[Part 2](#section3)

[Introduction](#section3-1)

[Cycle 1](#section3-2)

[Cycle 2](#section3-3)

[Cycle 3](#section3-4)

[Cycle 4](#section3-5)

[Conclusion](#section3-6)

[References](#section4)

![Shrimp](https://static.igem.wiki/teams/5549/headline/headline-shrimp.webp)

# Engineering Success

![Crab](https://static.igem.wiki/teams/5549/headline/headline-crab.webp)

## Overview

This year, XMU-China has focused on advancing the development of chitooligosaccharides (COS) as a novel preservative and achieved the expected outcomes in this field (see our [Results](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27results%27%29%20%7D%7D) for more details).

To ensure our design meets the intended goals, we have made substantial efforts. On one hand, we conducted engineering on the LMT signal peptide to further improve its secretion performance. On the other hand, we verified the antimicrobial effect of degradation products catalyzed by chitosanase through multiple rounds of screening, and finally obtained the chitosanase that can efficiently degrade chitosan into COS with stronger antimicrobial activity. All relevant data have been documented, and we hope they can contribute to the iGEM community.

## Part 1: Engineering LMT signal peptide for improving secretion performance

## Introduction

The secretion of cellular proteins is a core process in protein biosynthesis and secretion mechanisms, holding profound significance for cellular function regulation and the biopharmaceutical field (1). Current research widely suggests that the secretion of most proteins relies on the mediation of specific signal peptides. Therefore, exploring and optimizing signal peptide sequences to enhance the secretion efficiency of target proteins, thereby increasing protein production yields, has become a key strategy for improving the economic benefits of protein biopharmaceuticals (1). Based on the efforts of our previous teams ([XMU-China 2021 SALVAGE](https://2021.igem.org/Team%3AXMU-China), [XMU-China 2022 OMEGA](https://2022.igem.wiki/xmu-china/index.html), [XMU-China 2023 NAIADS](https://2023.igem.wiki/xmu-china/index.html), [XMU-China 2024 REPARO](https://2024.igem.wiki/xmu-china/index.html)), our study is dedicated to the improvement of secretion performance of the existing signal peptide LMT ([BBa\_K5136066](https://parts.igem.org/Part%3ABBa_K5136066)), achieving higher concentrations of secreted recombinant proteins.

## Cycle 1: Identifying sequence features of LMT signal peptide

### Design and Build

The secretion of most proteins is primarily achieved through the General Secretion (Sec) pathway (Figure 1A) (2). The typical sequence structure of signal peptides that guide secretion *via* this pathway is illustrated in Figure 1B. In this pathway, proteins first bind to SecB *via* the **positively charged N-region** of their signal peptide while SecB inhibits premature folding of the protein. Subsequently, SecA recognizes the **hydrophobic H-region** of the signal peptide and translocates the protein to the cell membrane, in which the SecYEG translocon acts as a channel, guiding the protein to the periplasmic space. After the signal peptidase (SPase) recognizes and cleaves at the **cleavage site (AXA) in the C-region** of signal peptide, the protein then is correctly folded in the periplasm (3).

![fig1](https://static.igem.wiki/teams/5549/engineeringsuccess/figure1.webp)

**Figure 1 Sec pathway-dependent secretion process and the corresponding sequence structures of signal peptide.** (**A**) Secretion of recombinant proteins through Sec pathway. (**B**) The typical sequence features of signal peptide can be identified in the sequence of LMT.

The LMT signal peptide we discovered has the typical sequence features of signal peptide, which can be divided into 3 regions described above as well (Figure 1B). Thus, we turned to investigate the influence of some specific residues in this signal peptide on protein (here, superfolder green fluorescent protein (sfGFP)) secretion. Two specific variants of the N-terminal positively charged region (presumed to be the SecB binding region) and the C-terminal AXA cleavage site (SPase recognition site) were then constructed, namely **LMT Δ2-4** and **LMT A19V**, respectively (Figure 2A). The **sfGFP**(BBa\_K5136028) was chosen as the **cargo** protein to characterize the secretion performance of different variants. Gibson Assembly was performed to construct the expression module ([BBa\_25G6VM6I](https://registry.igem.org/parts/bba-25g6vm6i) and [BBa\_25CWGZSX](https://registry.igem.org/parts/bba-25cwgzsx), respectively) of these variants at pSB1C3 vector in *E. coli* DH5α and positive transformants were selected and confirmed by colony PCR (Figure 2B) and sequencing.

![fig2](https://static.igem.wiki/teams/5549/engineeringsuccess/figure2.webp)

**Figure 2 Construction of the expression modules of LMT Δ2-4 and A19V.** (**A**) Sequence alignments of LMT and two variants. Mutated sites were colored in black. (**B**) DNA gel electrophoresis of the colony PCR products of expression modules of LMT Δ2-4 and A19V fused with *sfgfp* at pSB1C3 in DH5α. Target bands, 1383 bp and 1374 bp, can be observed at the position between 1500 bp and 1000 bp, respectively.

### Test

![fig3](https://static.igem.wiki/teams/5549/engineeringsuccess/figure3.avif)

**Figure 3 Probe the key *secA* and *secB* genes in *E. coli* DH5α *via* colony PCR.** DNA gel electrophoresis of colony PCR products to probe *secA* (**A**) and *secB* (**B**). Target bands, 2706 bp and 468 bp, can be observed at the position around 3000 bp and 500 bp, respectively. Specific primers were designed according to the genome sequence of DH5α ([GCF\_002899475.1](https://www.ncbi.nlm.nih.gov/datasets/genome/GCF_002899475.1/)).

Before characterization, we confirmed the presence of the key genes *secA* and *secB* of the Sec pathway in the chassis *E. coli* DH5α *via* colony PCR (Figure 3), which set the foundation of downstream works and tests.

![fig4](https://static.igem.wiki/teams/5549/engineeringsuccess/figure4.avif)

**Figure 4 Characterization of secretion performance of LMT and two variants.** The OD600 (**A**), fluorescence intensity of culture (**B**) and supernatant (**C**) was measured as time progressed. Relative fluorescence units (RFU) was defined as the fluorescence of bacterial culture subtracted the background fluorescence of growth media.

We then conducted the characterization experiments in *E. coli* DH5α. After inoculation, the fluorescence intensity (*λ*ex = 475 nm, *λ*em = 515 nm) of the culture and the supernatant after centrifugation as well as the OD600 of each group were monitored as time progressed. Our results showed that the fluorescence intensity of the two variants, no matter for the culture or supernatant, was much lower than that of the wild-type (WT) LMT group (Figure 4B and 4C), although no significant growth defect due to the modifications was observed (Figure 4A).

### Learn

Colony PCR yielded some non-specific bands, and the brightness of the *secB* bands were not that satisfying. Therefore, to obtain more unique *secA* and *secB* fragments in the future, the PCR conditions may need to be adjusted or the primers should be redesigned. Nevertheless, we successfully validated the presence of *secA* and *secB* genes.

Characterization experiments revealed that both the reduction of positive charges in N-region (LMT Δ2-4) and mutation on SPase cleavage site (A19V) are “**lethal**” to the function of LMT signal peptide, which highlights the importance of **critical interactions** between transportation machinery of the host and signal peptide during secretion process. Notably, the LMT Δ2-4 variant exhibited slightly higher culture and supernatant fluorescence intensity compared to A19V. Although the Δ2-4 variant deletes a significant portion of the positively charged amino acids in N-region, it only reduces, rather than completely blocks, the binding of SecB to the signal peptide. Therefore, the signal peptide may retain some degree of Sec pathway transport capability. However, the A19V variant disrupts the crucial AXA cleavage site, significantly impairing the specific recognition and cleavage by the SPase. This could even block the entire Sec secretion pathway's function, thereby greatly inhibiting the secretion of the cargo protein. Additionally, the folding of cargo sfGFP might be interfered due to these two modifications, which finally resulted in the lower fluorescence intensity of bacterial culture.

The importance of the N-terminal positively charged region and the AXA cleavage site of LMT signal peptide was validated. Based on the mechanistic understanding, we planned to design and construct a series of sequence mutants for the purpose of screening variant(s) with higher-performance on protein secretion.

## Cycle 2: Enhancing the hydrophobicity of the H-region of LMT signal peptide

### Design

Extensive literature reviews indicated that one of the crucial factors for signal peptides to successfully mediate protein secretion is their overall hydrophobicity, especially the hydrophobicity of the H-region core (2-4). This hydrophobicity is directly correlated with the efficiency of the signal peptide in traversing the hydrophobic biological membrane. Aiming to strengthen the interactions between the cell membrane and signal peptide to promote protein secretion, we focused on enhancing the hydrophobicity of the LMT signal peptide's H-region.

### Build

We designed 4 LMT variants: **G12L**, **M13L**, **C14L**, and **A15L**, which replaced the residue in H-region with the **hydrophobic leucine (L) residue** (Figure 5A). Using the same method mentioned before, we constructed a series of expression modules of single-point mutants ([BBa\_25PJKLRW](https://registry.igem.org/parts/bba-25pjklrw), [BBa\_25HA3X2Z](https://registry.igem.org/parts/bba-25ha3x2z), [BBa\_25IFJMYS](https://registry.igem.org/parts/bba-25ifjmys) and [BBa\_25A752GX](https://registry.igem.org/parts/bba-25a752gx)) and confirmed by colony PCR (Figure 5B) and sequencing.

![fig5](https://static.igem.wiki/teams/5549/engineeringsuccess/figure5.avif)

**Figure 5 Targeted mutations in H-region of LMT signal peptide.** (**A**) Sequence alignments of LMT and 4 variants. Mutated sites were colored in black. (**B**) DNA gel electrophoresis of the colony PCR products of expression modules of LMT variants fused with *sfgfp* at pSB1C3 in DH5α. Target bands (1383 bp) can be observed at the position between 2000 bp and 1000 bp. (**C**) Each variant's fluorescence intensity of supernatant of 10 h was normalized to LMT WT, for the first characterization. (**D**) Fluorescence intensity of supernatant of M13L variant with LMT WT was measured as time progressed, for the second characterization.

### Test

The same characterization method was utilized. For the first time of characterization, the LMT G12L and A15L variants showed lower fluorescence intensity of supernatant than wild-type LMT (Figure 5C). Given the poor reproducibility observed in C14L group, we evaluated M13L variant **only** for the second time of characterization with LMT as control, wondering whether the improvement was true or not. However, as time progressed, the M13L variant presented a little bit lower fluorescence intensity of supernatant than the wild-type (Figure 5D), inconsistent with the previous observation.

### Learn

It is evident that not all mutations that enhance hydrophobicity will certainly improve secretion efficiency. In addition, there are other hydrophobic residues to be tested. An optimal range of hydrophobicity might exist, and exceeding this range may negatively affect cargo protein's folding, transmembrane transportation, or other crucial steps. Taking together, the strategy of enhancing the hydrophobicity of the H-region core might not be so feasible and robust for the case of LMT signal peptide. Therefore, we turned to consider the mutations in the C-region of LMT.

## Cycle 3: Increasing the number of Q residues in C-region of LMT signal peptide

### Design and Build

Demonstrated through high-throughput experiments, the number of glutamine residues (Q) in the C-region of most signal peptides is positively correlated with secretion efficiency (4). Therefore, we designed to increase the number of glutamine residues in the C-region of LMT and constructed four C-terminal point mutants (Figure 6A), **A15Q**, **S16Q**, **A17Q**, and **F18Q** as well as the corresponding expression modules ([BBa\_25C7FG1O](https://registry.igem.org/parts/bba-25c7fg1o), [BBa\_25CR4ZPR](https://registry.igem.org/parts/bba_25cr4zpr), [BBa\_25MZ2ZUA](https://registry.igem.org/parts/bba_25mz2zua) and [BBa\_25JOOXIU](https://registry.igem.org/parts/bba_25jooxiu)). The same cloning method was implemented and positive transformants were confirmed by colony PCR (Figure 6B) and sequencing.

![fig6](https://static.igem.wiki/teams/5549/engineeringsuccess/figure6.avif)

**Figure 6 Construction of the expression modules of the variants increasing the number of glutamine residues in the C-region.** (**A**) Sequence alignments of LMT and four variants. Mutated sites were colored in black. (**B**) DNA gel electrophoresis of the colony PCR products of four variants' expression modules fused with *sfgfp* at pSB1C3 in DH5α. Target bands (1383 bp) can be observed at the position between 1500 bp and 1000 bp.

### Test

In characterization, none of the variants impaired the cell growth (Figure 7A). However, even though some variants (except A17Q) presented higher culture fluorescence intensity than the wild-type LMT (Figure 7B), all variants showed much lower fluorescence intensity of supernatant than LMT (Figure 7C), which means no improvements of these variants in secretion performance.

![fig7](https://static.igem.wiki/teams/5549/engineeringsuccess/figure7.avif)

**Figure 7 Characterization of secretion performance of LMT and the variants increasing the number of glutamine residues in the C-region.** The OD600 (**A**), fluorescence intensity of culture (**B**) and supernatant (**C**) was measured as time progressed.

### Learn

The current findings indicated that increasing the number of Q residues in C-region may not effectively improve the secretion performance of LMT signal peptide. Especially, A17Q mutation disrupts the AXA cleavage site like A19V variant, thus resulting in the same “abortive” phenomenon of fluorescence intensity. With the failure experience of increasing the number of glutamine residues in the C-region, we then turned to focus on the key factor for SPase cleavage efficiency–the composition of residues in C-region (4).

## Cycle 4: Increasing the number of A residues in LMT signal peptide

### Design and Build

It was reported that the number of alanine (A) residues at the C-terminus of the signal peptide might be closely related to the cleavage efficiency of SPase, thus influencing secretion efficiency (4). Therefore, we hypothesized that increasing the number of C-terminal alanine residues could effectively enhance SPase cleavage efficiency of the signal peptide, resulting in improved secretion performance. We designed three C-terminal point mutants, **M13A**, **C14A**, and **F18A** (Figure 8A) and constructed corresponding expression modules ([BBa\_257PMILQ](https://registry.igem.org/parts/bba_257pmilq), [BBa\_258JKPVF](https://registry.igem.org/parts/bba_258jkpvf) and [BBa\_25VUQPBL](https://registry.igem.org/parts/bba_25vuqpbl)) like before (Figure 8B).

![fig8](https://static.igem.wiki/teams/5549/engineeringsuccess/figure8.avif)

**Figure 8 Increase the number of alanine residues in LMT.** (**A**) Sequence alignments of LMT and 3 variants. Mutated sites were colored in black. (**B**) DNA gel electrophoresis of the colony PCR products of expression modules of LMT variants fused with *sfgfp* at pSB1C3 in DH5α. Target bands (1383 bp) can be observed at the position between 1500 bp and 1000 bp. (**C**) Fluorescence intensity of the supernatant was measured as time progressed. (**D**) Fluorescence intensity (at 10 h) of culture and supernatant of C14A and LMT WT were compared, for the second time of characterization. *p*-value: 0.0008 (\*\*\*) and 0.0098 (\*\*).

### Test

As time progressed, the M13A and F18A variants showed a little bit higher fluorescence intensity of supernatant than wild-type LMT, while it can be easily observed that the **C14A** variant's performance was **superior** to other variants and even the wild-type LMT (Figure 8C). To confirm whether the improvement was true or not, we conducted the characterization experiments of LMT C14A for the second time with LMT as control. Stronger fluorescence intensity of both bacterial culture and supernatant was observed for the C14A variant when compared to the wild-type LMT signal peptide (Figure 8D), validating the effectiveness of this mutation in improving secretion performance.

### Learn

The successful construction and characterization of the high-performance C14A variant confirmed the hypothesis that increasing the number of C-terminal A residues might effectively enhance the function of signal peptide to direct proteins out of the bacterial cell. The results (Figure 8C and 8D) suggested that the C14A mutation might promote protein secretion through mechanisms such as boosting SPase cleavage efficiency or optimizing the signal peptide conformation. Thus, C14A represents a promising optimization site for the LMT signal peptide with potential application values. Future studies should focus on an in-depth exploration of the SPase mechanism and the structure-activity relationship of C14A.

## Conclusion

Through engineering iterations, we have identified the sequence features of LMT signal peptide and conducted several rounds of targeted mutations for the purpose of screening higher-performance variant(s) that can be used to secrete more recombinant proteins produced by the microbial cell factories. Ultimately, we successfully obtained an outstanding variant LMT C14A, indicating that increasing the number of C-terminal alanine residues can effectively promote protein secretion to some extent. In addition, this outstanding variant was further applied in secreting chitosanase for food preservation (see [Proof of Concept](https://2025.igem.wiki/xmu-china/poc) for details), tightly linking the idea of iteration with our project.

In summary, engineering a biological system cannot be easy. We need to put our heart into it and move towards success through experiments and feedbacks step by step. Here, we have tried to demonstrate **how we engineered LMT signal peptide for improving secretion performance** through successive iterations of the **DBTL** (Design-Build-Test-Learn) cycle (Figure 9).

![fig9](https://static.igem.wiki/teams/5549/engineeringsuccess/figure9.webp)

**Figure 9 Summary of the different engineering steps completed before reaching the final goal of obtaining higher-performance variant(s) of LMT signal peptide.**

## Part 2: Screening for chitosanase to degrade chitosan to the products with the optimal antibacterial effect

## Introduction

COS can act as a natural preservative (5), as they inhibit the growth of microorganisms on food surfaces, thereby delaying the spoilage process of aquatic products. In our experiments, we found that COS with different degrees **of polymerization (DP) exhibit distinct antibacterial activities** (6). Based on this feature, we aim to engineer chitosanase to ultimately obtain chitosanase that degrades chitosan to the products with the optimal antibacterial effect, which will be used in further preservation experiments.

## Cycle 1: Evaluation of wild-type chitosanases

### Design

To screen for high-efficiency chitosanases, we selected 5 reported endo-chitosanases (BamCsn, CsnMY002, McChoA, SaCsn46A, CsnCA) (7-10) and 1 exo-chitosanase (GlmATk) (11) based on the characteristics of chitosan hydrolysis products (see our [Design](https://2025.igem.wiki/xmu-china/design) for details). We aimed to obtain the initial antimicrobial activity of the degradation products from each enzyme through preliminary toxicity tests, and then select one or several of these enzymes for random mutagenesis.

