iGEM 2024 — 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/5136/load-new.gif)

# . NoteBook .

Welcome to explore our iGEM 2024 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.

November 22, 2023

- We are starting to select new members of XMU-China 2024! So excited to start
a
New Year of synthetic biology - post a photo of the introductory lecture!

![](https://static.igem.wiki/teams/5136/notebook-img/nb-fig1.png)

December 31, 2023

- The eve of the 2023 to 2024 New Year's Eve coincided
with the deadline for
submitting our second selection assignment. While everyone was celebrating the New Year's Eve, a group
of students who were aspiring and expecting to be selected into iGEM, namely us, were studying hard. We
were so busy during this wonderful time that we even declined an invitation from our classmates to go to
the playground to watch a meteor shower, what a pity! But fortunately, finally, after successfully
completing it, we managed to get together with everyone in the middle of the night and spent a few hours
having fun with them. But it also means that we're also coming up to the final week of the semester, and
time is tight!

![](https://static.igem.wiki/teams/5136/notebook-img/nb-fig2.png)

January 26, 2024

- Wow, we can put these DNA fragment together through the sticky ends of the
enzyme cutting site, amazing! That's a real boost to our knowledge!

![](https://static.igem.wiki/teams/5136/notebook-img/nb-fig3.png)

February 26, 2024

- At last! After such a long time of selection, the new team of XMU-China is completed. Next is to
determine the project, go! ! !

March 3, 2024

- Today was our first in-person team meeting, and it was so exciting to finally meet everyone! After our discussions, we headed to the Cat Street Courtyard for some food and drinks. It was such a joy to gather together, share laughs, and enjoy each other's company. We were really looking forward to our upcoming competition and all the great moments we'll create as a team. What a fantastic start to our journey!

![](https://static.igem.wiki/teams/5136/notebook-img/nb-fig4.png)

March 27, 2024

- It was so difficult to do our endnote homework
today.
Some of us totally
didn't know how to insert the literature into manuscript! Through all kinds of learning and consulting,
we finally completed it after fighting for several hours!

April 14, 2024

- This week, all of the members of the lab group in
our team had wiki
assignments. This was our first exposure to computer programming, and the process of writing code was
a little bit fun but also hard to learn.

![](https://static.igem.wiki/teams/5136/notebook-img/nb-fig5.png)

April 20, 2024

- Recently, we started to draw schematics of our
project, We try to use our
imagination to describe the principle vividly, such as enzyme dolls, scissor hands, grasping hands and
so on.

April 21, 2024

- Today, We conducted an online interview with Prof.
Liang.

April 25, 2024

- We worked together on interviewing Xiamen citizens
to learn their demand for
writing correction products, and it was very rewarding.

![](https://static.igem.wiki/teams/5136/notebook-img/nb-fig6.png)

May 1, 2024

- On this day, all of us from Xiang'an Campus woke up early and headed to Siming Campus for our
experimental
training session. It was challenging yet incredibly enjoyable. Before we realized what was going to
happen, we first heard the following words and thought them very funny. For example, "If you feel
stressed, then go to the lab, because there are autoclaves that are more stressing than you"......

![](https://static.igem.wiki/teams/5136/notebook-img/nb-fig7.png)

May 3, 2024

- In this team meeting, we reported on our whole
project design. We were
looking forward to the experimental days ahead to start a new chapter!

May 3, 2024

- What an atmospheric picture! Following the
experimental training and the team
meeting, everyone celebrated Chenxin Xu's birthday in grand style, much to her pleasant surprise. Even
the online members sent their warmest wishes. It was a truly unforgettable and memorable experience that
we will cherish forever. Chenxin Xu expressed her sincerest thanks to everyone and XMU-China, this big
warm family.

![](https://static.igem.wiki/teams/5136/notebook-img/nb-fig8.png)

May 12, 2024

- It's raining tonight, and we are quite exhausted. However, when thinking
about the interesting human practice
activities we participated in and the valuable information we gained in Zhangzhou, we feel very happy!

May 18, 2024

- It's all been so bumpy recently. Finally, at this day we completed a 17-page, 7701-word lab protocol
on enzymes and deinking!

![](https://static.igem.wiki/teams/5136/notebook-img/nb-fig9.png)

May 19, 2024

- During DNA Gel Electrophoresis experiment, one of us
found that the DNA bands were not migrating during electrophoresis. After three attempts, the issue was
identified as using 50x TAE stock instead of 1x TAE buffer. The experiment ended successfully after
correcting the buffer concentration to 1x TAE.