### Build

**Table 1 The part information of six wild-type chitosanases.**

| Name | Type | Description |
| --- | --- | --- |
| [BBa\_25E8EHJF](https://registry.igem.org/parts/bba-25e8ehjf) | coding | His tag-*BamCsn* |
| [BBa\_25JJYAF6](https://registry.igem.org/parts/bba-25jjyaf6) | coding | His tag-*CsnMY002*-His tag |
| [BBa\_25JA5N69](https://registry.igem.org/parts/bba-25ja5n69) | coding | His tag-*Tk Glm* |
| [BBa\_25RYX5T9](https://registry.igem.org/parts/bba-25ryx5t9) | coding | His tag-*CsnCA WT*-His tag |
| [BBa\_25WTOTOC](https://registry.igem.org/parts/bba-25wtotoc) | coding | *ChoA WT* |
| [BBa\_252ULD2I](https://registry.igem.org/parts/bba-252uld2i) | coding | *SaCsn46A WT* |

The targeted sequences BBa\_25E8EHJF, BBa\_25JJYAF6, BBa\_25JA5N69, BBa\_25RYX5T9, BBa\_25WTOTOC, BBa\_252ULD2I were individually inserted into pET-28a(+) to construct expression plasmids (Table 1), which were then transformed into *E. coli* BL21(DE3) to express each enzyme. The positive transformants were confirmed by kanamycin, colony PCR (Figure 10), and sequencing.

![fig10](https://static.igem.wiki/teams/5549/engineeringsuccess/es-figure-10.avif)

**Figure 10 DNA gel electrophoresis of the colony PCR products of BamCsn (A), CsnMY002 (BA), GlmATk (C), CsnCA (D), McChoA (E) and SaCsn46A (F) at pET-28a(+) in *E. coli* BL21(DE3).** Target bands of BBa\_25JA5N69 can be observed at the position between 2000 bp and 3000 bp, the other target bands can be observed at the position between 1000 bp and 2000 bp.

### Test

The expression of chitosanases was confirmed by SDS-PAGE analysis (Figure 11). Chitosan powder was added to the lysate supernatant of the expression system, followed by incubation at 37 °C for 18 h. The resulting supernatant was collected as the degradation product. The degradation product was co-cultured with the test strain (*E. coli* BL21(DE3), OD600 ~0.6) at a 1:1 ratio. The OD600 of the co-culture system was measured at 2 h and 8 h. The antibacterial activity of the degradation products treated by the enzymes was evaluated using the **relative ΔOD600 value**, calculated as (OD600 at 8 h − OD600 at 2 h) / OD600 at 2 h. The empty vector pET-28a(+) without expressing any chitosanases was set as control.

![fig11](https://static.igem.wiki/teams/5549/engineeringsuccess/figure11.avif)

**Figure 11 SDS-PAGE analysis of BamCsn (A), CsnMY002 (B), GlmATk (C), CsnCA (D), McChoA (E) and SaCsn46A (F).** Target bands can be observed between 25 kDa and 35 kDa, while the target bands of GlmATk can be observed between 80 kDa and 90 kDa.

Based on the results (Figure 12), we evaluated the antibacterial activity of the enzymatic degradation products from all six chitosanases. Among them, the enzymatic degradation products of CsnMY002, CsnCA, McChoA, SaCsn46A displayed relatively higher antibacterial activity.

![fig12](https://static.igem.wiki/teams/5549/engineeringsuccess/figure12.avif)

**Figure 12 Antibacterial activity of chitosan degradation products of wild-type BamCsn, CsnMY002, GlmATK, CsnCA, McChoA and SaCsn46A.** Relative ΔOD600 values were normalized to that of the empty vector pET-28a(+) group.

### Learn

From the results of the first-round test, we found that the degradation products *via* two chitosanases (BamCsn and GlmATk) showed no antimicrobial effect. In the GlmATk group, the growth of the test bacteria was even stronger than that in the control group. We speculated that this was because the exo-chitosanase property of GlmATk produces a large amount of glucosamine, which promotes the growth of the bacterial strain instead of inhibition. Therefore, we subjected SaCsn46A, CsnCA, CsnMY002 and McChoA, which showed good performance in the first-round test to **error-prone PCR (EP-PCR)** to obtain a large library of mutants, followed by a second-round screening.

Meanwhile, we optimized some conditions for our toxicity test: we adjusted the OD600 value of the bacterial strain to 0.8 before incubating with the degradation products, and fixed the detection time points at 1.5 h and 8 h. These adjustments ensured the standardized operation of the experiment and avoided random errors.

## Cycle 2: Random mutagenesis of chitosanase and large-scale preliminary screening

### Design and Build

After the first-round test, we selected SaCsn46A, CsnCA, CsnMY002, and McChoA for EP-PCR followed by screening (see our [Experiments](https://2025.igem.wiki/xmu-china/experiments) for more details), aiming to accumulate extensive data on the toxicity of enzymatic degradation products. During this stage, we generated a large library of mutants, and the relevant agarose gel electrophoresis results can be found in our [Results](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27results%27%29%20%7D%7D) section.

### Test

The resulting colonies from EP-PCR were all picked and the antibacterial assay was performed again with some adjusted conditions as described in Cycle 1.

### Learn

The results showed that the degradation products of some mutants exhibited **strong antibacterial activity**
(Figure 13), which was consistent with our expectations. We selected five mutants, namely [SaCsn46A mutant 2](https://registry.igem.org/parts/bba-25c3no7w) (SaCsn46A E151G D222G),
[SaCsn46A mutant 14](https://registry.igem.org/parts/bba-25w5agtg) (SaCsn46A N29S K50R D150G D189G F247L),
[SaCsn46A mutant 16](https://registry.igem.org/parts/bba-25vruxtc) (SaCsn46A V52A D134G K165I F175L S190C G210D K250E N252D),
[SaCsn46A mutant 60](https://registry.igem.org/parts/bba-25uuairi) (SaCsn46A D40G Q125R H234R), and
[SaCsn46A mutant 94](https://registry.igem.org/parts/bba-254p0m23) (SaCsn46A T249A L259P) (framed in Figure 13A). When their degradation products were mixed with test bacterial culture, the ratio of the system was significantly lower than that of the wild-type SaCsn46A enzyme, showing the optimal antibacterial effect (Figure 13A). Therefore, we subjected these five mutants to secondary screening.

![fig13](https://static.igem.wiki/teams/5549/engineeringsuccess/figure13.avif)

**Figure 13 Antibacterial activity of enzymatic degradation products from wild-type (WT) and mutant chitosanases.** (**A**) SaCsn46A. (**B**) McChoA. (**C**) CsnCA. BL: sterile water, EV: empty vector pET-28a(+). Relative ΔOD600 values were normalized to that of the empty vector pET-28a(+) group.

## Cycle 3: Secondary screening of chitosanases

### Design and Build

The mutation sites of every mutant we selected were confirmed *via* **sequencing**: [SaCsn46A mutant 2](https://registry.igem.org/parts/bba-25c3no7w) (E151G D222G), SaCsn46A mutant 14 (N29S K50R D150G D189G F247L), SaCsn46A mutant 16 (V52A D134G K165I F175L S190C G210D K250E N252D), SaCsn46A mutant 60 (D40G Q125R H234R), and SaCsn46A mutant 94 (T249A L259P). In order to check the antibacterial activity as well as functional change of these mutants, we decided to obtain the **purified** mutants.

### Test

We then purified these enzyme mutants through an AKTA pure™ chromatography system. After normalizing the protein concentration, we performed chitosan degradation experiments and toxicity tests following the same procedure as described above.

![fig14](https://static.igem.wiki/teams/5549/engineeringsuccess/figure14.avif)

**Figure 14 Antibacterial activity of enzymatic degradation products of SaCsn46A WT and selected mutants.** Relative ΔOD600 values were normalized to that of the empty vector pET-28a(+) group.

The relative ΔOD600 values of [SaCsn46A mutant 2](https://registry.igem.org/parts/bba-25c3no7w) (E151G D222G) and SaCsn46A mutant 60 (D40G Q125R H234R) were lower than other mutants as well as the wild-type SaCsn46A (Figure 14), indicating their enzymatic degradation products possessing **superior antibacterial activity** thus promising to further food preservation.

### Learn

Based on the results of all rounds of screening, the chitosan degradation products through [SaCsn46A mutant 2](https://registry.igem.org/parts/bba-25c3no7w) (E151G D222G) and SaCsn46A mutant 60 (D40G Q125R H234R) exhibited the optimal activity. We therefore selected these two mutants for more detailed enzymatic characterizations.

## Cycle 4: Enzymatic characterizations of outstanding mutants

### Design and Build

After identifying SaCsn46A mutant 2 (E151G D222G) and SaCsn46A mutant 60 (D40G Q125R H234R) as the most effective chitosanases in our library, we aimed to obtain more detailed enzymatic parameters to guide subsequent degradation experiments and efficiently produce COS with strong antibacterial activity.

We chose to test the activity of these mutants under **different temperature and pH conditions** to determine their optimal degradation temperature and pH, as well as their stability profiles.

### Test

Chitosanase activity was measured *via* the 3,5-dinitrosalicylic acid (DNS) method. Using D-glucose as the standard, reducing sugar content was determined by measuring the optical density (7) at 540 nm, with the reaction mixture incubated at 100 °C for 5 min. One unit (U) of activity was defined as the enzyme amount needed to release 1 mmol of D-glucose-equivalent reducing sugars per minute under these conditions.

![fig15](https://static.igem.wiki/teams/5549/engineeringsuccess/figure15.avif)

**Figure 15 Enzymatic properties of [SaCsn46A mutant 2](https://registry.igem.org/parts/bba-25c3no7w) and 60.** (**A**) Effects of temperature on enzyme activity. (**B**) Effects of pH on enzyme activity. (**C**) Enzyme inactivation at different temperatures. (**D**) pH stability. The 100% relative activity of mutant 2 was 2.385 U/mg and the 100% relative activity of mutant 60 was 2.124 U/mg.

For pH profiling, assays were conducted at 45 °C across pH 3.0~9.0 using 0.05 M acetate (pH 3.0~6.0), phosphate (pH 6.0~8.0), or Tris-HCl (pH 9.0) buffers. pH stability was assessed by pre-incubating enzymes in ice bath (pH 3.0~9.0), then measuring residual activity under optimal conditions (mutant 2: pH 5, 30 min; mutant 60: pH 4, 30 min). Temperature profiling used different temperatures, while thermostability was tested by pre-incubating at 0~60 °C for 2 h, followed by measuring the residual activity at 45 °C (see our [Experiments](https://2025.igem.wiki/xmu-china/experiments) for more details).

Additionally, we analyzed the degradation products of both mutants using HPLC and TLC as well, and the relevant TLC and HPLC results can be found in our [Results](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27results%27%29%20%7D%7D) section.

### Learn

SaCsn46A mutant 60 and mutant 2 presented different optimal reaction conditions and stabilities. Mutant 60 has an optimal temperature of approximately 40 °C (slight activity decreases but high retention at 45 °C) and an optimal pH condition of 4.0. While Mutant 2 showed unique temperature characteristics—activity drops sharply at 40 °C but recovers at 45 °C (similar to the activity at 35 °C)—with an optimal pH condition of 5.0. Both maintained good thermostability below 50 °C (minor activity increases around 40 °C), while mutant 2 is stable at pH 6~8 and mutant 60 at pH 5 (Figure 15).

## Conclusion

To efficiently obtain COS with strong antibacterial activity for aquatic products preservation, XMU-China implemented a multi-cycle strategy for chitosanase screening and engineering. Initial evaluation of 6 wild-type chitosanases identified 4 (SaCsn46A, CsnCA, CsnMY002, McChoA) with the degradation products showed effective antibacterial activity, excluding BamCsn (weak activity) and GlmATk (exo-chitosanase property promotes bacterial growth). After screening from the large library generated *via* EP-PCR, [SaCsn46A mutant 2](https://registry.igem.org/parts/bba-25c3no7w) (E151G D222G) and [mutant 60](https://registry.igem.org/parts/bba-25uuairi) (D40G Q125R H234R) were selected as optimal candidates—both have good thermostability below 50 °C, distinct optimal pH/temperature, and chitosan degradation products that efficiently inhibit bacterial growth. These two outstanding mutants were further employed for food preservation experiments with the LMT C14A variant (see our [Proof of Concept](https://2025.igem.wiki/xmu-china/poc) for more details).

Engineering a biological system requires iterative efforts. This study demonstrated the development of high-performance COS-producing chitosanases through successive DBTL (Design-Build-Test-Learn) cycles (Figure 16). Each step was guided by experimental feedback, ultimately forming a reliable technical route for low-cost, high-efficiency antimicrobial COS production—critical for advancing natural preservatives in the aquatic product industry.

![fig17](https://static.igem.wiki/teams/5549/engineeringsuccess/esfigure-16-3.avif)

**Figure 16 Summary of the different engineering steps in screening for chitosanase to degrade chitosan to the products with the optimal antibacterial effect.**

## References

1. F.R. Schmidt, Recombinant expression systems in the pharmaceutical industry, Appl Microbiol Biotechnol 65, 363-372 (2004).
2. S. Kaushik, H. He, R.E. Dalbey, Bacterial Signal Peptides- Navigating the Journey of Proteins, Front Physiol 13, 933153 (2022).
3. H. Owji, N. Nezafat, M. Negahdaripour, A. Hajiebrahimi, Y. Ghasemi, A comprehensive review of signal peptides: Structure, roles, and applications, Eur J Cell Biol 97, 422-441 (2018).
4. S. Grasso, V. Dabene, M.M.W.B. Hendriks, P. Zwartjens, R. Pellaux, M. Held, S. Panke, J.M. van Dijl, A. Meyer, T. van Rij, Signal Peptide Efficiency: From High-Throughput Data to Prediction and Explanation, ACS Synth Biol 12, 390-404 (2023).
5. J. Wang, H. Chen, N. Wang, X. Pan, B. Xia, K. Xu, B. Zhong, Chitosan Oligosaccharides: A Natural Rich, High-Efficiency, and Safe Frontrunner for the Futural Fruits/Vegetables Preservation, Food Bioprocess Technol 18, 1104-1124 (2025).
6. L. Zhang, Y. Wang, S. Ling, M. Yuan, Q. Sun, X. Dong, Antibacterial mechanism of chitooligosaccharides against specific spoilage organisms in chilled processed fish paste products, Food Control 172, 111148 (2025).
7. B. Bhuvanachandra, D. Sivaramakrishna, S. Alim, G. Preethiba, S. Rambabu, M.J. Swamy, A.R. Podile, New Class of Chitosanase from Bacillus amyloliquefaciens for the Generation of Chitooligosaccharides, J Agric Food Chem 69, 78-87 (2021).
8. Y. Li, Y. Gou, Z. Liu, T. Xie, G. Wang, Structure-based rational design of chitosanase CsnMY002 for high yields of chitobiose, Colloids Surf B Biointerfaces 202, 111692 (2021).
9. J. Guo, Y. Wang, W. Gao, X. Wang, X. Gao, Z. Man, Z. Cai, Q. Qing, Gene Cloning, Functional Expression, and Characterization of a Novel GH46 Chitosanase from Streptomyces avermitilis (SaCsn46A), Appl Biochem Biotechnol 194, 813-826 (2022).
10. H. Su, J. Sun, C. Guo, Y. Wang, F. Secundo, H. Dong, X. Mao, Structure-based mining of a chitosanase with distinctive degradation mode and product specificity, Appl Microbiol Biotechnol 107, 6859-6871 (2023).
11. W.-S. Xia, D.-X. Lee, Purification and characterization of exo-β-d-glucosaminidase from commercial lipase, Carbohydr Polym 74, 544-551 (2008).

{% endblock %}

{% extends "layout.html" %}
{% block title %}Experiments{% endblock %}
{% block lead %}Describe the research, experiments, and protocols you used in your iGEM project.{% endblock %}
{% block page\_content %}

![](https://static.igem.wiki/teams/5549/icon/loading.webp)

[Commom Experiment](#section1)

[Screening of Chitosanase](#section2)

[Biochemical Characterization of Chitosanase](#section3)

[Characterization of the Secretion System](#section4)

[Characterization for the Combination of Secretion System and Chitosanase](#section5)

[Preservation Efficacy Validation](#section6)

[Biosafety](#section7)

[Others](#section8)

![Shrimp](https://static.igem.wiki/teams/5549/headline/headline-shrimp.webp)

# Experiments

![Crab](https://static.igem.wiki/teams/5549/headline/headline-crab.webp)

## Commom Experiment

### Molecular Cloning Handbook

#### [Handbook](#collapse2024)

[molecular-cloning-handbook-10-0.pdf](https://static.igem.wiki/teams/5549/experiment/2025-xmu-igem-molecular-cloning-handbook.pdf)

## Screening of Chitosanase

Protein Purification

#### [Materials & Apparatus](#collapse1)

— AKTA pure™ chromatography system
— LB liquid medium
— Kanamycin (50 mg/mL)
— IPTG (Isopropyl β-D-1-thiogalactopyranoside) (1 mol/L)
— Ultrasonic Cell Crusher
— 0.22-µm Micron Pore Filter Membrane
— Tecan Infinite M200 microplate reader
— Elution Solution A (20 mM Tris-HCl, 0.5 M NaCl, pH 8)
— Elution Solution B (20 mM Tris-HCl, 0.5 M NaCl, 0.08 M imidazole, pH 8)
— HisTrap HP His tag protein purification columns
— Centrifuge

#### [Protocol](#collapse2)

1.Bacteria culture is inoculated into LB liquid mediums at 2% (v/v)
and kept at 37°C, 200 rpm and last 12-16 h.
2.Bacteria culture from step 1 is inoculated into LB liquid mediums
at 2% (v/v) and kept at 37°C, 200 rpm until the OD600 of bacteria
culture reaches the OD600 specified in the table below.

**Table 1 OD600 for Inducing Chitosanase Expression**

![fig1](https://static.igem.wiki/teams/5549/experiment/table1.webp)
3.Corresponding inducer is added into the bacteria culture from step 2, which is then incubated under appropriate conditions for the specified duration.

**Table 2 Conditions for Inducing Chitosanase Expression**

![fig2](https://static.igem.wiki/teams/5549/experiment/table2-1.webp)
4.Bacteria culture from step 3 is centrifuged at 6500 rpm at
4°C for 10 min to collect the cell and resuspended with 25-30 mL Elution Solution A.
5.Repeat step 4 once.
6.The liquid from step 5 is treated with ultrasonication to lyse the cell.
7.The lysis is centrifuged at 9000 rpm at 4°C for 10 min to remove
the cell debris and then the supernatant is filtered through a 0.22-µm Micron Pore Filter Membrane.
8.The target protein is purified from the supernatant using a
HisTrap column with AKTA pure™ chromatography system.
9.The eluate containing the target proteins is dialyzed
with Elution Solution A overnight to remove imidazole for subsequent uses.

Antibacterial Assay

#### [Materials & Apparatus](#collapse3)

— Chitosanase Solution
— 0.22-µm Micron Pore Filter Membrane
— LB liquid medium
— Tecan Infinite M200 microplate reader
— UV-Sterilized Chitosan powder

#### [Protocol](#collapse4)

1. Add 2 mL chitosanase solution to a centrifuge tube pre-loaded with 0.01 g of chitosan powder, and keep it at 37°C, 200 rpm and last 18 h.
2. Hydrolysate from step 1 is centrifuged at 12000 rpm for 3 min to collect the supernatant and performs membrane filtration. Seal with parafilm and store at -20°C.
3. Bacteria culture is inoculated into LB liquid mediums at 2% (v/v) and kept at 37°C, 200 rpm and last 12-16 h.
4. Bacteria culture from step 3 is inoculated into LB liquid mediums at 2% (v/v) and kept at 37°C, 200 rpm until the OD600 of bacteria culture reached 0.8.
5. Bacterial culture from step 4 is mixed with the supernatant from step 2 at a 1:1 (v/v) ratio and incubated at 37°C, 200 rpm. Reaction solution is collected at 1.5 h and 8 h.
6. The OD600 of the reaction solution is measured immediately by Tecan Infinite M200 microplate reader.

## Biochemical Characterization of Chitosanase

Preparation of Chitosan Solution

#### [Materials & Apparatus](#collapse5)

— Chitosan powder
— 30% hydrogen peroxide(H2O2)
— 2.0% (w/w) acetic acid (HAc)
— Sterile water
— Catalase (CAT)
— Water bath
— Centrifuge

#### [Protocol](#collapse6)

1. Mix the reagents according to the table 3 to prepare a 2% chitosan solution.

**Table 3 Composition of Chitosan Solution**

![fig3](https://static.igem.wiki/teams/5549/experiment/table3.webp)
2. Incubate the mixed solution from step 1 at 65°C for 15 min.
3. Add catalase in small aliquots multiple times, and incubate at 40°C for 30 min until no further bubble formation is observed.
*Note: Swirl the flask while adding catalase.*

Determination of Reducing Sugar Content

#### [Materials & Apparatus](#collapse7)

— DNS chromogen reagent (MY73006)
— Tecan Infinite M200 microplate reader
— Glucose
— Test sample solution
— Sterile water
— Water bath
— Centrifuge

#### [Protocol](#collapse8)

1. Glucose solutions are prepared at concentrations of 0.2 mg/mL, 0.4 mg/mL, 0.6 mg/mL, 0.8 mg/mL, 1 mg/mL in a 15 mL centrifuge tube.
2. Separately take equal volumes of the glucose solution from step 1, sterile water (blank control), and the test sample solution, and mix each with an equal volume of DNS chromogen reagent.
3. Incubate the mixed solution from step 2 at 100°C for 5 min, then immediately transfer it to an ice-water bath for rapid cooling.
4. Dilute the reaction solution from step 3 with sterile water at a 1:2 (v/v) ratio and then measure OD540 of the diluted solution by Tecan Infinite M200 microplate reader.
5. Plot a standard curve with glucose standard concentration as the x-axis and the corresponding OD540 values as the y-axis, and calculate the reducing sugar content in the test sample solution based on this curve.

Determination of the Optimal pH for Chitosanase Activity

#### [Materials & Apparatus](#collapse9)

— 2% Chitosan solution
— Chitosanase solution
— 2.0% (w/w) acetic acid (HAc)
— 11% Sodium hydroxide solution
— DNS chromogen reagent (MY73006)
— Tecan Infinite M200 microplate reader
— Water bath
— Centrifuge

#### [Protocol](#collapse10)

1. Mix the 2% chitosan solution with the chitosanase solution at a 1:1 (v/v) ratio, and add amounts of acetic acid or sodium hydroxide solution to adjust the pH to 3.0, 4.0, 5.0, 6.0, 7.0, 8.0 and 9.0.
2. Incubate the mixed solution from step 1 at 45°C for 30 min.
3. Hydrolysate from step 2 is subjected to reducing sugar content determination.
4. The enzyme activity is calculated according to the following formula, and a curve of enzyme activity versus pH is plotted to determine the optimal pH for the enzyme reaction.
![fig4](https://static.igem.wiki/teams/5549/experiment/formula.webp)
with *C* the reducing sugar concentration calculated from the standard curve (mg/mL), *Vt* the total volume of the enzymatic reaction system (mL),
*n* the dilution factor of the reaction solution after adding DNS reagent (if undiluted, n = 1), *Ce* the concentration (mg/mL) of enzyme solution, *M* the molecular weight of glucose (180.16 g/mol), *t* the enzymatic reaction time (min), and *Ve* the volume (mL) of enzyme solution added.

Investigation of pH Stability of Chitosanase

#### [Materials & Apparatus](#collapse11)

— 2% Chitosan solution
— Chitosanase solution
— 2.0% (w/w) acetic acid (HAc)
— 1% Ammonia water
— 0.05 M acetate buffer (pH 3.0-6.0)
— 0.05 M phosphate buffer (pH 6.0-8.0)
— 0.05 M Tris-HCl buffer (pH 9.0)
— DNS chromogen reagent (MY73006)
— Tecan Infinite M200 microplate reader
— Water bath
— Centrifuge

#### [Protocol](#collapse12)

1. Adjust the pH of the chitosanase solution to 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, and 9.0 using 0.05 M acetate buffer (pH 3.0-6.0), 0.05 M phosphate buffer (pH 6.0-8.0), and 0.05 M Tris-HCl buffer (pH 9.0).
2. Pre-incubate the chitosanase solution from step 1 at 0°C for 2 h.
3. Mix the pre-incubated chitosanase solution from step 2 with the 2% chitosan solution at a 1:1 (v/v) ratio, adjust the temperature and pH to the optimal values.
4. Incubate the mixed solution from step 3 at 45°C for 30 min.
5. Hydrolysate from step 4 is subjected to reducing sugar content determination and then the pH stability curve of the enzyme is plotted.

Determination of the Optimal Temperature for Chitosanase Activity

#### [Materials & Apparatus](#collapse13)

— 2% Chitosan solution
— Chitosanase solution
— 2.0% (w/w) acetic acid (HAc)
— 1% Ammonia water
— DNS chromogen reagent (MY73006)
— Tecan Infinite M200 microplate reader
— Water bath
— Centrifuge

#### [Protocol](#collapse14)

1. Mix the 2% chitosan solution with the chitosanase solution at a 1:1 (v/v) ratio, and adjust the pH to approximately 5.0.
2. Incubate the mixed solution from step 1 at 25°C, 30°C, 35°C, 40°C, and 45°C for 30 min.
3. Hydrolysate from step 2 is subjected to reducing sugar content determination and then the temperature-activity curve of the enzyme is plotted.

Investigation of Thermal Stability of Chitosanase

#### [Materials & Apparatus](#collapse15)

— 2% Chitosan solution
— Chitosanase solution
— 2.0% (w/w) acetic acid (HAc)
— 1% Ammonia water
— 0.05 M acetate buffer (pH 3.0-6.0)
— 0.05 M phosphate buffer (pH 6.0-8.0)
— 0.05 M Tris-HCl buffer (pH 9.0)
— DNS chromogen reagent (MY73006)
— Tecan Infinite M200 microplate reader
— Water bath
— Centrifuge

#### [Protocol](#collapse16)

1. Adjust the pH of the chitosanase solution to 7.5 using 0.05 M phosphate buffer (pH 6.0-8.0).
2. Pre-incubate the chitosanase solution (pH 7.5) from step 1 at 30°C, 35°C, 40°C, 45°C, and 50°C for 2 h.
3. Mix the pre-incubated chitosanase solution from step 2 with the 2% chitosan solution at a 1:1 (v/v) ratio, and adjust the temperature to the optimal values.
4. Incubate the mixed solution from step 3 at 45°C for 30 min.
5. Hydrolysate from step 4 is subjected to reducing sugar content determination and then the thermal stability curve of the enzyme is plotted.

Characterization of Enzymatic Hydrolysis Products

#### Thin Layer Chromatography (TLC)

#### [Materials & Apparatus](#collapse17)

— Test sample
— Developing solvent (Isopropanol: sterile water: 1% Ammonia Water = 60: 30: 4, v/v/v)
— TLC silica gel plate
— Microcapillary tube
— 0.5% Ninhydrin

#### [Protocol](#collapse18)

1. Using a microcapillary tube, spot test sample onto the TLC plate, positioning the spots approximately 1.0 cm above the bottom edge along the starting line.
2. Add an appropriate amount of developing solvent to the TLC chamber until the liquid depth reaches approximately 0.5 cm. Carefully place the spotted TLC plate into the chamber, ensuring the starting line is positioned above the solvent level. Remove the TLC plate when the solvent front has migrated to approximately 1.0 cm from the top edge, and immediately mark the solvent front position with a pencil.
3. The TLC plate from step 2 is air-dried, sprayed with 0.5% ninhydrin, and then heated to visualize the separated chitooligosaccharides.
4. Compare the spot positions between the hydrolysate and the standard, and analyze the ability of each mutant to produce low-degree polymerization COSs; simultaneously, observe the color and intensity of each spot to preliminarily quantify the COSs of different polymerization degrees in the hydrolysate.

#### High Performance Liquid Chromatography (HPLC)

#### [Materials & Apparatus](#collapse19)

— Test sample
— Shodex Asahipak NH2P-50 4E Column (4.6 mm × 250 mm,5 μm)
— Mobile phase (acetonitrile : 1% ammonia solution (v/v) = 7:3)
— Methanol (HPLC Grade)
— 0.22-µm Micron Pore Filter Membrane
— Chitooligosaccharides (DP 3-7)
— Essentia Prep LC-16P (Shimadzu, Japan)
— Glucosamine
— Chitobiose solution

#### [Protocol](#collapse20)

1. Chitooligosaccharides solutions are prepared at concentrations of 5 mg/mL, 10 mg/mL, 15 mg/mL, 25 mg/mL, and 50 mg/mL.
2. Analytical-grade glucosamine and chitobiose solutions are prepared at a concentration of 5 mg/mL.
3. The test sample, chitooligosaccharides, glucosamine and chitobiose solution from step 1 and step 2 are filtered through a 0.22-µm Micron Pore Filter Membrane into a sample vial for HPLC analysis.
4. The HPLC parameters are set as follows:

**Table 4 HPLC Parameters**

![fig5](https://static.igem.wiki/teams/5549/experiment/table4.webp)
5. The standard curve is generated by recording the retention times and peak areas for the chitooligosaccharides standards with various polymerization degrees. Subsequently, the retention times corresponding to chitooligosaccharides with different polymerization degrees are identified, and a linear standard curve is constructed for each polymerization degree based on the relationship between concentration and peak area.
6. The filtered test sample from step 3 is analyzed to determine the peak area and retention time.
7. Data analysis involves qualitative analysis of the polymerization degree of the main products by comparing retention times with standard references, and quantitative analysis is performed using integrated peak areas to calculate the yields of chitooligosaccharides with different polymerization degrees in the degradation products.

## Characterization of the Secretion System

#### [Materials & Apparatus](#collapse21)

— Chloramphenicol (34 mg/mL)
— LB liquid medium
— Tecan Infinite M200 microplate reader
— Centrifuge

#### [Protocol](#collapse22)

1. Bacteria culture is inoculated into LB liquid mediums at 2% (v/v) and kept at 37°C, 200 rpm and last 12-16 h.
2. Bacteria culture from step 1 is inoculated into LB liquid mediums at 2% (v/v) and kept at 37°C, 200 rpm until the OD600 of bacteria culture reached 0.6.
3. Bacteria culture from step 2 is inoculated into LB liquid mediums at 2% (v/v) and kept at 37°C, 200 rpm.
4. 1 mL of the bacterial cultures is collected per hour until 10 h.
5. The fluorescence intensity of the bacteria culture is measured immediately by Tecan Infinite M200 microplate reader.
6. The secretion efficiency is calculated by dividing the fluorescence intensity of the supernatant by the fluorescence intensity of the bacteria culture.

## Characterization for the Combination of Secretion System and Chitosanase

#### [Materials & Apparatus](#collapse23)

— Tecan Infinite M200 microplate reader
— Centrifuge
— Kanamycin (50 mg/mL)
— LB liquid medium
— sterile water
— IPTG (Isopropyl β-D-1-thiogalactopyranoside) (1 mol/L)
— Anhydrous ethanol

#### [Protocol](#collapse24)

1. Bacteria culture is inoculated into LB liquid mediums at 2% (v/v) and kept at 37°C, 200 rpm and last 12-16 h.
2. Bacteria culture from step 1 is inoculated into LB liquid mediums at 2% (v/v) and kept at 37°C, 200 rpm until the OD600 of bacteria culture reached at 0.6.
3. IPTG is added into the bacterial culture to a final concentration of 1 mM, and expression is induced at 16°C, 200 rpm for 36 hours.
4. 2 mL of the bacterial cultures is collected at 0 h, 5 h, 8 h, 12 h, 16 h and 20 h.
5. Bacteria culture from step 4 is centrifuged at 13000 rpm at 4°C for 10 min to collect the supernatant.
6. The collected supernatant from step 5 is mixed with 2 mL of anhydrous ethanol by gentle inversion, and stored at 4°C for 12 h.
7. Mixture from step 6 is centrifuged at 13000 rpm at 4°C for 10 min and then discard the supernatant.
8. 400 µL sterile water is added into the precipitate from step 7 to dissolve and obtain the extracellular supernatant, which is then subjected to SDS-PAGE.
9. The supernatant from the 18 h is taken for preservation experiment.

## Preservation Efficacy Validation

It should be noted that the fruits and fish used in our preservation experiment have been approved by the Safety and Security Committee ([**Safety Form**](https://teams.igem.org/5549/safety)).

#### [Materials & Apparatus](#collapse25)

— Tryptone powder
— Yeast extract powder
— D-glucose
— Agar powder
— sterile water
— Fresh back meat of fish
— Tomato
— Sterile sampling bag
— Normal saline solution (0.9% sodium chloride)
— Chitooligosaccharides (MW < 1 kDa)
— Chitooligosaccharides (MW < 2 kDa)
— Chitooligosaccharides (MW < 3 kDa)
— Potassium sorbate

#### [Protocol](#collapse26)

#### [A. Preparation of PCA Solid Plates](#collapse27)

1. Prepare the PCA medium by mixing reagents according to the table 5, and sterilize it at 121°C for 15 min.

**Table 5 Composition of PCA Medium**

![fig6](https://static.igem.wiki/teams/5549/experiment/table5.webp)
2. Pour the medium into plates while in liquid state, allow it to cool and solidify, and store at 4°C.

#### [B. Testing the Freshness Preservation Effectiveness of Fish Meat](#collapse28)

1. Fresh back meat of fish is cut into uniformly sized and equal-weight pieces, immersed in different solutions for 10 minutes, drained of excess liquid, packaged in sterile sampling bags, and stored at 4°C for refrigeration.
2. Samples are retrieved and weighed on day 0, day 1, day 2, day 3, and day 4.
3. In a biosafety cabinet, 10 mL of sterile physiological saline is added to the samples retrieved in step 2. After sealing the sampling bags, the fish meat is homogenized by grinding.
4. 10 µL of the homogenized fish sample is mixed thoroughly with 90 µL of sterile physiological saline.
5. 10 µL of the diluted homogenate is mixed with 90 µL of sterile physiological saline. This dilution process is repeated 5 times, resulting in 6 dilution levels.
6. 10 µL of each diluted homogenate is taken and spotted onto pre-prepared PCA solid plates. The plates are then incubated at 37°C for 48 hours.
7. Viable bacterial count is calculated. The colony forming units (CFU)/g is calculated as follows: CFU/g = colony count × dilution factor × 10 / sample weight (g) (a single dilution factor is 10, two dilutions correspond to 102, and so on).
*Note: When the viable bacterial count reaches 108 CFU/g or higher, the food is considered to be in the initial stage of spoilage.*

#### [C. Testing the Freshness Preservation Effectiveness of Tomato](#collapse29)

1. Fresh tomatoes are immersed in different solutions for 10 minutes, drained of excess liquid, packaged in sterile sampling bags (one tomato per bag), and stored at room temperature.
2. Samples are retrieved on day 0, day 1, day 2, day 3, and day 4, weighed, and the diameter measured to calculate surface area.
3. In a biosafety cabinet, 10 mL of sterile water is added to the samples retrieved in step 2 to collect the surface bacteria of the tomato.
4. 10 µL of the water containing surface bacteria is mixed thoroughly with 90 µL of sterile water.
5. 10 µL of the diluted solution is mixed with 90 µL of sterile water. This dilution process is repeated 5 times, resulting in 6 dilution levels.
6. 10 µL of each diluted solution is taken and spotted onto pre-prepared PCA solid plates. The plates are then incubated at 37°C for 48 hours.
7. After water collection in step 4, the tomato is mashed to prepare a homogenate.
8. 10 µL of the tomato homogenate is mixed thoroughly with 90 µL of sterile water.
9. 10 µL of the diluted homogenate is mixed with 90 µL of sterile water. This dilution process is repeated 5 times, resulting in 6 dilution levels.
10. 10 µL of each diluted homogenate is taken and spotted onto pre-prepared PCA solid plates. The plates are then incubated at 37°C for 48 hours.
11. Viable bacterial count is calculated.
Surface bacterial count of tomato: The colony forming units (CFU)/cm2 is calculated as follows: CFU/cm² = colony count × dilution factor × 10 / sample surface area (cm2) (a single dilution factor is 10, two dilutions correspond to 102, and so on).
Total bacterial count of tomato: The colony forming units (CFU)/g is calculated as follows: CFU/g = colony count × dilution factor × 10 / sample weight (g).

## Biosafety

Verification for the Cytotoxicity of TyrS

#### [Materials & Apparatus](#collapse30)

— Chloramphenicol (34 mg/mL)
— LB liquid medium
— LB solid plates
— IPTG (Isopropyl β-D-1-thiogalactopyranoside) (1 mol/L)

#### [Protocol](#collapse31)

1. Bacteria culture is inoculated into LB liquid mediums at 2% (v/v) and kept at 37°C, 200 rpm and last 12-16 h.
2. Bacteria culture from step 1 is inoculated into LB liquid mediums at 2% (v/v) and kept at 37°C, 200 rpm. Two groups are set: one group (IPTG) is added IPTG to reach the concentration of 1 mM and the other group (NO IPTG) is without IPTG.
*Note: Each group should have three parallel cultures.*
3. After culturing for 6 h, 20 μL of the bacteria culture is taken and mixed thoroughly with 180 μL of fresh LB medium containing the same antibiotic concentration.
4. 20 μL of the diluted culture is taken and mixed with another 180 μL of fresh LB medium with the same antibiotic concentration. Repeat this dilution process 7 times, resulting in 8 dilution levels.
5. 5 μL of each diluted culture is taken and spotted onto pre-prepared solid plates containing the same antibiotic concentration. Then the plates are incubated at 37°C for 12 h.
6. The survival ratio is calculated. First, the colony forming units (CFU)/mL is calculated: CFU/mL = colony count × dilution factor / 0.005 mL (a single dilution has a dilution factor of 10, two dilutions would be 102, and so on). The survival ratio (log10) is calculated as: log10[(CFU/mL of the IPTG group)/(CFU/mL of the NO IPTG group)].