May 22, 2024

- At this day, one of us made the most of her time and tried making competent
cells by herself in the laboratory in the Tuoyuan Building for the first time. Fortunately, what's truly
worth celebrating is that the competent cells she made over these two days were both successful!

![](https://static.igem.wiki/teams/5136/notebook-img/nb-fig10-1.png)
![](https://static.igem.wiki/teams/5136/notebook-img/nb-fig10-2.png)

May 28, 2024

- Yesterday when one of us extracted the plasmid from bacteria, she found that the bacterial content was
horribly low, mainly because the bacteria in the Tuoyuan Building grow surprisingly slowly. Well,
we'll just have to continue today. Fortunately, the final concentration we measured was ideal, saving us
from repeating the experiment.

![](https://static.igem.wiki/teams/5136/notebook-img/nb-fig11.png)

May 31, 2024

- Our strain carrying the constitutive GFP expression plasmid has grown up. It seems that cells with a
single plasmid tend to grow more smoothly.

![](https://static.igem.wiki/teams/5136/notebook-img/nb-fig13.png)

June 1, 2024

- Take a look at our group meeting routine!

![](https://static.igem.wiki/teams/5136/notebook-img/nb-fig14.png)

June 2, 2024

- At this day, we had an exchange and learning session with HUBU-China. We not only introduced our
respective projects but also shared the experiences accumulated by our teams. It was the first time that
we exchanged ideas with other iGEM teams during this season. Therefore, our team
took it very seriously, with everyone participating, making for a grand scene. Happily, the overall
communication went smoothly.

![](https://static.igem.wiki/teams/5136/notebook-img/nb-fig15.png)

June 4, 2024

- Everyone celebrated birthday for Mingzheng Fang and Jingwen Wang!!!!!

June 6, 2024

- Some of our trivial experimental routines - determining OD600 values.

![](https://static.igem.wiki/teams/5136/notebook-img/nb-fig16.png)

June 19, 2024

- The final exam week is over. Next, all team members, please dive into the team work immediately, ha ha!

June 23, 2024

- After the team meeting, we had a dinner together to relax, and everyone showed off their parrot-like
singing voices without stinting. In the midst of the fun, everyone deepened their impression of each
other.

![](https://static.igem.wiki/teams/5136/notebook-img/nb-fig17-1.png)

![](https://static.igem.wiki/teams/5136/notebook-img/nb-fig17-2.png)

June 24, 2024

- Come and take a look at our homemade series of pulp reaction devices! Isn't it very interesting? There
is no doubting that they can be definitely considered our original creation!

![](https://static.igem.wiki/teams/5136/notebook-img/nb-fig18-1.png)

June 28, 2024

- The third semester began! Being the first team member to shuttle between Xiang'an and Siming campuses
to do experiments, it was also a challenge for ChenXin Xu. As you can see in the picture, at this
point she hadn't even started working with sfGFP, an amazing fluorescent protein, and she was still
conducting experiments with constitutive fluorescent protein expression vetor.

![](https://static.igem.wiki/teams/5136/notebook-img/nb-fig19.png)

June 29, 2024

- Today's experimental phenomenon seems to be working well, and the contrast is relatively obvious.

![](https://static.igem.wiki/teams/5136/notebook-img/nb-fig20.png)

June 30, 2024

- It were the recycled paper products we produced after working tirelessly without rest.

![](https://static.igem.wiki/teams/5136/notebook-img/nb-fig21.png)

July 1, 2024

- Let's leave a record of our today's achievement.

![](https://static.igem.wiki/teams/5136/notebook-img/nb-fig22-1.png)

![](https://static.igem.wiki/teams/5136/notebook-img/nb-fig22-2.png)

July 2, 2024

- Today, one of us went to visit another lab, which is doing experiments on *Pseudomonas
putida*.
And she found that the smell of *E. coli* is still very good.

July 4, 2024

- Recently, we have been holding our lab group meeting per Thursday at the Siming laboratory, and our
experimental schedules have become so detailed that we outline specific tasks for each day. It has to be
said, the coming weeks are going to be very busy.

July 5, 2024

- Today, Jia Li and ChenXin Xu went to Huaqiao University to learn how to make 3D printed
hydrogels. It was so hard to explore, but overall we felt fresh.

![](https://static.igem.wiki/teams/5136/notebook-img/nb-fig23-1.png)

![](https://static.igem.wiki/teams/5136/notebook-img/nb-fig23-3.png)

July 6, 2024

- After discovering a new experimental direction, this is our first time conducting a validation
experiment related to an inducible sfGFP-based autolysis system. We hope that the experiment goes
smoothly.

![](https://static.igem.wiki/teams/5136/notebook-img/nb-fig24.png)

July 7, 2024

- At this day, Jia Li and Chenxin Xu stayed up all night in Lounge 494 and Lab 561 to write our
lectures, which is the first time we've worked so late together, and also kicked off our team's first
all-night trip.

July 8, 2024

- Let us experience the scenery — the morning light from outside our experimental base.

![](https://static.igem.wiki/teams/5136/notebook-img/nb-fig26.png)

July 9, 2024

- BBa\_K5136201, BBa\_K5136202, BBa\_K5136203, BBa\_K5136204, BBa\_K5136205 were constructed successfully.

July 11-14, 2024

- The CCiC journey has left me incredibly excited! We met many like-minded friends and had in-depth
discussions about our topics, gaining valuable advice. This experience made us realize that iGEM is not
just a competition; it's a warm and supportive family. We truly feel the passion and strength here.
iGEMers, let's keep pushing forward together and make a positive change in the world with our efforts!

![](https://static.igem.wiki/teams/5136/notebook-img/nb-fig27.png)

July 15, 2024

- After the new primers were delivered, we immediately started the experiments. The PCR results for
constructing the new circuit were really great with the bright and thick bands, indicating that it's a
very successful experiment! Indeed, when experiments go well, everything around us seems beautiful, even
the color of the gel used for recovery looks particularly nice.

![](https://static.igem.wiki/teams/5136/notebook-img/nb-fig28-1.png)

July 16, 2024

- Tonight, it's another all-nighter in the lab. Let me show you some green fluorescent proteins. They
are so brilliant that they almost hurt my eyes! I'd like to temporarily call them "the brightest
proteins of the season." (PS: The one on the left is GFP, and the other one on the right is sfGFP.)

![](https://static.igem.wiki/teams/5136/notebook-img/nb-fig29-1.png)
![](https://static.igem.wiki/teams/5136/notebook-img/nb-fig29-2.png)

July 16, 2024

- Looking at the fluorescence from a different angle, it's so cute!

![](https://static.igem.wiki/teams/5136/notebook-img/nb-fig30-1.png)

![](https://static.igem.wiki/teams/5136/notebook-img/nb-fig30-2.png)

July 17, 2024

- BBa\_K5136224, BBa\_K5136225 were constructed successfully.

July 18, 2024

- BBa\_K5136226 was constructed successfully.

July 19, 2024

- Due to participating in this competition, a part of us have their own passports for the first time!

![](https://static.igem.wiki/teams/5136/notebook-img/nb-fig31.png)

July 19, 2024

- After trying countless ways to separate the ink from the pulp, today we finally found that we can
extract it with limonene. To our surprise, the effect is very good, and finally we are not heartbroken persons!

July 21, 2024

- Today is one of us first time staying overnight in the lab, and she felt a little excited! She is not
sure if she'll have more nights like this in the future, but she is committed to finishing all her tasks
for this season. She hope there aren't too many more late nights ahead, haha, because health is really
important! The atmosphere tonight is great, and She is looking forward to the results!

![](https://static.igem.wiki/teams/5136/notebook-img/nb-fig32-1.png)
![](https://static.igem.wiki/teams/5136/notebook-img/nb-fig32-2.png)

July 22, 2024

- We came up with the idea of creating our promotional video in the form of a conversation between a witch and team members.

July 24, 2024

- BBa\_K5136230 was constructed successfully.

July 28, 2024

- Discussing topics between our teammates until dawn, rising not only the sun but also our hope for the
future!

July 29, 2024

- BBa\_K5136227 was constructed successfully.

July 30, 2024

- BBa\_K5136228 was constructed successfully.

July 31, 2024

- BBa\_K5136229 was constructed successfully.

August 1, 2024

- Recording the basic operation of pouring plates in the lab.

![](https://static.igem.wiki/teams/5136/notebook-img/nb-fig33.png)

August 1, 2024

- After such a long time of design, experiments and human practices, our project design is finally
determined! The push that we send received a lot of people's attention. Ah, so happy!