Improvement of the tyrS Overexpression System

#### [Materials & Apparatus](#collapse32)

— Chloramphenicol (34 mg/mL)
— LB liquid medium
— LB solid plates
— IPTG (Isopropyl β-D-1-thiogalactopyranoside) (1 mol/L)

#### [Protocol](#collapse33)

1. Bacteria culture is inoculated into LB liquid mediums at 2% (v/v) and kept at 37°C, 200 rpm and last 12-16 h.
2. Bacteria culture from step 1 is inoculated into LB liquid mediums at 2% (v/v) and kept at 37°C, 200 rpm. Groups are set: groups (IPTG) are added IPTG to reach the concentration of 0.001 mM, 0.003 mM, 0,01 mM, 0.03 mM, 0.1 mM, 0.5 mM, 1 mM and the control group (NO IPTG) is without IPTG.
*Note: Each group should have three parallel cultures.*
3. Bacterial culture is collected at 0 h, 2 h, 4 h, 6 h and 8 h.
4. 20 μL of the bacteria culture is taken and mixed thoroughly with 180 μL of fresh LB medium containing the same antibiotic concentration.
5. 20 μL of the diluted culture is taken and mixed with another 180 μL of fresh LB medium with the same antibiotic concentration. Repeat this dilution process 7 times, resulting in 8 dilution levels.
6. 5 μL of each diluted culture is taken and spotted onto pre-prepared solid plates containing the same antibiotic concentration. Then the plates are incubated at 37°C for 12 h.
7. The survival ratio is calculated. First, the colony forming units (CFU)/mL is calculated: CFU/mL = colony count × dilution factor / 0.005 mL (a single dilution has a dilution factor of 10, two dilutions would be 102, and so on). The survival ratio (log10) is calculated as: log10[(CFU/mL of the IPTG group)/(CFU/mL of the NO IPTG group)].

Verification for the Cytotoxicity of CcdB

#### [Materials & Apparatus](#collapse34)

— Chloramphenicol (34 mg/mL)
— LB liquid medium
— LB solid plates
— Cuminic acid stock solution (0.1 mol/L)

#### [Protocol](#collapse35)

1. Bacteria culture is inoculated into LB liquid mediums at 2% (v/v) and kept at 37°C, 200 rpm and last 12-16 h.
2. Bacteria culture from step 1 is inoculated into LB liquid mediums at 2% (v/v) and kept at 37°C, 200 rpm. Two groups are set: one group (Cuma) is added Cuminic acid stock solution to reach the concentration of 0.1 mM and the other group (NO Cuma) is without Cuminic acid.
*Note: Each group should have three parallel cultures.*
3. After culturing for 4 h, 20 μL of the bacteria culture is taken and mixed thoroughly with 180 μL of fresh LB medium containing the same antibiotic concentration.
4. 20 μL of the diluted culture is taken and mixed with another 180 μL of fresh LB medium with the same antibiotic concentration. Repeat this dilution process 7 times, resulting in 8 dilution levels.
5. 5 μL of each diluted culture is taken and spotted onto pre-prepared solid plates containing the same antibiotic concentration. Then the plates are incubated at 37°C for 12 h.
6. The survival ratio is calculated. First, the colony forming units (CFU)/mL is calculated: CFU/mL = colony count × dilution factor / 0.005 mL (a single dilution has a dilution factor of 10, two dilutions would be 102, and so on). The survival ratio (log10) is calculated as: log10[(CFU/mL of the Cuma group)/(CFU/mL of the NO Cuma group)].

Spot Assay for Kill Switch

#### [Materials & Apparatus](#collapse36)

— Chloramphenicol (34 mg/mL)
— LB liquid medium
— LB solid plates
— IPTG (Isopropyl β-D-1-thiogalactopyranoside) (1 mol/L)
— Cuminic acid stock solution (0.1 mol/L)

#### [Protocol](#collapse37)

1. Bacteria culture is inoculated into LB liquid mediums at 2% (v/v) and kept at 37°C, 200 rpm and last 12-16 h.
2. Bacteria culture from step 1 is inoculated into LB liquid mediums at 2% (v/v) and kept at 37°C, 200 rpm. Four groups are set:

**Table 6 Kill Switch Condition Setting**

![fig7](https://static.igem.wiki/teams/5549/experiment/table6.webp)
*Note: Each group should have three parallel cultures.*
3. After culturing for 4 h, 20 μL of the bacteria culture is taken and mixed thoroughly with 180 μL of fresh LB medium containing the same antibiotic concentration.
4. 20 μL of the diluted culture is taken and mixed with another 180 μL of fresh LB medium with the same antibiotic concentration. Repeat this dilution process 7 times, resulting in 8 dilution levels.
5. 5 μL of each diluted culture is taken and spotted onto pre-prepared solid plates containing the same antibiotic concentration. Then the plates are incubated at 37°C for 12 h.
6. The survival ratio is calculated. First, the colony forming units (CFU)/mL is calculated: CFU/mL = colony count × dilution factor / 0.005 mL (a single dilution has a dilution factor of 10, two dilutions would be 102, and so on). The survival ratio (log10) is calculated as: log10[(CFU/mL of the other groups)/(CFU/mL of the NO IPTG and NO Cuma group)].

## Others

Toxicity Assessment of Chitooligosaccharides standards against *Escherichia coli*

#### [Materials & Apparatus](#collapse38)

— LB liquid medium
— Tecan Infinite M200 microplate reader
— UV-Sterilized chitooligosaccharides (MW < 1 kDa)
— UV-Sterilized chitooligosaccharides (MW < 2 kDa)
— UV-Sterilized chitooligosaccharides (MW < 3 kDa)
— sterile water
— Kanamycin (50 mg/mL)

#### [Protocol](#collapse39)

1. Chitooligosaccharides with different degrees of molecular weights (MW) is weighed and added into an appropriate amount of sterile water to prepare 50% (w/v) stock solution.
2. Bacteria culture is inoculated into LB liquid mediums at 2% (v/v) and kept at 37°C, 200 rpm and last 12-16 h.
3. Bacteria culture from step 2 is inoculated into LB liquid mediums at 2% (v/v) and kept at 37°C, 200 rpm until the OD600 of bacteria culture reached 0.6.
4. Bacterial culture from step 3 is added chitooligosaccharides stock solution from step 1 to reach the concentration of 7%, 1.2%, 0.012%, 0% (w/v) and kept at 37°C, 200 rpm. Reaction solution is collected at different time points.
5. The OD600 of the reaction solution is measured immediately by Tecan Infinite M200 microplate reader.

Spot Assay for Chitooligosaccharides

#### [Materials & Apparatus](#collapse40)

— LB liquid medium
— LB solid plates
— UV-Sterilized chitooligosaccharides (MW < 1 kDa)
— UV-Sterilized chitooligosaccharides (MW < 2 kDa)
— UV-Sterilized chitooligosaccharides (MW < 3 kDa)
— Kanamycin (50 mg/mL)
— IPTG (Isopropyl β-D-1-thiogalactopyranoside) (1 mol/L)

#### [Protocol](#collapse41)

1. After autoclaving and cooling to 50°C, the LB solid medium is supplemented with chitooligosaccharides standards (MW < 1 kDa, MW < 2 kDa and MW < 3 kDa) at 0%, 0.25%, 0.5%, 1%, 1.25%, 1.5% (w/v), Kanamycin (50 µg/mL) and IPTG (0.1 mM) prior to pouring plates.
2. Bacteria culture is inoculated into LB liquid mediums at 2% (v/v) and kept at 37°C, 200 rpm and last 12-16 h.
3. Bacteria culture from step 2 is inoculated into LB liquid mediums at 2% (v/v) and kept at 37°C, 200 rpm until the OD600 of bacteria culture reached 0.6.
4. Bacteria culture from step 3 is diluted 104-fold and plated onto LB solid plates containing Kanamycin (50 µg/mL) and IPTG (0.1 mM). Then the plates are incubated at 37°C for 12 h.
5. Colonies from the plate in step 4 are picked and spotted onto the plates containing different concentrations of chitooligosaccharides from step 1, using the same colony for all plates. Then the plates are incubated at 37°C for 12 h.
6. The survival frequency (%) is calculated as: (the number of colonies formed)/(total number of colonies).

{% endblock %}

{% extends "layout.html" %}
{% block title %}Results{% endblock %}
{% block lead %}You can describe the results of your project and your future plans here.{% endblock %}
{% block page\_content %}

![](https://static.igem.wiki/teams/5549/icon/loading.webp)

[Overview](#section1)

[Highlight](#section2)

[Characterization of Commercial COS](#section3)

[Chitosanase Screening](#section4)

[Mutants Characterization](#section5)

[Improvement of the Extracellular Secretion System](#section6)

[Preservation Experiments](#section7)

[Kill Switch](#section8)

[Conclusion](#section9)

[Future Prospects](#section10)

[Reference](#section11)

![Shrimp](https://static.igem.wiki/teams/5549/headline/headline-shrimp.webp)

# Results

![Crab](https://static.igem.wiki/teams/5549/headline/headline-crab.webp)

## 1 Overview

The spoilage and deterioration of food, especially aquatic products, pose significant challenges to global food safety. Shortly after harvest, seafood often experiences a decline in quality, which not only decreases its shelf life but also cause symptoms such as diarrhea, vomiting, and acute poisoning in consumers, primarily due to the accumulation of histamine toxins and proliferation of pathogens. Although the industry predominantly employs chemical preservatives and cold chain systems to preserve product freshness, the carcinogenic risks associated with additives like sulfites and the environmental impact from carbon emissions of cold chain systems reliant on fossil fuels necessitate a trade-off between economic expense and environmental health. Hence, the development of safe and efficient natural preservation techniques is of paramount importance.

Chitosan is the second most abundant natural polysaccharide in nature, predominantly found in the exoskeletons of crustaceans such as shrimp and crab. However, it is often discarded due to the high costs associated with its processing. Chitooligosaccharides (COS), a degradation product of chitosan, demonstrate enhanced water solubility and antibacterial properties, which can effectively damage bacterial cell membranes and offering potential preservatives for seafood and fruits (1). According to the HP survey (see [**IHP**](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27human-practices%27%29%20%7D%7D) for more details), challenges include the complexity of the process, high costs, environmental concerns, low efficiency, and difficulties in product control, particularly in the biology, chemical and physical conversion of chitosan into chitosan oligosaccharide. Hence, our CRUSTA project seeks to produce COS using a route developed through synthetic biology, which can serve as alternative to chemical preservatives.

➤ Initiated by the discovery that COS with higher degrees of polymerization possess enhanced antibacterial properties, we established a chitosanase screening method
that relies on the antibacterial efficiency of COS degraded by mutants. At last, we obtained the [SaCsn46A mutant 2](https://registry.igem.org/parts/bba-25c3no7w) (SaCsn46A E151G D222G) from the various mutants
whose degradation products exhibited the best antibacterial properties.

➤ To reduce the subsequent enzyme extraction cost, we semi-rationally designed the signal peptide LMT and obtained the mutant LMT C14A with higher secretion efficiency.

➤ We achieved chitosan degradation *via* a secretion system, enhancing the antibacterial performance of the degradation products by 7.96-fold compared to that of wild-type.

➤ To prevent engineered bacteria in the chitosanase production chain from contaminating other processes and posing biosafety risks, we designed two independent suicide switches. The first is a *ccdB/ccdA* toxin-antitoxin system regulated by cuminic acid, and the second is an IPTG-inducible system that triggers overexpression of the *tyrS* gene. Together, we integrated these two systems into a same plasmid, they can operate independently, thus guarantee rapid cell death, keeping the engineered bacteria in a controllable state.

When the viable bacterial count reaches 108 CFU/g or higher, the food is considered to be in the initial stage of spoilage. The results showed that the degradation products had an excellent fresh-keeping effect and could effectively inhibit microbial growth. This outcome confirms the rationality and feasibility of our designed solution, paving the way for its application in practical production.

## 2 Highlights

##### We established a chitosanase screening method that relies on the antibacterial efficiency of COS degraded by mutants, by which obtained the [SaCsn46A mutant 2](https://registry.igem.org/parts/bba-25c3no7w) (SaCsn46A E151G D222G) from the various mutants whose degradation products exhibited the best antibacterial properties.

##### We semi-rationally designed the signal peptide LMT and obtained the mutant LMT C14A with higher secretion efficiency.

##### We achieved extracellular chitosan degradation *via* a secretion system, enhancing the antibacterial performance of the degradation products by 7.96-fold compared to that of wild type.

##### Our route saves about 61.0% of CO2 emissions and 71.7% of cost compared to the chemical production of traditional preservative, potassium sorbate, in the same functional performance.

##### We integrated *ccdB/ccdA* toxin-antitoxin with *tyrS* system into a kill switch circuit, which can operate independently, keeping the engineered bacteria in a controllable state.

## 3 Determination of the Antibacterial Activity and Preservation Performance of commercialized COS

To investigate the potential of COS with different average molecular weights as preservatives, we purchased COS standards with different average molecular weights, and conducted a series of experiments to test their antibacterial activity and preservation performance at different concentrations (3).

### 3.1 The Antibacterial Activity of commercialized COS

We purchased three varieties of commercial COS products. These are mixtures comprising COS of varying polymerization degrees, with average molecular weights under 1 kDa (Beyotime, Y261047), 2 kDa (Beyotime, Y261046), and 3 kDa (Beyotime, Y261045), respectively. We employed Escherichia coli BL21(DE3) as the indicator strain, culturing it until the optical density (OD₆₀₀) reached 0.6. Subsequently, we introduced three types of commercial COS solutions at varying concentrations and measured the 24-hour growth curves of the strains, as shown in Figures 1 A-C, to evaluate their antibacterial efficacy. The experimental results showed that all three types of COS exhibited significant antibacterial effects when their concentration exceeded 1.2% (w/v). Furthermore, higher concentrations of COS resulted in bacterial cell death and flocculation. Additionally, we prepared LB solid media containing COS with different type and concentrations, onto which we inoculated single colonies of *E. coli* BL21(DE3). After 14 hours, the survival rates were calculated to assess the antibacterial properties (Figure 1 D). The results showed a general decline in survival rates as COS concentrations increased; when the concentration exceeded 1.5% (w/v), it completely inhibited the growth of the strain.

![](https://static.igem.wiki/teams/5549/results/figure-1-2.avif)

**Figure 1 Antibacterial activity of COS with different average molecular
weights and concentrations.** (**A-C**) OD600 values of *E. coli* BL21(DE3) cultures containing
COS with average molecular weights < 1 kDa (**A**), < 2 kDa (**B**), and < 3 kDa (**C**) at indicated concentrations over 24 h.
(**D**) Bacterial survival rates on LB solid media containing COS with different average molecular weights and concentrations.

Therefore, our experiments demonstrate that COS with varying average molecular weights exhibit significant antibacterial effects at concentrations exceeding 1.5% (w/v). Notably, comparative analysis under identical conditions revealed that COS with average molecular weights below 3 kDa exhibits the strongest antibacterial activity.

### 3.2 The Preservation Performance of COS

Based on the results of the antibacterial activities of COS, we have preliminarily confirmed that COS effectively inhibit microbial reproduction and possesses considerable potential for food preservation. To further validate the COS's efficacy in food preservation, subsequent experiments will focus on evaluating its preservation performance. After approval by the Safety and Security Committee ([**Safety Form**](https://teams.igem.org/5549/safety)), we will measure the total bacterial colony count to assess the ability of COS to delay the spoilage in aquatic products and fruits, thus providing a comprehensively evaluation of its utility in food preservation.

#### 3.2.1 Aquatic Product Preservation

##### 3.2.1.1 Effects of COS Concentration and Average Molecular weights on Aquatic Product Preservation

To evaluate the preservation performance of COS with various average molecular weights under 1 kDa, 2 kDa, and 3 kDa on aquatic products, we immersed fish samples in sterile solutions of the commercial COS. These treated fish samples were then sealed in sterile bags and stored at 4°C for the number of days. After storage period, the fish samples were mashed within these sterile bags. We added sterile water to the mashed samples, collected the resultant leachate, diluted it at a specified ratio, and then plated it on plate count agar (PCA) medium. The medium was then incubated at 37°C for 48 hours, after which colonies were counted. Colony counts were normalized to colony-forming units per gram of fish meat (CFU/g) (Figure 2A).

The results indicated that by the second day of the experiment, all groups treated with COS of various average molecular weights exhibited significant preservation effects on aquatic product, with marked differences from the sterile water control group (Figure 2B). However, no statistically significant differences in preservation efficacy were observed among the COS-treated groups with different average molecular weights (Figure 2C).

![](https://static.igem.wiki/teams/5549/results/figure-2-2.avif)

**Figure 2 Aquatic product preservation performance
of COS with different average molecular weights in fish samples.** (**A**) Treatment of fish samples
with COS of different average molecular weights (< 1 kDa, < 2 kDa, < 3 kDa). (**B**) On Day 2 post-treatment,
all COS-treated groups showed significant aquatic product preservation performance, with obvious
differences from the sterile water control group. (**C**) No significant differences in aquatic product
preservation performance between COS-treated groups with average molecular weights.

We evaluated the preservation performance of COS with average molecular weights under 3 kDa on fish samples using three concentrations: 15, 20, and 25 mg/mL. This experimental design allowed us to systematically assess the impact of varying COS concentrations on aquatic product preservation. The results indicated a decreasing trend in colony count across all COS-treated samples, in contrast to a marked increase observed in the sterile water control group (Figure 3A). Although a dose-dependent trend was apparent, there was no significant difference in preservation performance among the COS-treated groups (Figure 3B). By the third day of the experiment, all COS-treated groups exhibited statistically significant differences compared to the control group (Figure 3B). We observed an interesting phenomenon that the initial bacterial count in fish purchased at varying times shows significant variation, with differences reaching an 1-2 of magnitude. At an initial CFU of 105, a concentration of 20 mg/mL of COS demonstrates a robust inhibitory effect lasting three days or longer. However, when the initial CFU increases to 106, this inhibitory effect diminishes.

![](https://static.igem.wiki/teams/5549/results/figure-3-2.avif)

**Figure 3 The impact of varying COS concentrations on fish samples
preservation.** (**A**) Treatment of fish samples with COS (< 3 kDa) of different concentrations (25 mg/mL, 20 mg/mL, 15 mg/mL).
(**B**) Significant differences between COS-treated groups and the sterile water control group on Day 3 post-treatment.
(**C**) Comparison of preservation performance between COS (< 3 kDa) and potassium sorbate in fish samples on Day 1
post-treatment.

##### 3.2.1.2 Comparison of Aquatic Product Preservation Performance Between COS and Potassium Sorbate

To evaluate the practicality of COS, its preservation efficacy was compared with that of potassium sorbate, a widely used chemical preservative. Preservation experiments were conducted on fish samples using 20 mg/mL of COS (average molecular weights < 3 kDa) and 0.2% (w/v) potassium sorbate. At these concentrations, COS exhibited superior preservation efficacy to potassium sorbate on the first day post-treatment (Figure 3C).

The findings from both methods showed relative consistency. On the second day, the < 3 kDa COS group exhibited significant fruit preservation efficacy, whereas the < 1 kDa and < 2 kDa COS groups demonstrated lesser effectiveness. Additionally, experimental outcomes suggested a correlation between the preservation efficacy of COS on tomatoes and its average molecular weights (Figure 4C, D).

![](https://static.igem.wiki/teams/5549/results/figure-4-1.avif)

**Figure 4 Fruit preservation performance of COS with different average
molecular weights (< 1 kDa, < 2 kDa, and < 3 kDa) on tomatoes.** (**A**) Colony counts after direct treatment.
(**B**) Colony counts after integral crushing treatment. (**C**) Comparison between the COS-treated group
and the sterile water control group on Day 2 post-direct-treatment. (**D**) Comparison among COS-treated groups on
Day 2 post-direct-treatment. (**E**) Comparison of fruit preservation performance between COS (< 3 kDa) and potassium
sorbate in the tomato sample on Day 3 post-treatment.

#### 3.2.2 Fruit Preservation

##### 3.2.2.1 Preservation Effects of COS on Fruit Preservation

To evaluate the preservation efficacy of COS with various average molecular weights (< 3 kDa, < 2 kDa, < 1 kDa) on fruits, tomatoes were selected as the test subject. The initial method entailed a direct approach to isolate microorganisms present on the surface, while the second method, known as integral crushing treatment, was designed to gather both surface and internal microorganisms (see [Experiments](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27experiments%27%29%20%7D%7D) for details). By integrating these two methods, a comprehensive assessment of the overall microbial contamination on tomatoes was achieved.

The findings from both methods showed relative consistency. On the second day, the < 3 kDa COS group exhibited significant fruit preservation efficacy, whereas the < 1 kDa and < 2 kDa COS groups demonstrated lesser effectiveness. Additionally, experimental outcomes suggested a correlation between the preservation efficacy of COS on tomatoes and its average molecular weights (Figure 4C, D).

##### 3.2.2.2 Comparison of Fruit Preservation Performance Between COS and Potassium Sorbate

Similarly, we compared the efficacy of COS in preserving fruits with that of potassium sorbate. Preservation trials were performed on tomato samples using 20 mg/mL of COS (average molecular weights < 3 kDa) and 0.2% (w/v) potassium sorbate. By the third day post-treatment, both COS and potassium sorbate demonstrated significant and comparable antibacterial properties, showing significant differences from the sterile water control group (Figure 4E).

## 4 Screening for superior chitosanase

Current production methods for COS involve the degradation of chitosan, a deacetylated derivative of crustacean waste, and fall into three primary categories: chemical, physical, and enzymatic degradation (3). Chemical degradation utilizes acids, alkalis, or oxidizers, providing low cost and high efficiency but resulting in significant environmental pollution (4). In contrast, physical degradation methods, such as ultrasound or microwave-assisted techniques, are eco-friendly but less efficient (5).

Enzymatic degradation, which employs chitosanase and other enzymes to specifically cleave glycosidic bonds under mild conditions (ambient temperature and near-neutral pH), is the preferred method due to its eco-friendliness and efficient. This method produces products with uniform degree of polymerization (DP) and preserved biological activity, which are ideal for high-value industries like food and pharmaceuticals. Nonetheless, the high cost of enzymes limits its scalability (3).

In the CRUSTA project, aiming for rapid and sustainable production of COS, enzymatic degradation has been selected. After reviewing the literature, we chose 6 kinds of chitosanases and constructed a mutant library, targeting the identification of a chitosanase mutant that optimally degrades chitosan into COS with enhanced antibacterial properties (see our [Design](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27design%27%29%20%7D%7D) page for details).

### 4.1 Preliminary Screening of Chitosanases

The targeted sequences BBa\_25E8EHJF, BBa\_25JJYAF6, BBa\_25JA5N69, BBa\_25RYX5T9, BBa\_25WTOTOC, BBa\_252ULD2I were individually inserted in pET-28a(+) to construct circuits (Table 1), which were transformed into *E. coli* BL21(DE3) to express each enzyme. The positive transformants were confirmed by kanamycin, colony PCR (Figure 5), and sequencing.

**Table 1 The part information of six wild-type chitosanases.**

| Name | Type | Description |
| --- | --- | --- |
| [BBa\_25E8EHJF](https://registry.igem.org/parts/bba-25e8ehjf) | coding | His tag-*BamCsn* |
| [BBa\_25JJYAF6](https://registry.igem.org/parts/bba-25jjyaf6) | coding | His tag-*CsnMY002*-His tag |
||  |  |  |
| --- | --- | --- |
| [BBa\_25JA5N69](https://registry.igem.org/parts/bba-25ja5n69) | coding | His tag-*TK Glm* |
| [BBa\_25RYX5T9](https://registry.igem.org/parts/bba-25ryx5t9) | coding | His tag-*CsnCA WT*-His tag |
| [BBa\_25WTOTOC](https://registry.igem.org/parts/bba-25wtotoc) | coding | *ChoA WT* |
| [BBa\_252ULD2I](https://registry.igem.org/parts/bba-252uld2i) | coding | *SaCsn46A WT* |

![](https://static.igem.wiki/teams/5549/engineeringsuccess/es-figure-10.avif)

**Figure 5 DNA gel electrophoresis of the colony PCR products
of BamCsn (A), CsnMY002 (B), GlmTk (C), CsnCA WT (D), McChoA WT (E) and SaCsn46A WT
(F)\_pET-28a(+) in *E. coli* BL21(DE3).** Target bands of BBa\_25JA5N69 can be observed at the position
between 2000 bp and 3000 bp, the others target can be observed at the position between 1000 bp and 2000 bp.

After being cultivated and induced under responsive conditions (see our [Experiments](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27experiments%27%29%20%7D%7D) page for details), we lysed the cells *via* sonication and collected the supernatant of the lysate. SDS-PAGE analysis and Coomassie blue staining were used to verify the expression of the target protein (Figure 6).

![](https://static.igem.wiki/teams/5549/results/figure-6.avif)

**Figure 6 SDS-PAGE analysis**.
BamCsn (**A**), CsnMY002 (**B**), GlmTk (**C**), CsnCA (**D**), McChoA (**E**), and SaCsn46A (**F**).
Target bands can be observed between 25 kDa and 35 kDa, and the GlmTk can be observed between 80 kDa and 150 kDa.

Chitosan powder was added to the supernatant of lysate, followed by incubation at 37°C for 18 h. The supernatant obtained thereafter was designated as the degradation product. The degradation product was then co-cultured with the test strain (*E. coli* BL21(DE3), OD600 ~0.6) in a 1:1 ratio. The OD600 of the co-culture was measured at 2 h and 8 h. The antibacterial activity of the enzymatic degradation product assessed by calculating the relative ΔOD600 value as (OD600 at 8 h - OD600 at 2 h) / OD600 at 2 h. The results were subsequently plotted (Figure 7).

![](https://static.igem.wiki/teams/5549/results/figure-7.avif)

**Figure 7 Antibacterial activity of degradation products of BamCsn, CsnMY002,
GlmATK, CsnCA WT, McChoA WT, SaCsn46A WT.**

Based on the preliminary experiment results, we evaluated the antibacterial efficacy of the enzymatic degradation products from six chitosanases. Notably, the enzymatic degradation products of CsnMY002, CsnCA, McChoA, and SaCsn46A displayed significant antibacterial effects. Nevertheless, the antibacterial activity of their degradation products was inadequate for optimal preservation that we had to conducted random mutagenesis on these four chitosanases to generate mutants for further screening.

### 4.2 Secondary Screening of Chitosanases

Detailed steps for circuit construction and induced expression are outlined on our Experiments page. Ultimately, we successfully obtained 103 mutants of SaCsn46A, 18 mutants of CsnCA, and 19 mutants of McChoA (Table 2). Unfortunately, despite multiple attempts, we were unable to successfully construct genetic circuits containing fragments of CsnMY002 mutant. Therefore, we try to focuses on evaluating the antibacterial activity of the degradation products from the SaCsn46A, CsnCA, and McChoA.

**Table 2 The part information of CsnCA, choA and SaCsn46A and their mutants.**

| Name | Type | Description |
| --- | --- | --- |
| [BBa\_25RYX5T9](https://registry.igem.org/parts/bba-25ryx5t9) | coding | His tag-*CsnCA WT*-His tag |
| [BBa\_25LX1A6G](https://registry.igem.org/parts/bba-25lx1a6g) | coding | His tag-*CsnCA mutant 1*-His tag |
| [BBa\_25DI3KWQ](https://registry.igem.org/parts/bba-25di3kwq) | coding | His tag-*CsnCA mutant 2*-His tag |
| [BBa\_252D8O5O](https://registry.igem.org/parts/bba-252d8o5o) | coding | His tag-*CsnCA mutant 3*-His tag |
| [BBa\_25E80BM9](https://registry.igem.org/parts/bba-25e80bm9) | coding | His tag-*CsnCA mutant 4*-His tag |
| [BBa\_25K6ON5C](https://registry.igem.org/parts/bba-25k6on5c) | coding | His tag-*CsnCA mutant 5*-His tag |
| [BBa\_259U04NM](https://registry.igem.org/parts/bba-259u04nm) | coding | His tag-*CsnCA mutant 6*-His tag |
| [BBa\_25799RU6](https://registry.igem.org/parts/bba-25799ru6) | coding | His tag-*CsnCA mutant 7*-His tag |
| [BBa\_25XKCUE1](https://registry.igem.org/parts/bba-25xkcue1) | coding | His tag-*CsnCA mutant 8*-His tag |
| [BBa\_25YI9KB2](https://registry.igem.org/parts/bba-25yi9kb2) | coding | His tag-*CsnCA mutant 9*-His tag |
| [BBa\_259WUUDL](https://registry.igem.org/parts/bba-259wuudl) | coding | His tag-*CsnCA mutant 10*-His tag |
| [BBa\_25A60H32](https://registry.igem.org/parts/bba-25a60h32) | coding | His tag-*CsnCA mutant 11*-His tag |
| [BBa\_25M545CS](https://registry.igem.org/parts/bba-25m545cs) | coding | His tag-*CsnCA mutant 12*-His tag |
| [BBa\_25KI55JT](https://registry.igem.org/parts/bba-25ki55jt) | coding | His tag-*CsnCA mutant 13*-His tag |
| [BBa\_25GYNC23](https://registry.igem.org/parts/bba-25gync23) | coding | His tag-*CsnCA mutant 14*-His tag |
| [BBa\_25T16YJ9](https://registry.igem.org/parts/bba-25t16yj9) | coding | His tag-*CsnCA mutant 15*-His tag |
| [BBa\_25M63R0Q](https://registry.igem.org/parts/bba-25m63r0q) | coding | His tag-*CsnCA mutant 16*-His tag |
| [BBa\_25KPZ427](https://registry.igem.org/parts/bba-25kpz427) | coding | His tag-*CsnCA mutant 17*-His tag |
| [BBa\_25WM872F](https://registry.igem.org/parts/bba-25wm872f) | coding | His tag-*CsnCA mutant 18*-His tag |
| [BBa\_25WTOTOC](https://registry.igem.org/parts/bba-25wtotoc) | coding | *choA WT* |
| [BBa\_25A1A11L](https://registry.igem.org/parts/bba-25a1a11l) | coding | *choA mutant 1* |
| [BBa\_25MAOZJ0](https://registry.igem.org/parts/bba-25maozj0) | coding | *choA mutant 2* |
| [BBa\_25Y19CZ9](https://registry.igem.org/parts/bba-25y19cz9) | coding | *choA mutant 3* |
| [BBa\_25P8LM1P](https://registry.igem.org/parts/bba-25p8lm1p) | coding | *choA mutant 4* |
| [BBa\_253P7MX6](https://registry.igem.org/parts/bba-253p7mx6) | coding | *choA mutant 5* |
| [BBa\_259O41R8](https://registry.igem.org/parts/bba-259o41r8) | coding | *choA mutant 6* |
| [BBa\_253UIYME](https://registry.igem.org/parts/bba-253uiyme) | coding | *choA mutant 7* |
| [BBa\_254UDM1Z](https://registry.igem.org/parts/bba-254udm1z) | coding | *choA mutant 8* |
| [BBa\_25ZVSFWR](https://registry.igem.org/parts/bba-25zvsfwr) | coding | *choA mutant 9* |
| [BBa\_25NYZ8AH](https://registry.igem.org/parts/bba-25nyz8ah) | coding | *choA mutant 10* |
| [BBa\_25AKH39Z](https://registry.igem.org/parts/bba-25akh39z) | coding | *choA mutant 11* |
| [BBa\_25A9P137](https://registry.igem.org/parts/bba-25a9p137) | coding | *choA mutant 12* |
| [BBa\_25HIZKM3](https://registry.igem.org/parts/bba-25hizkm3) | coding | *choA mutant 13* |
| [BBa\_25Z6C7H3](https://registry.igem.org/parts/bba-25z6c7h3) | coding | *choA mutant 14* |
| [BBa\_250TKTAH](https://registry.igem.org/parts/bba-250tktah) | coding | *choA mutant 15* |
| [BBa\_25ZU8KJS](https://registry.igem.org/parts/bba-25zu8kjs) | coding | *choA mutant 16* |
| [BBa\_25ZOLOG5](https://registry.igem.org/parts/bba-25zolog5) | coding | *choA mutant 17* |
| [BBa\_256KNZ1M](https://registry.igem.org/parts/bba-256knz1m) | coding | *choA mutant 18* |
| [BBa\_25PYE6JQ](https://registry.igem.org/parts/bba-25pye6jq) | coding | *choA mutant 19* |
| [BBa\_252ULD2I](https://registry.igem.org/parts/bba-252uld2i) | coding | *SaCsn46A WT* |
| [BBa\_25XD48E3](https://registry.igem.org/parts/bba-25xd48e3) | coding | *SaCsn46A mutant 1* |
| [BBa\_25C3NO7W](https://registry.igem.org/parts/bba-25c3no7w) | coding | *SaCsn46A mutant 2* |
| [BBa\_25E0NREY](https://registry.igem.org/parts/bba-25e0nrey) | coding | *SaCsn46A mutant 3* |
| [BBa\_25B647RD](https://registry.igem.org/parts/bba-25b647rd) | coding | *SaCsn46A mutant 4* |
| [BBa\_25Q1WAOS](https://registry.igem.org/parts/bba-25q1waos) | coding | *SaCsn46A mutant 5* |
| [BBa\_250A7JXW](https://registry.igem.org/parts/bba-250a7jxw) | coding | *SaCsn46A mutant 6* |
| [BBa\_25JNYQPJ](https://registry.igem.org/parts/bba-25jnyqpj) | coding | *SaCsn46A mutant 7* |
| [BBa\_252VKL28](https://registry.igem.org/parts/bba-252vkl28) | coding | *SaCsn46A mutant 8* |
| [BBa\_25UE49BR](https://registry.igem.org/parts/bba-25ue49br) | coding | *SaCsn46A mutant 9* |
| [BBa\_25W38YVQ](https://registry.igem.org/parts/bba-25w38yvq) | coding | *SaCsn46A mutant 10* |
| [BBa\_25RFSTGN](https://registry.igem.org/parts/bba-25rfstgn) | coding | *SaCsn46A mutant 11* |
| [BBa\_258HOJLV](https://registry.igem.org/parts/bba-258hojlv) | coding | *SaCsn46A mutant 12* |
| [BBa\_257CYVFF](https://registry.igem.org/parts/bba-257cyvff) | coding | *SaCsn46A mutant 13* |
| [BBa\_25W5AGTG](https://registry.igem.org/parts/bba-25w5agtg) | coding | *SaCsn46A mutant 14* |
| [BBa\_25682DOT](https://registry.igem.org/parts/bba-25682dot) | coding | *SaCsn46A mutant 15* |
| [BBa\_25VRUXTC](https://registry.igem.org/parts/bba-25vruxtc) | coding | *SaCsn46A mutant 16* |
| [BBa\_25IE36XD](https://registry.igem.org/parts/bba-25ie36xd) | coding | *SaCsn46A mutant 17* |
| [BBa\_25L5FGZS](https://registry.igem.org/parts/bba-25l5fgzs) | coding | *SaCsn46A mutant 18* |
| [BBa\_25YWISOC](https://registry.igem.org/parts/bba-25ywisoc) | coding | *SaCsn46A mutant 19* |
| [BBa\_25X1GS95](https://registry.igem.org/parts/bba-25x1gs95) | coding | *SaCsn46A mutant 20* |
| [BBa\_25T0LH3A](https://registry.igem.org/parts/bba-25t0lh3a) | coding | *SaCsn46A mutant 21* |
| [BBa\_25R4MUWT](https://registry.igem.org/parts/bba-25r4muwt) | coding | *SaCsn46A mutant 22* |
| [BBa\_254NCO54](https://registry.igem.org/parts/bba-254nco54) | coding | *SaCsn46A mutant 23* |
| [BBa\_25WQW2YO](https://registry.igem.org/parts/bba-25wqw2yo) | coding | *SaCsn46A mutant 24* |
| [BBa\_258RQEX5](https://registry.igem.org/parts/bba-258rqex5) | coding | *SaCsn46A mutant 25* |
| [BBa\_25M9X69O](https://registry.igem.org/parts/bba-25m9x69o) | coding | *SaCsn46A mutant 26* |
| [BBa\_25I3F15Z](https://registry.igem.org/parts/bba-25i3f15z) | coding | *SaCsn46A mutant 27* |
| [BBa\_25KXYX3Y](https://registry.igem.org/parts/bba-25kxyx3y) | coding | *SaCsn46A mutant 28* |
| [BBa\_257L0RTR](https://registry.igem.org/parts/bba-257l0rtr) | coding | *SaCsn46A mutant 29* |
| [BBa\_25LAIPPV](https://registry.igem.org/parts/bba-25laippv) | coding | *SaCsn46A mutant 30* |
| [BBa\_25B21OLA](https://registry.igem.org/parts/bba-25b21ola) | coding | *SaCsn46A mutant 31* |
| [BBa\_25SAKIFU](https://registry.igem.org/parts/bba-25sakifu) | coding | *SaCsn46A mutant 32* |
| [BBa\_257W6LLR](https://registry.igem.org/parts/bba-257w6llr) | coding | *SaCsn46A mutant 33* |
| [BBa\_25L960X7](https://registry.igem.org/parts/bba-25l960x7) | coding | *SaCsn46A mutant 34* |
| [BBa\_25MY1OIP](https://registry.igem.org/parts/bba-25my1oip) | coding | *SaCsn46A mutant 35* |
| [BBa\_25RU8PZ1](https://registry.igem.org/parts/bba-25ru8pz1) | coding | *SaCsn46A mutant 36* |
| [BBa\_25ADM8S2](https://registry.igem.org/parts/bba-25adm8s2) | coding | *SaCsn46A mutant 37* |
| [BBa\_25HJZ0VQ](https://registry.igem.org/parts/bba-25hjz0vq) | coding | *SaCsn46A mutant 38* |
| [BBa\_25TA048W](https://registry.igem.org/parts/bba-25ta048w) | coding | *SaCsn46A mutant 39* |
| [BBa\_25WXF73A](https://registry.igem.org/parts/bba-25wxf73a) | coding | *SaCsn46A mutant 40* |
| [BBa\_25T3AL3W](https://registry.igem.org/parts/bba-25t3al3w) | coding | *SaCsn46A mutant 41* |
| [BBa\_25E1S0EF](https://registry.igem.org/parts/bba-25e1s0ef) | coding | *SaCsn46A mutant 42* |
| [BBa\_256H3PNY](https://registry.igem.org/parts/bba-256h3pny) | coding | *SaCsn46A mutant 43* |
| [BBa\_255TVPWF](https://registry.igem.org/parts/bba-255tvpwf) | coding | *SaCsn46A mutant 44* |
| [BBa\_25GAM8A7](https://registry.igem.org/parts/bba-25gam8a7) | coding | *SaCsn46A mutant 45* |
| [BBa\_25ZLOQ2M](https://registry.igem.org/parts/bba-25zloq2m) | coding | *SaCsn46A mutant 46* |
| [BBa\_25Z07ACA](https://registry.igem.org/parts/bba-25z07aca) | coding | *SaCsn46A mutant 47* |
| [BBa\_256DW4H3](https://registry.igem.org/parts/bba-256dw4h3) | coding | *SaCsn46A mutant 48* |
| [BBa\_251DHXHJ](https://registry.igem.org/parts/bba-251dhxhj) | coding | *SaCsn46A mutant 49* |
| [BBa\_25L6N3YO](https://registry.igem.org/parts/bba-25l6n3yo) | coding | *SaCsn46A mutant 50* |
| [BBa\_25HZGD5R](https://registry.igem.org/parts/bba-25hzg-d5r) | coding | *SaCsn46A mutant 51* |
| [BBa\_25SEHU78](https://registry.igem.org/parts/bba-25seh-u78) | coding | *SaCsn46A mutant 52* |
| [BBa\_258KPVTK](https://registry.igem.org/parts/bba-258kp-vtk) | coding | *SaCsn46A mutant 53* |
| [BBa\_259M7QOX](https://registry.igem.org/parts/bba-259m7-qox) | coding | *SaCsn46A mutant 54* |
| [BBa\_255GCY7H](https://registry.igem.org/parts/bba-255gc-y7h) | coding | *SaCsn46A mutant 55* |
| [BBa\_2503GTSJ](https://registry.igem.org/parts/bba-2503g-tsj) | coding | *SaCsn46A mutant 56* |
| [BBa\_25LIHI5W](https://registry.igem.org/parts/bba-25lih-i5w) | coding | *SaCsn46A mutant 57* |
| [BBa\_25CKVDJK](https://registry.igem.org/parts/bba-25ckv-djk) | coding | *SaCsn46A mutant 58* |
| [BBa\_256X6YL0](https://registry.igem.org/parts/bba-256x6-yl0) | coding | *SaCsn46A mutant 59* |
| [BBa\_25UUAIRI](https://registry.igem.org/parts/bba-25uua-iri) | coding | *SaCsn46A mutant 60* |
| [BBa\_25Q796Q5](https://registry.igem.org/parts/bba-25q79-6q5) | coding | *SaCsn46A mutant 61* |
| [BBa\_25QH5I0Y](https://registry.igem.org/parts/bba-25qh5-i0y) | coding | *SaCsn46A mutant 62* |
| [BBa\_2590JOGY](https://registry.igem.org/parts/bba-2590j-ogy) | coding | *SaCsn46A mutant 63* |
| [BBa\_25DU2G7L](https://registry.igem.org/parts/bba-25du2-g7l) | coding | *SaCsn46A mutant 64* |
| [BBa\_25JT9PV3](https://registry.igem.org/parts/bba-25jt9-pv3) | coding | *SaCsn46A mutant 65* |
| [BBa\_2554HT9A](https://registry.igem.org/parts/bba-2554h-t9a) | coding | *SaCsn46A mutant 66* |
| [BBa\_25VPJ432](https://registry.igem.org/parts/bba-25vpj-432) | coding | *SaCsn46A mutant 67* |
| [BBa\_25GLU39E](https://registry.igem.org/parts/bba-25glu-39e) | coding | *SaCsn46A mutant 68* |
| [BBa\_25OIP5EL](https://registry.igem.org/parts/bba-25oip-5el) | coding | *SaCsn46A mutant 69* |
| [BBa\_25ANZHXL](https://registry.igem.org/parts/bba-25anz-hxl) | coding | *SaCsn46A mutant 70* |
| [BBa\_2585F9UN](https://registry.igem.org/parts/bba-2585f-9un) | coding | *SaCsn46A mutant 71* |
| [BBa\_252BO8IV](https://registry.igem.org/parts/bba-252bo-8iv) | coding | *SaCsn46A mutant 72* |
| [BBa\_253NFEFP](https://registry.igem.org/parts/bba-253nf-efp) | coding | *SaCsn46A mutant 73* |
| [BBa\_25EBHMXR](https://registry.igem.org/parts/bba-25ebh-mxr) | coding | *SaCsn46A mutant 74* |
| [BBa\_25U8RXOH](https://registry.igem.org/parts/bba-25u8r-xoh) | coding | *SaCsn46A mutant 75* |
| [BBa\_25ZNEWQ9](https://registry.igem.org/parts/bba-25zne-wq9) | coding | *SaCsn46A mutant 76* |
| [BBa\_25CDNYR9](https://registry.igem.org/parts/bba-25cdn-yr9) | coding | *SaCsn46A mutant 77* |
| [BBa\_25POD1FL](https://registry.igem.org/parts/bba-25pod-1fl) | coding | *SaCsn46A mutant 78* |
| [BBa\_252EYBZ1](https://registry.igem.org/parts/bba-252ey-bz1) | coding | *SaCsn46A mutant 79* |
| [BBa\_25X95C47](https://registry.igem.org/parts/bba-25x95-c47) | coding | *SaCsn46A mutant 80* |
| [BBa\_2594Q7VY](https://registry.igem.org/parts/bba-2594q-7vy) | coding | *SaCsn46A mutant 81* |
| [BBa\_251Q8TAO](https://registry.igem.org/parts/bba-251q8-tao) | coding | *SaCsn46A mutant 82* |
| [BBa\_253ZGO14](https://registry.igem.org/parts/bba-253zg-o14) | coding | *SaCsn46A mutant 83* |
| [BBa\_253Z6R8Q](https://registry.igem.org/parts/bba-253z6-r8q) | coding | *SaCsn46A mutant 84* |
| [BBa\_259HL2YZ](https://registry.igem.org/parts/bba-259hl-2yz) | coding | *SaCsn46A mutant 85* |
| [BBa\_252H9N6O](https://registry.igem.org/parts/bba-252h9-n6o) | coding | *SaCsn46A mutant 86* |
| [BBa\_25TRDY7X](https://registry.igem.org/parts/bba-25trd-y7x) | coding | *SaCsn46A mutant 87* |
| [BBa\_25LWIXHH](https://registry.igem.org/parts/bba-25lwi-xhh) | coding | *SaCsn46A mutant 88* |
| [BBa\_25VB41VH](https://registry.igem.org/parts/bba-25vb4-1vh) | coding | *SaCsn46A mutant 89* |
| [BBa\_25T2LPXB](https://registry.igem.org/parts/bba-25t2l-pxb) | coding | *SaCsn46A mutant 90* |
| [BBa\_252ATZCM](https://registry.igem.org/parts/bba-252at-zcm) | coding | *SaCsn46A mutant 91* |
| [BBa\_25ERWOEW](https://registry.igem.org/parts/bba-25erw-oew) | coding | *SaCsn46A mutant 92* |
| [BBa\_25UBLCW3](https://registry.igem.org/parts/bba-25ubl-cw3) | coding | *SaCsn46A mutant 93* |
| [BBa\_254P0M23](https://registry.igem.org/parts/bba-254p0-m23) | coding | *SaCsn46A mutant 94* |
| [BBa\_2546QW2Q](https://registry.igem.org/parts/bba-2546q-w2q) | coding | *SaCsn46A mutant 95* |
| [BBa\_251RV8Q8](https://registry.igem.org/parts/bba-251rv-8q8) | coding | *SaCsn46A mutant 96* |
| [BBa\_25LJVK8C](https://registry.igem.org/parts/bba-25ljv-k8c) | coding | *SaCsn46A mutant 97* |
| [BBa\_25QBEBAY](https://registry.igem.org/parts/bba-25qbe-bay) | coding | *SaCsn46A mutant 98* |
| [BBa\_250OH2JX](https://registry.igem.org/parts/bba-250oh-2jx) | coding | *SaCsn46A mutant 99* |
| [BBa\_25XVN534](https://registry.igem.org/parts/bba-25xvn-534) | coding | *SaCsn46A mutant 100* |
| [BBa\_25RAKHSZ](https://registry.igem.org/parts/bba-25rak-hsz) | coding | *SaCsn46A mutant 101* |
| [BBa\_256X475P](https://registry.igem.org/parts/bba-256x4-75p) | coding | *SaCsn46A mutant 102* |
| [BBa\_25EGHZXZ](https://registry.igem.org/parts/bba-25egh-zxz) | coding | *SaCsn46A mutant 103* |

The targeted sequences were inserted in pET-28a(+) to construct circuits, which were transformed into *E. coli* JM109(DE3) to express each enzyme. The positive transformants were confirmed by kanamycin, colony PCR (see our [Parts](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27parts%27%29%20%7D%7D) page for details), and sequencing. After being cultivated and induced under responsive conditions (see our [Experiments](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27experiments%27%29%20%7D%7D) page for details), we lysed the cells vis sonication and collected the lysate supernatant. Chitosan powder was added to the lysate supernatant, followed by incubation at 37°C for 18 h. The resulting supernatant was collected as the degradation product.

The degradation products of SaCsn46A, McChoA, and CsnCA mutants were separately co-cultured with *E. coli* BL21(DE3) at a 1:1 ratio. The initial optical density (OD600) of the test strains used for SaCsn46A mutants, McChoA mutants and CsnCA mutants was 0.8, 0.8, and 0.6, respectively. OD600 measurements in co-culture systems were recorded at 1.5 h and 8 h for SaCsn46A and CsnCA mutants, and at 2 h and 8 h for McChoA mutants. Antibacterial activity of degradation products from SaCsn46A mutants was evaluated using the relative ΔOD600 value, and calculated by the following formula:

Relative ΔOD600 value = (OD600 at 8 h - OD600 at t0) / OD600 at t0

where t₀ refers to the earlier measurement time point.

Through the exploration of the antibacterial effects of SaCsn46A WT, McChoA WT, CsnCA WT and their mutants,
we successfully obtained mutants with different antibacterial efficiencies of degradation products.
The relative OD₆₀₀ values of most McChoA mutants fluctuates slightly around the EV baseline (Figure 8B),
with no mutants showing extremely enhanced or weakened antibacterial activity of their enzymatic degradation products,
and no mutants exhibit extreme changes in the antibacterial activity of their enzymatic degradation products.
The overall antibacterial performance of CsnCA mutants is stable (Figure 8C), and mutations do not induce substantial alterations
in their antibacterial efficacy. Notably, SaCsn46A mutants demonstrated two key advantages. Firstly, we obtained more SaCsn46A mutants,
and these mutants exhibited greater diversity in mutation sites and a broader range of antibacterial activity in their enzymatic
degradation products. Secondly, they exhibited better antibacterial activities (Figure 8A). Therefore, we selected the six SaCsn46A
mutants that showed the most promising results for further analysis. These included:
[SaCsn46A mutant 2](https://registry.igem.org/parts/bba-25c3no7w) (SaCsn46A E151G D222G),
[SaCsn46A mutant 14](https://registry.igem.org/parts/bba-25w5agtg) (SaCsn46A N29S K50R D150G D189G F247L),
[SaCsn46A mutant 16](https://registry.igem.org/parts/bba-25vruxtc) (SaCsn46A V52A D134G K165I F175L S190C G210D K250E N252D),
[SaCsn46A mutant 60](https://registry.igem.org/parts/bba-25uuairi) (SaCsn46A D40G Q125R H234R), and
[SaCsn46A mutant 94](https://registry.igem.org/parts/bba-254p0m23) (SaCsn46A T249A L259P).

![](https://static.igem.wiki/teams/5549/results/figure-8.avif)

**Figure 8 Antibacterial activity of enzymatic degradation products
from wild-type (WT) and mutant chitosanases.** (**A**) SaCsn46A mutants. (**B**) McChoA mutants. (**C**)
CsnCA mutants. BL: sterile water, EV: empty vector pET-28a(+), WT: SaCsn46A WT, McChoA WT and CsnCA WT respectively.
Relative OD600 values were normalized using empty vector pET-28a(+) as the baseline value of 1.

### 4.3 Tertiary Screening of Chitosanases

At this stage, following cultivation and induction, the GE AKTA Prime Plus FPLC System was employed to purify proteins
from the lysate supernatant of these six SaCsn46A mutants. Ultimately, these six SaCsn46A mutants were purified and normalized to
a uniform concentration of 0.2207 mg/mL. In contrast, the protein concentration of [SaCsn46A mutant 2](https://registry.igem.org/parts/bba-25c3no7w) (SaCsn46A E151G D222G) was 0.0167
mg/mL, and that of [SaCsn46A WT](https://registry.igem.org/parts/bba-252uld2i) was 0.1607 mg/mL. The expression of the target protein was confirmed using SDS-PAGE and Coomassie blue
staining (Figure 9).

![](https://static.igem.wiki/teams/5549/results/figure-9.avif)

**Figure 9 SDS-PAGE analysis of SaCsn46A and its mutants.** Target
bands (about 30.5 kDa) can be observed between 25 kDa and 35 kDa.

Employing the same screening method as in the previous section, we evaluated the antibacterial activity of their degradation products.
As shown in Figure 10, [SaCsn46A mutant 2](https://registry.igem.org/parts/bba-25c3no7w)
(E151G D222G) and [SaCsn46A mutant 60](https://registry.igem.org/parts/bba-25uuairi) (D40G Q125R H234R) exhibited superior performance, with their enzymatic degradation products demonstrating superior antibacterial activity. Consequently, these two mutants were therefore selected for subsequent analysis of fundamental enzymatic properties.

![](https://static.igem.wiki/teams/5549/results/figure-10.avif)

**Figure 10 Antibacterial activity of enzymatic degradation products
of SaCsn46A WT and its mutants under fixed protein concentration conditions.**

## 5 Enzymatic Characterization of Chitosanase Mutants

### 5.1 Enzymatic property characterization experiments of Purified Chitosanase

Following the selection of [SaCsn46A mutant 2](https://registry.igem.org/parts/bba-25c3no7w) (E151G D222G) and [SaCsn46A mutant 60](https://registry.igem.org/parts/bba-25uuairi) (D40G Q125R H234R),
further analysis was carried out to assess their optimal temperature, pH and stability.

Chitosanase activity was measured using the 3,5-dinitrosalicylic acid (DNS) method. The quantity of reducing
sugar was determined by measuring optical density (OD) at 540 nm using D-glucose as standard. The reaction
mixture was incubated at 100°C for 5 min. One unit (U) of chitosanase activity is defined as the amount of
enzyme that liberates 1 mmol of D-glucose-equivalent reducing sugars per minute under these conditions.

Details on the characterization methods for chitosanase properties can be found in the [Experiments](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27experiments%27%29%20%7D%7D) section.

The optimal reaction temperature of [SaCsn46A mutant 60](https://registry.igem.org/parts/bba-25uuairi) (D40G Q125R H234R) is approximately 40°C, where it
maintains relatively high activity even at 45°C, albeit with a slight decrease. In contrast, SaCsn46A mutant
2 (E151G D222G) displays a different temperature profile, experiencing a significant decline in activity at
40°C, which unexpectedly recovers at 45°C, returning to levels observed at 35°C (Figure 11A). The peak
chitosanase activity for [SaCsn46A mutant 2](https://registry.igem.org/parts/bba-25c3no7w) (E151G D222G) and [SaCsn46A mutant 60](https://registry.igem.org/parts/bba-25uuairi) (D40G Q125R H234R) occur at
pH 5.0 and pH 4.0, respectively (Figure 11B). Thermostability analysis indicates that both mutants exhibited
greater stability below 50°C. A minor increase in activity was observed around 40°C, which generally remains
stable across this temperature spectrum (Figure 11C). Regarding pH stability, [SaCsn46A mutant 2](https://registry.igem.org/parts/bba-25c3no7w) (E151G
D222G) shows robust stability between pH 6 and 8, whereas [SaCsn46A mutant 60](https://registry.igem.org/parts/bba-25uuairi) (D40G Q125R H234R) is notably
stable at pH 5 (Figure 11D).

![](https://static.igem.wiki/teams/5549/results/figure-11.avif)

**Figure 11 Enzymatic properties of SaCsn46A mutant 2 and 60.**
(**A**) Effects of temperature conditions on enzyme activity. (**B**) Effects of pH conditions on enzyme activity.
(**C**) Thermostability. (**D**) pH stability. The 100% relative activity of mutant 2 was 2.385 U/mg, while the 100%
relative activity of mutant 60 was 2.124 U/mg.

### 5.2 hydrolytic properties of chitosanase

#### 5.2.1 Characterization Results of the DP for COS Mixture Standards

To facilitate subsequent characterization, we utilized COS standards for High Performance Liquid
Chromatography (HPLC) and Thin-Layer Chromatography (TLC) analysis. The objective was to pinpoint the
standard peak positions of COS with various DPs, thereby establishing a basis for characterizing the
degradation products of chitosanase in subsequent experiments.

We purchased COS mixture standards which consists of a mixture of COS with DPs ranging from 3 to 7.
Subsequently, we prepared a series of COS standard solutions at concentrations of 0.62, 1.24, 2.49, 4.98 and
9.95 mg/mL, which were then filtered through a 0.22 μm microporous membrane. Then, a Shodex Asahipak NH₂P-50
4E column (4.6 mm × 250 mm, 5 μm) was used to quantitative analyze the content of COS with different DPs in
the standard solutions (see [Experiments](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27experiments%27%29%20%7D%7D) for specific parameters). This procedure generated standard curves
plotted the peak area of COS (with varied DPs) against mass concentration (Figure 12), establishing a
foundational basis for the subsequent determination of COS with different DPs in degradation products.

![](https://static.igem.wiki/teams/5549/results/figure-12.avif)

**Figure 12 Standard curves of COS with different DPs.**
Relationship between peak area and mass concentration for (**A**) Glucosamine (DP1), (**B**) Chitobiose (DP2),
(**C**) Chitotriose (DP3), (**D**) Chitotetraose (DP4), (**E**) Chitopentaose (DP5), (**F**) Chitohexaose (DP6),
and (**G**) Chitoheptaose (DP7).

A series of COS mixture standard solutions with concentrations of 1.24, 2.49, 4.98, and 9.95 mg/mL were
prepared for TLC analysis, using a developing solvent system comprising isopropanol: water: ammonia in a 60:
30: 4 volume ratio. The COS mixture standard solutions with different concentrations were spotted on a
silica gel plate for separation. After spraying with ninhydrin solution and heating for color development,
fuchsin spots were observed at different Rf (retardation factor) values (Figure 13), indicating the presence
of amino-containing carbohydrates in all detected components. The standards showed uniform color
development, with clear spots, reasonable distribution, and no obvious tailing or impurity bands. This
confirms that the used developing system is suitable for the separation and detection of COS with different
DPs.

![](https://static.igem.wiki/teams/5549/results/figure-13.avif)

**Figure 13 TLC analysis of COS standards.** Lanes A-F
correspond to COS concentrations of 1.24, 2.49, 4.98 (duplicate), and 9.95 (duplicate) mg/mL,
respectively.

#### 5.2.2 Characterization Results of the DP for Commercial COS

After preliminary protein purification, we obtained six wild-type chitosanases. Subsequently, we conducted
degradation experiments and analyzed the DP of the degradation products from these enzymes. This analysis
aimed to identify the product spectrum of each enzyme, which will provide essential guidance for subsequent
research.

During conducting antimicrobial experiments using commercial COS with average molecular weights under 1 kDa,
2 kDa, and 3 kDa, we performed HPLC analysis on these COS solutions to investigate the effect of COS
composition on their antimicrobial activity. Solutions of 50 mg/mL for each COS type were prepared, filtered
through a 0.22 μm microporous membrane to yield samples, and then analyzed using HPLC with the same method
as that for the standard curves (Figure 14).

![](https://static.igem.wiki/teams/5549/results/figure-14.avif)

**Figure 14 HPLC analysis of commercial COS with different
DPs.** Chromatograms of COS with average molecular weights of (**A**) < 1 kDa, (**B**) < 2 kDa, and (**C**) < 3 kDa.

Among the commercial COS with average molecular weights < 1, < 2, and < 3 kDa, concentration
of COS with DPs ranging from 1 to 3 progressively decreased, while those with DPs ranging from 4 to 6
progressively increased. Moreover, the commercial COS with average molecular weights < 3 kDa had the lowest
content of low DPs COS.

Meanwhile, we performed TLC analysis on commercial COS samples with average molecular weights of < 1
kDa, < 2 kDa, and < 3 kDa to assess variations in COS composition across different DPs. Each sample
were developed using the same solvent system and subsequently visualized by spraying with ninhydrin
solution (Figure 15).

Results showed that multiple spots with different mobilities corresponding to COS components of
varying polymerization degrees Notably, as average molecular weights increased, the intensity of
spots associated with lower DPs diminished or disappeared altogether in the TLC profiles. This
phenomenon indicates that the sample with average molecular weights < 3 kDa encompasses a larger
proportion of high DPs COS, whereas the sample with average molecular weights < 1 kDa
predominantly contains lower polymerization components. These findings validate significant
disparities in COS composition ratios and imply a potential link to the observed variations in
biological activity in later antimicrobial experiments.

![](https://static.igem.wiki/teams/5549/results/figure-15.avif)

**Figure 15 TLC separation of COS with different
average molecular weights.** Lanes A, B, and C correspond to < 1 kDa, < 2 kDa, and < 3 kDa
fractions.

#### 5.2.3 Characterization Results of the DP of Degradation Products

After determining the optimal temperature and pH for [SaCsn46A mutant 2](https://registry.igem.org/parts/bba-25c3no7w) (SaCsn46A E151G D222G)
and [SaCsn46A mutant 60](https://registry.igem.org/parts/bba-25uuairi) (SaCsn46A D40G Q125R H234R), we purified the enzymes and obtained 0.1392
mg/mL of [SaCsn46A mutant 2](https://registry.igem.org/parts/bba-25c3no7w) (SaCsn46A E151G D222G) and 0.121 mg/mL of [SaCsn46A mutant 60](https://registry.igem.org/parts/bba-25uuairi)
(SaCsn46A D40G Q125R H234R). Degradation was then carried out for 30 minutes under optimal
conditions using pretreated chitosan solution as the substrate. We first performed TLC,
observing sample spots near the application point with minimal visibility elsewhere, leading us
to infer the presence of high-degree polymerization COS in the products (Figure 16). To further
characterize the distribution of oligomeric COS, we conducted HPLC analysis, obtaining the
chromatograms shown (Figure 17). Based on retention times, the product peaks of SaCsn46A mutant
2 (SaCsn46A E151G D222G) included glucosamine, chitobiose, chitotriose, among others, while
[SaCsn46A mutant 60](https://registry.igem.org/parts/bba-25uuairi) (SaCsn46A D40G Q125R H234R) product peaks consisted of glucosamine,
chitobiose, etc. Analysis of peak areas indicated that [SaCsn46A mutant 2](https://registry.igem.org/parts/bba-25c3no7w) (SaCsn46A E151G D222G)
exhibits higher degradation efficiency.

![](https://static.igem.wiki/teams/5549/part/tlc-enzyme-update-10-9.avif)

**Figure 16 TLC separation of products from the
degradation experiment under optimal conditions.** Lanes A, B, C, D correspond to glucosamine,
COS standards (DP 3-7) of equal mass at 50 mg/mL, degradation products of [SaCsn46A mutant 2](https://registry.igem.org/parts/bba-25c3no7w)
(SaCsn46A E151G D222G), degradation products of [SaCsn46A mutant 60](https://registry.igem.org/parts/bba-25uuairi) (SaCsn46A D40G Q125R
H234R).

![](https://static.igem.wiki/teams/5549/results/figure-17.avif)

**Figure 17 HPLC analysis of degradation products of [SaCsn46A mutant 2](https://registry.igem.org/parts/bba-25c3no7w) (SaCsn46A E151G D222G)
(A), [SaCsn46A mutant 60](https://registry.igem.org/parts/bba-25uuairi) (SaCsn46A D40G Q125R H234R) (B).**

**Table 3 The concentration (mg/mL) of COS with different DPs in degradation products of SaCsn46A mutant 2 and SaCsn46A mutant 60. Note: ND, not detected**

| Degree of Polymerization | 1 | 2 | 4 | 5 | 6 |
| --- | --- | --- | --- | --- | --- |
| SaCsn46A mutant 2 | 17.09 | 9.39 | 0.86 | ND | 10.23 |
| SaCsn46A mutant 60 | 5.72 | 3.33 | ND | 2.71 | ND |

## 6 Improvement of the Extracellular Secretion System

LMT, a Sec pathway signal peptide ([BBa\_K5136066](http://parts.igem.org/Part%3Abba-k5136066)), was first identified by our team from
the upstream region
of the lytic murein transglycosylase gene of Vibrio natriegens in 2021 ([SALVAGE](https://2021.igem.org/Team%3AXMU-China/Design)). Then in the following 2022
([OMEGA](https://2022.igem.wiki/XMU-China/design#T-P)), 2023 ([NAIADS](https://2023.igem.wiki/xmu-china/design)), 2024 ([REPARO](https://2024.igem.wiki/xmu-china/design)) projects, LMT was successfully utilized to
enable the extracellular
secretion of functional proteins. Further characterization of LMT alongside several other signal peptides in
2024 ([REPARO](https://2024.igem.wiki/xmu-china/design)) revealed that LMT achieved our secretion
requirements while minimizing metabolic burden, thus
demonstrating its superior performance in comparison to other signal peptides.

Compared to intracellular expression systems that might form inclusion bodies, secretion-expression systems
offer potential advantages for the production of recombinant proteins . Therefore, the signal peptide we
identified holds significant application values. In this year's work, we aim to improve the secretion
performance of LMT signal peptide to produce more recombinant proteins extracellularly, thus providing more
robust tools and deeper insights for future iGEM teams and even SynBio community.

We fused sfGFP ([BBa\_K5136028](https://registry.igem.org/parts/bba-k5136028)) as a reporter to the LMT signal peptide
(variants) and tested it in *E. coli*
DH5α (Figure 18). The fluorescence intensity (λex = 475 nm, λem = 515 nm) of bacterial
culture and
supernatant as well as OD600 were monitored as time progressed during characterizations (see our [Experiments](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27experiments%27%29%20%7D%7D)
for details).

![](https://static.igem.wiki/teams/5549/results/figure-18-1.avif)

**Figure 18 Characterization device for LMT signal peptide or
variants in *E. coli* DH5α.**

### 6.1 Basic of Sec secretion pathway and sequence features of LMT signal peptide

![](https://static.igem.wiki/teams/5549/results/figure19.avif)

**Figure 19 Sec pathway-dependent secretion process and the
corresponding sequence structures of signal peptide.** (**A**) Secretion of recombinant proteins through Sec
pathway. (**B**) The typical sequence features of signal peptide can be identified in the sequence of
LMT.

The Sec pathway facilitates the translocation of many proteins in bacteria, archaea, and eukaryotes across
the cytoplasmic or endoplasmic reticulum membrane (2). A classical Sec pathway signal peptide comprises
three distinct regions: an N-terminal region enriched in positive charges (N-region), a central hydrophobic
core (H-region), and a C-terminal region exhibiting strong polarity (C-region). In Sec pathway, proteins
first bind to SecB *via* the positively charged N-region of their signal peptide while SecB inhibits premature
folding of the protein. Subsequently, SecA recognizes the hydrophobic H-region of the signal peptide and
translocates the protein to the cell membrane, in which the SecYEG translocon acts as a channel, guiding the
protein to the periplasmic space. After the signal peptidase (SPase) recognizes and cleaves at the cleavage
site (AXA) in the C-region of signal peptide, the protein then correctly folds in the periplasm (Figure 19A)
(3-5).

Based on our previous work, to better understand the sequence features of LMT, we used [ProtParam](https://web.expasy.org/protparam/)
to calculate the positive charge intensity, hydrophobicity, and polarity
of the LMT sequence, thereby dividing the LMT signal peptide into N-region, H-region, and C-region (Figure
19B). It should be noted that these region partitions are not absolute but are based on a qualitative
analysis of the LMT sequence features.

![](https://static.igem.wiki/teams/5549/results/figure-20.avif)

**Figure 20 Probe the key *secA* and *secB* genes in *E. coli* DH5α
*via* colony PCR.** DNA gel electrophoresis of colony PCR products to probe *secA* (**A**) and *secB* (**B**). Target
bands, 2706 bp and 468 bp, can be observed at the position around 3000 bp and 500 bp, respectively.
Specific primers were designed according to the genome sequence of DH5α ([GCF\_002899475.1](https://www.ncbi.nlm.nih.gov/datasets/genome/GCF_002899475.1/)).

Before investigating the influence of some specific residues in this signal peptide on protein (here, sfGFP)
secretion, we firstly confirmed the presence of the key genes *secA* and *secB* of the Sec pathway in the
chassis *E. coli* DH5α *via* colony PCR (Figure 20), which set the foundation of downstream works and
characterizations. Two specific variants of the N-terminal positively charged region (presumed to be the
SecB binding region) and the C-terminal AXA cleavage site (SPase recognition site) were then constructed,
namely LMT Δ2-4 and LMT A19V, respectively (Figure 21A). Gibson Assembly was performed to construct the
expression module (BBa\_25G6VM6I and BBa\_25CWGZSX, respectively) of these variants at pSB1C3 vector and
positive transformants were selected and confirmed by colony PCR (Figure 21B) and sequencing.

![](https://static.igem.wiki/teams/5549/results/figure-21.avif)

**Figure 21 Construction of the expression modules of LMT
Δ2-4 and A19V.** (**A**) Sequence alignments of LMT and two variants. Mutated sites were colored in black. (**B**)
DNA gel electrophoresis of the colony PCR products of expression modules of LMT Δ2-4 and A19V fused with
sfgfp at pSB1C3 in DH5α. Target bands, 1383 bp and 1374 bp, can be observed at the position between 1500
bp and 1000 bp, respectively.

The fluorescence intensity of the two variants, no matter for the culture or supernatant, was much lower
than that of the wild-type (WT) LMT group (Figure 22B and 22C), although no significant growth defects due
to the modifications were observed (Figure 22A). Characterization experiments revealed that both the
reduction of positive charges in N-region (LMT Δ2-4) and mutation on SPase cleavage site (A19V) are “lethal”
to the function of LMT signal peptide, which highlights the importance of critical interactions between
transportation machinery of host and signal peptide during secretion process. Notably, the LMT Δ2-4 variant
exhibited slightly higher culture and supernatant fluorescence intensity compared to A19V. Although the Δ2-4
variant deletes a significant portion of the positively charged amino acids in N-region, it only reduces,
rather than completely blocks, the binding of SecB to the signal peptide. Therefore, the signal peptide may
retain some degree of Sec pathway transport capability. However, the A19V variant disrupts the crucial AXA
cleavage site, significantly impairing the specific recognition and cleavage by the SPase. This could even
block of the entire Sec secretion pathway′s function, thereby greatly inhibiting the secretion of the cargo
protein. Additionally, the folding of cargo sfGFP might be interfered due to these two modifications, which
finally resulted in the lower fluorescence intensity of bacterial culture.

![](https://static.igem.wiki/teams/5549/results/figure-22.avif)

**Figure 22 Characterization of secretion performance of LMT
and two variants.** The OD600 (**A**), fluorescence intensity of culture (**B**) and supernatant (**C**)
was measured
as time progressed. Relative fluorescence units (RFU) was defined as the fluorescence of bacterial
culture subtracted the background fluorescence of growth medium.

### 6.2 Targeted mutations and screening of high-performance variants

The importance of the N-terminal positively charged region and the AXA cleavage site of LMT signal peptide
was validated. Based on the mechanistic understanding, and inspired by the high-throughput experiments and
analysis by Gresso *et al.* (3), we planned to design and construct a series of sequence mutants (especially
mutations in H-region and C-region) for the purpose of screening variant(s) with higher-performance on
protein secretion.

#### 6.2.1 Enhancing the hydrophobicity of the H-region of LMT signal peptide

Extensive literature reviews indicated that one of the crucial factors for signal peptides to successfully
mediate protein secretion is their overall hydrophobicity, especially the hydrophobicity of the H-region
core (3-5). This hydrophobicity is directly correlated with the efficiency of the signal peptide in
traversing the hydrophobic biological membrane. Aiming to strengthen the interactions between the cell
membrane and signal peptide to promote protein secretion, we focused on enhancing the hydrophobicity of the
LMT signal peptide's H-region.

![](https://static.igem.wiki/teams/5549/results/figure-23.avif)

**Figure 23 Targeted mutations in H-region of LMT signal
peptide.** (**A**) Sequence alignments of LMT and 4 variants. Mutated sites were colored in black. (**B**) DNA gel
electrophoresis of the colony PCR products of expression modules of LMT variants fused with sfgfp at
pSB1C3 in DH5α. Target bands (1383 bp) can be observed at the position between 2000 bp and 1000 bp. (**C**)
Each variant's fluorescence intensity of supernatant of 10 h was normalized to LMT WT, for the first
characterization. (**D**) Fluorescence intensity of supernatant of M13L variant with LMT WT was measured as
time progressed, for the second characterization.

Thus, we designed 4 LMT variants: G12L, M13L, C14L, and A15L, which replaced the residue in H-region with
the hydrophobic leucine (L) residue (Figure 23A) and constructed corresponding expression modules (Figure
23B) using the same method mentioned above. For the first time of characterization, two variants, M13L and
C14L showed higher fluorescence intensity of supernatant than LMT (Figure 23C). Given the poor
reproducibility observed in C14L group, we evaluated M13L only for the second time of characterization with
LMT as control. However, as time progressed, the M13L variant presented a little bit lower fluorescence
intensity of supernatant than the wild-type (Figure 23D), inconsistent with the previous observation.

It is evident that not all mutations that enhance hydrophobicity will certainly improve secretion
efficiency. In addition, there are other hydrophobic residues to be tested. An optimal range of
hydrophobicity might exist, and exceeding this range may negatively affect cargo protein's folding,
transmembrane transportation, or other crucial steps. Taking together, the strategy of enhancing the
hydrophobicity of the H-region core might not be so feasible and robust for the case of LMT signal peptide.
Therefore, we turned to consider the mutations in the C-region of LMT.

#### 6.2.2 Increasing the number of Q residues in C-region of LMT signal peptide

![](https://static.igem.wiki/teams/5549/results/figure-24.avif)

**Figure 24 Construction of the expression modules of the
variants increasing the number of glutamine residues in the C-region.** (**A**) Sequence alignments of LMT and
four variants. Mutated sites were colored in black. (**B**) DNA gel electrophoresis of the colony PCR
products of four variants' expression modules fused with sfgfp at pSB1C3 in DH5α. Target bands (1383 bp)
can be observed at the position between 1500 bp and 1000 bp.

Demonstrated through high-throughput experiments, the number of glutamine residues (Q) in the C-region of
most signal peptides is positively correlated with secretion efficiency (3). Therefore, we designed to
increase the number of glutamine residues in the C-region of LMT and constructed four C-terminal point
mutants (Figure 24), named LMT A15Q, LMT S16Q, LMT A17Q, and LMT F18Q. In characterization, none of the
variants impaired the cell growth (Figure 25A). However, even though some variants presented higher culture
fluorescence intensity than the wild-type LMT (Figure 25B), all variants showed much lower fluorescence
intensity of supernatant than LMT (Figure 25C), which means no improvements of these variants in secretion
performance. Especially, A17Q mutation disrupts the AXA cleavage site like A19V variant, thus resulting in
the same “abortive” phenomenon of fluorescence intensity. With the failure experience of increasing the
number of glutamine residues in the C-region, we then turned to focus on the key factor for SPase cleavage
efficiency-the composition of residues in C-region (3).

![](https://static.igem.wiki/teams/5549/results/figure-25.avif)

**Figure 25 Characterization of secretion performance of LMT
and the variants increasing the number of glutamine residues in the C-region.** The OD600 (**A**),
fluorescence intensity of culture (**B**) and supernatant (**C**) was measured as time progressed.

#### 6.2.3 Increasing the number of A residues in LMT signal peptide

It was reported that the number of alanine (A) residues at the C-terminus of the signal peptide might be
closely related to the cleavage efficiency of SPase, thus influencing secretion efficiency (3). Therefore,
we hypothesized that increasing the number of C-terminal alanine residues could effectively enhance SPase
cleavage efficiency of the signal peptide, resulting in improved secretion performance. This time, we
designed to increase the number of alanine residues in LMT and constructed three C-terminal point mutants,
named LMT M13A, LMT C14A, and LMT F18A (Figure 26A and 26B). As time progressed, the M13A and F18A variants
showed a little bit higher fluorescence intensity of supernatant than wild-type LMT, while it can be easily
observed that the C14A variant's performance was superior to other variants and even the wild-type LMT
(Figure 26C). To confirm whether the improvement was true or not, we conducted the characterization
experiments of LMT C14A for the second time with LMT as control. Stronger fluorescence intensity of both
bacterial culture and supernatant was observed for the C14A variant when compared to the wild-type LMT
signal peptide (Figure 26D), validating the effectiveness of this mutation in improving secretion
performance.

![](https://static.igem.wiki/teams/5549/results/figure-26.avif)

**Figure 26 Increasing the number of alanine residues in LMT.**
(**A**) Sequence alignments of LMT and 3 variants. Mutated sites were colored in black. (**B**) DNA gel
electrophoresis of the colony PCR products of expression modules of LMT variants fused with sfgfp at
pSB1C3 in DH5α. Target bands (1383 bp) can be observed at the position between 1500 bp and 1000 bp. (**C**)
Fluorescence intensity of the supernatant was measured as time progressed. (**D**) Fluorescence intensity
(at 10 h) of culture and supernatant of C14A and LMT WT were compared, for the second time of
characterization. *p*-value: 0.0008 (\*\*\*) and 0.0098 (\*\*).

The successful construction and characterization of the high-performance C14A variant confirmed the
hypothesis that increasing the number of C-terminal A residues might effectively enhance the function of
signal peptide to direct proteins out of the bacterial cell. The results (Figure 26C and 26D) suggested that
the C14A mutation might promote protein secretion through mechanisms such as boosting SPase cleavage
efficiency or optimizing the signal peptide conformation. Thus, C14A represents a promising optimization
site for the LMT signal peptide with potential application values. Future studies should focus on an
in-depth exploration of the SPase mechanism and the structure-activity relationship of C14A.

### 6.3 Modeling and analysis of the secretion mechanism

With the help of [the model implemented by XMU-China 2024](https://2024.igem.wiki/xmu-china/model) that describes the relationship between the amount
of secreted recombinant protein and time in the natural state of our engineered bacteria, rather than the
growth arrest state in pulse-chase experiments, the performance difference of C14A and LMT can be revealed
at the microlevel quantitatively. By fitting the model with our experimental data, two important parameters,
the translocation rate from cytoplasm space to periplasm space (α, 1/h) and the translocation rate from
periplasm space to medium space (β, 1/h), are shown in Figure 27. A 24% increase in the translocation rate
from cytoplasmic space to periplasmic space by C14A mutation proves that the improvement is attributed to
the interaction between secretion machine in the Sec pathway and mutant, with the translocation rate from
periplasm space to medium space showing non-significant changes. With the help of the model implemented by
XMU-China 2024 that describes the relationship between the amount of secreted recombinant protein and time
in the natural state of our engineered bacteria, rather than the growth arrest state in pulse-chase
experiments, the performance difference of C14A and LMT can be revealed at the microlevel quantitatively. By
fitting the model with our experimental data, two important parameters, the translocation rate from
cytoplasm space to periplasm space (α, 1/h) and the translocation rate from periplasm space to medium space
(β, 1/h), are shown in Figure 27. A 24% increase in the translocation rate from cytoplasmic space to
periplasmic space by C14A mutation proves that the improvement is attributed to the interaction between
secretion machine in the Sec pathway and mutant, with the translocation rate from periplasm space to medium
space showing non-significant changes.

![](https://static.igem.wiki/teams/5549/results/figure-27.avif)

**Figure 27 Model analysis for C14A and LMT.** (**A**) The
translocation rate from cytoplasmic space to periplasmic space. (**B**) The translocation rate from
periplasmic space to medium space.

In summary, this mutant (LMT C14A, [BBa\_25LUSMUT](https://registry.igem.org/parts/bba-25lusmut)) significantly improved the secreted amount of the target
protein. This finding provides new strategies and tools for signal peptide engineering and research on the
extracellular secretion mechanism of enzymes. In our subsequent work, we will apply it to the secretion
process of chitosanase, aiming to further increase the production yield of COS.

## 7 Enzymatic Hydrolysis Products Used in Preservation Experiments

### 7.1 Circuit Construction and Characterization

We demonstrated that the chitosanase mutants SaCsn46A mutant 2 (SaCsn46A E151G D222G) and SaCsn46A mutant 60
(SaCsn46A D40G Q125R H234R) exhibit excellent performance, whereas the LMT C14A shows high secretion
efficiency. Consequently, we attempted to validate the degradation capabilities of these chitosanase mutants
when secreted into the supernatant by LMT C14A.

The targeted sequences BBa\_2507E4TO, BBa\_25B08G08, BBa\_25PUHM7F were individually inserted in pET-28a(+) to
construct circuits (Table 4), which were transformed into *E. coli* BL21(DE3) to express each enzyme. The
positive transformants were confirmed by kanamycin, colony PCR (Figure 28), and sequencing.

**Table 4 The part information of LMT C14A-linker-SaCsn46A
mutants.**

| Name | Type | Description |
| --- | --- | --- |
| [BBa\_2507E4TO](https://registry.igem.org/parts/bba-2507e4to) | coding | LMT C14A-linker-SaCsn46A WT |
| [BBa\_25PUHM7F](https://registry.igem.org/parts/bba-25puhm7f) | coding | LMT C14A-linker-SaCsn46A mutant 2 |
||  |  |  |
| --- | --- | --- |
| [BBa\_25B08G08](https://registry.igem.org/parts/bba-25b08g08) | coding | LMT C14A-linker-SaCsn46A mutant 60 |

![](https://static.igem.wiki/teams/5549/results/figure-28.avif)

**Figure 28 DNA gel electrophoresis of the colony PCR
products of BBa\_2507E4TO (A), BBa\_25B08G08 (B) and BBa\_25PUHM7F (C)\_pET-28a(+) in *E. coli* BL21(DE3).**
Target bands (1143 bp) can be observed at the position between 1000 bp and 2000 bp.

The engineered bacteria were cultured at 16°C, and supernatants were collected at 0, 5, 8, 12, 16 and 20
hours. SDS-PAGE analysis confirmed the successful secretion of fusion proteins into the supernatant (Figure
29 A-C).

![](https://static.igem.wiki/teams/5549/results/figure-29.avif)

**Figure 29 SDS-PAGE analysis of LMT C14A-linker-SaCsn46A WT
(A), [LMT C14A-linker-SaCsn46A mutant 2](https://registry.igem.org/parts/bba-25puhm7f) (B),
and [LMT C14A-linker-SaCsn46A mutant 60](https://registry.igem.org/parts/bba-25b08g08) (C).**
Target bands can be observed about 30 kDa.

### 7.2 Characterization of the DP of Degradation Products from SaCsn46A Mutants with LMT C14A

Meanwhile, the supernatant collected at 20 h was used for degradation experiments (see
[Experiments](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27experiments%27%29%20%7D%7D) for details). After degradation, the supernatant was collected for TLC and HPLC analyses, following the same methods as described above. The TLC and HPLC results of the degradation products are shown in Figures 30 and 31, respectively.

The TLC results indicated that the chitosan degradation products catalyzed by the wild-type enzyme and its mutants produced apparent magenta spots. The COS in these degradation products exhibited relatively high degrees of polymerization (DP), making clear separation challenging (Figure 30).

![](https://static.igem.wiki/teams/5549/results/figure-31.avif)

**Figure 30 TLC analysis of degradation products of SaCsn46A
WT and its mutants.** Lanes A-E correspond to: glucosamine (**A**) 9.95 mg/mL COS products (**B**) Degradation
products of LMT C14A-linker-SaCsn46A WT (**C**) Degradation products of [LMT C14A-linker-SaCsn46A mutant 2](https://registry.igem.org/parts/bba-25puhm7f)
(**D**) Degradation products of [LMT C14A-linker-SaCsn46A mutant 60](https://registry.igem.org/parts/bba-25b08g08) (**E**).

To further verify the results, HPLC analysis was subsequently performed on the same samples. The HPLC results revealed that the degradation products were
primarily concentrated as glucosamine and chitobiose (Figure 31), with concentration trends consistent with the earlier TLC findings.
Therefore, the HPLC results provided more direct evidence for the functional changes of the mutants.

![](https://static.igem.wiki/teams/5549/results/figure-30.avif)

**Figure 31 HPLC analysis of degradation products of SaCsn46A
WT and its mutants.** (**A**) Degradation products of SaCsn46A WT. (**B**) Degradation products of LMT
C14A-linker-SaCsn46A mutant 2. (**C**) Degradation products of [LMT C14A-linker-SaCsn46A mutant 60](https://registry.igem.org/parts/bba-25b08g08).

### 7.3 Preservation Experiments

After obtaining the degradation products of the SaCsn46A and its mutants, we subjected these products to
membrane filtration sterilization. Subsequently, we treated fish samples with the sterile degradation
products, stored the samples at 4°C, and counted daily counts of bacterial colonies. Figure 32 shows a
comparison of bacterial colony counts among different groups on the second day, along with a significance
analysis. Notably, the degradation products of the [LMT C14A-linker-SaCsn46A mutant 2](https://registry.igem.org/parts/bba-25puhm7f) exhibited more
excellent antimicrobial activity.

![](https://static.igem.wiki/teams/5549/results/figure-32-1.avif)

**Figure 32 Fish preservation performance of degradation
products from LMT C14A-linker-SaCsn46A WT, [LMT C14A-linker-SaCsn46A mutant 2](https://registry.igem.org/parts/bba-25puhm7f), and LMT C14A-linker-SaCsn46A
mutant 60.** (**A**) Colony counts after direct treatment. (**B**) Comparison between the degradation products
group and the LB control group on Day 2 post-direct-treatment.

## 8 Kill Switch

### 8.1 tyrS Overexpression System

*tyrS* is an essential gene in *E. coli* that encodes tyrosyl-tRNA synthetase. We have developed a regulatory
system using the T5 promoter and *tyrS*, resulting in the composite component [BBa\_2567UPRQ](https://registry.igem.org/parts/bba-2567uprq), which was
subsequently assembled into the expression vector pCA24N. In the uninduced state (OFF state), *tyrS* maintains
cell viability by providing a low level of enzymatic activity through leaky expression. In the induced state
(ON state), excessive expression of *tyrS* leads to an enzymatic overload, which results in cell death. The
killing effect was characterized *via* a spot assay (see our [Experiments](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27experiments%27%29%20%7D%7D) for details), and cell viability was
measured by colony-forming unit (CFU) counting—expressed as the ratio of the number of cells in the
IPTG-treated group to that in the non-IPTG-treated group (survival rate).

We characterized this IPTG-inducible system using *E. coli* BL21-AI (Figure 33A) and selected positive transformants
verified by colony PCR and sequencing (Figure 33B). After approximately 6 h of inoculation with or without
IPTG (final concentration: 1 mM), CFUs were counted and survival rates were calculated. The CFU in the
IPTG-treated group was significantly lower than that in the untreated group, which implied that IPTG-induced
*tyrS* overexpression exhibited cytotoxicity (Figure 33C).

![](https://static.igem.wiki/teams/5549/results/figure-33-1.avif)

**Figure 33 Characterization of IPTG-induced *tyrS*
overexpression.** (**A**) The gene circuit of IPTG-induced kill switch (BBa\_2567UPRQ) on pCA24N vector. (**B**)
Agarose gel electrophoresis of the colony PCR products of BBa\_2567UPRQ in *E. coli* BL21-AI. (**C**) Cell
viability was measured by CFU count and is displayed as a ratio of cells treated with IPTG to cells
maintained without IPTG. *p*-value: 0.0005 (\*\*\*).

Although we have confirmed the lethal effect of the *tyrS* overexpression system, we continue to optimize the
induction conditions to enhance this effect further. A spot assay was conducted at 0 h, 2 h, 4 h, 6 h, and 8
h, with IPTG concentrations ranging from 0 mM to 1 mM, to assess *tyrS* cytotoxicity. The survival rate
decreased significantly at 4 h and reached its nadir at 6 h. The relationship between survival rate and IPTG
concentration exhibited a Hill curve pattern; notably, the survival rates at 0.5 mM and 1 mM IPTG were
nearly identical.

![](https://static.igem.wiki/teams/5549/results/figure-34-3.avif)

**Figure 34 Characterization of the *tyrS* overexpression
system under different induction conditions.** Cell viability was measured by CFU count and is displayed
as a ratio of cells treated with IPTG to cells maintained without IPTG. (**A**) Survival rate over time. (**B**)
Survival rate over IPTG concentration.

Meanwhile, the *tyrS* overexpression system was applied to and characterized in *E. coli* strains including
BL21-AI, Top10, MG1655 and DH5α. IPTG-induced *tyrS* expression demonstrated significant cytotoxic
effects across all tested strains, indicating the broad applicability of the *tyrS* overexpression system.

![](https://static.igem.wiki/teams/5549/results/figure-35.avif)

**Figure 35 Characterization of IPTG-induced *tyrS*
overexpression in different bacterial strains.** *p*-values: 0.0002 (\*\*\*) for ,.*E.coil* BL21-AI, 0.0024 (\*\*) for
*E.coli* Top10, 0.0001 (\*\*\*\*) for *E.coli* DH5α, and 0.0002 (\*\*\*) for *E.coli* MG1655.

### 8.2 CcdB/CcdA Toxin-Antitoxin System

The second kill switch system employs the *ccdB* gene, encoding a toxin that inhibits DNA gyrase
(topoisomerase II) and consequently induces cell death (6). The expression of *ccdB* is regulated by the pCymR
promoter and is induced by cuminic acid (7). To reduce cellular burden caused by leaky toxin expression, the
antitoxin gene *ccdA* is expressed at a low level *via* a weak constitutive promoter. When cuminic acid is
added, the expression of CcdB surpasses the neutralizing capacity of CcdA, effectively triggering cell
death.

We characterized this cuminic acid-inducible system using *E. coli* Top10 (Figure 36A) and selected positive
transformants, which were verified by colony PCR and sequencing (Figure 36B). Approximately 4 hours after
inoculation with or without cuminic acid (final concentration: 0.1 mM), CFUs were counted and survival rates were
calculated. The CFU in the cuminic acid-treated group was significantly lower than that in the untreated
group, suggesting that cuminic acid-induced CcdB toxin exhibited cytotoxicity (Figure 36C).

![](https://static.igem.wiki/teams/5549/results/figure-36-1.avif)

**Figure 36 Characterization of cuminic acid-induced CcdB
toxin.** (**A**) The gene circuit of cuminic acid-induced kill switch (BBa\_25464RFQ) on pSB3C5 vector. (**B**)
Agarose gel electrophoresis of the colony PCR products of BBa\_25464RFQ in *E. coli* Top10. (**C**) Cell
viability was measured by CFU count and is displayed as a ratio of cells treated with cuminic acid to
cells maintained without cuminic acid. 0.0114 (\*). (**D**) Bacterial culture at 4 h with and without the
addition of cuminic acid.

### 8.3 Integrated Kill Switch System

We integrated these two IPTG and cuminic acid-inducible systems into the same plasmid (Figure 37A) and
transformed into *E. coli* Trans10. Then, the positive transformants were selected and verified by colony PCR
and sequencing (Figure 37B). After approximately 4 h of induction, the CFU count in the inducer-treated
group was significantly lower than in the untreated group, suggesting that the two independent suicide
switches effectively enhance biosafety (Figure 37C).

![](https://static.igem.wiki/teams/5549/results/figure-37-1.avif)

**Figure 37 Characterization of IPTG and cuminic acid-induced
kill switch.** (**A**) The gene circuit of IPTG and cuminic acid-induced kill switch (BBa\_25V3X0FU) on pSB3C5
vector. (**B**) Agarose gel electrophoresis of the colony PCR products of BBa\_25V3X0FU in *E. coli* Trans10.
(**C**) Cell viability was measured by CFU count.

### 8.4 Conclusion

To prevent engineered bacteria in the chitosanase production chain from contaminating other processes and posing
biosafety risks, we designed two independent suicide switches. The first is a *ccdB/ccdA* toxin-antitoxin system
regulated by cuminic acid, and the second is an IPTG-inducible system that triggers overexpression of the *tyrS*
gene. Together, we integrated these two systems into a same plasmid, they can operate independently and provide
mutual backup for each other, thus guarantee rapid cell death, keeping the engineered bacteria in a controllable
state.

In summary, our modular verification and engineering iteration of the IPTG-inducible killing switch have
contributed to the biosafety of our CRUSTA project (see our [Proposed Implementation](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27implementation%27%29%20%7D%7D) for details). We hope
this design of biocontainment system will provide valuable experience to other iGEM teams and the synthetic
biology community.

## 9 Conclusion

In our CRUSTA project, we designed and developed an innovative COS-based bio-preservation technology using principles of synthetic biology. This technology provides a green and efficient alternative to traditional chemical preservatives in the food industry. In our project, we achieved chitosan degradation *via* a secretion system, enhancing the antibacterial performance of the degradation products by 7.96-fold compared to the wild-type. In the aspect of production of chitosan oligosaccharides, the enzymatic approach decreases about 99.1% of CO2 emissions and 96.4% of cost compared to the chemical one. Additionally, our method saves about 61.0% of CO2 emissions and 71.7% of cost compared to the chemical production of traditional preservative, potassium sorbate, in the same functional performance. This project not only has the potential to propel technological innovation and catalyze industrial advancement in food preservation but also aims to introduce advanced concepts of synthetic biology and eco-friendly preservation. These initiatives are expected to improve preservation efficiency, elevate food safety standards, and enhance environmental sustainability, thus bolstering market competitiveness and fostering the sustainable growth of the green food industry and the circular economy.

## 10 Future Prospects

In the future, we aim to leverage AI conduct the learning step in the final stage of the DBTL cycle to
obtain new LMT and chitosanase with enhanced efficacy. We aim to comprehensively characterize the
synergistic efficiency of high-performance chitosanase mutants and the optimized LMT C14A secretion system,
and explore the optimal reaction conditions (such as temperature, pH, and substrate concentration) that
maximize the yield of bioactive COS. Concurrently, we aim to validate the model-predicted properties of
novel chitosanase sequences (*e.g.*, catalytic activity, thermostability) through systematic experimental
testing, and conduct in-depth analysis of degradation products to clarify the correlation between COS
molecular weight, polymerization degree, and antibacterial/preservation efficacy. Importantly, we are
committed to expanding the application of COS-based preservation technology to more food scenarios (such as
aquatic products, fruits, and vegetables) and optimizing the formulation of COS preservatives to enhance
their stability and applicability. Ultimately, we dedicate ourselves to advancing the development of a
convenient, efficient, and eco-friendly green preservative product, providing practical solutions for the
industrialization of natural biological preservation technology.

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