![](https://static.igem.wiki/teams/5136/notebook-img/nb-fig34.png)
![](https://static.igem.wiki/teams/5136/notebook-img/nb-fig35.png)

August 2, 2024

- The chitosan adsorption experiment showed exciting result! The ink solution is much cleaner after
chitosan adsorption compared to the control group that uses paper to adsorb.

August 4, 2024

- Recording of beautiful fluorescent proteins.

![](https://static.igem.wiki/teams/5136/notebook-img/nb-fig36.png)

August 4, 2024

- Great! The transformation of so many plates was very successful, and the construction of the new
circuit for our autolytic system is about to welcome the dawn of victory!

![](https://static.igem.wiki/teams/5136/notebook-img/nb-fig37.png)

August 4, 2024

- Following our team meeting, we wandered around and ended up at the Midnight Diner. Here, we put aside
our research topics and experiments, enjoying a pleasant time together. After dinner, members of our two
groups (the Printing Group and the Life Group) engaged in a heated discussion. It really was a thorough
and intense brainstorming session. In the end, we exchanged well wishes and cheered each other on with a
hearty "Go, go, go! Let's ace those experiments". Onward and upward!

![](https://static.igem.wiki/teams/5136/notebook-img/nb-fig38-1.png)
![](https://static.igem.wiki/teams/5136/notebook-img/nb-fig38-2.png)

August 5, 2024

- BBa\_K5136220, BBa\_K5136221, BBa\_K5136222 were constructed successfully.

August 6, 2024

- The experiment with biplasmid cells is still in progress. Today, we tried to see if there could be any
other experimental protocols to validate the autolytic system, such as assessing it solely by measuring
OD600. However, after three days of hard work, we found that this method is not feasible.

August 7, 2024

- At Siming Campus, three of us stayed up all night doing experiments, and we all felt quite exhausted. However, taking a break from the experiments, we enjoyed a lively and inspiring conversation,
and we all share a strong hope for a fruitful season ahead.

August 9, 2024

- Today, Zimo Liu and Chenxin Xu used a new experimental method, and the effect is so obvious that we
wonder what will happen to the results of the later measurement.

![](https://static.igem.wiki/teams/5136/notebook-img/nb-fig39-1.png)

![](https://static.igem.wiki/teams/5136/notebook-img/nb-fig39-2.png)

August 9, 2024

- After the last batch of experiments was done, it was time to start the next set of experiments without
stopping. I have to say, the neatly arranged vials look pretty good when they are placed inside the
shaker.

![](https://static.igem.wiki/teams/5136/notebook-img/nb-fig40.png)
![](https://static.igem.wiki/teams/5136/notebook-img/nb-fig41.png)

August 10, 2024

- The printer showed up today, and it's so cool to see it running! Now we can finally do some proper
quantification of the ink. Can't wait to get started with our experiments!

August 10, 2024

- Today we had a collaboration with two other teams, where we showed each other the human practices we
had carried out and exchanged experiences. It is worthwhile for us to learn from each team as they are
so good at human practice!

![](https://static.igem.wiki/teams/5136/notebook-img/nb-fig42.png)

- This is our new modified reaction unit for deinking pulp experiments.

![](https://static.igem.wiki/teams/5136/notebook-img/nb-fig43.png)

August 11, 2024

- Such experiments have been done many times, even in many different kinds. We don't know how much
longer we will continue to do this in the future before we can wrap it up successfully, but we are
looking forward to it.

![](https://static.igem.wiki/teams/5136/notebook-img/nb-fig44.png)

August 11, 2024

- They are glowing! Look how bright the superfolder green fluorescent protein is! They truly live up to their reputation as proteins with fluorescence values in the tens of thousands. There's no doubt that their brightness is enough to leave a deep impression on everyone.

![](https://static.igem.wiki/teams/5136/notebook-img/nb-fig45-1.png)

![](https://static.igem.wiki/teams/5136/notebook-img/nb-fig45-2.png)

August 12, 2024

- Today, after completing the cross-linking chitosan experiment, Zhengyang Mo had some leftover chitosan
solution and he injected glutaraldehyde solution directly into the chitosan solution. To his amazement,
the solution gradually transformed into a golden-yellow gel!

![](https://static.igem.wiki/teams/5136/notebook-img/nb-fig46.png)

August 15, 2024

- Daily sleep has been seriously insufficient, leaving us both physically and mentally exhausted, but fortunately, the heavens have not failed us and our autolytic system experiment has received fairly ideal results. Let us show you our "Army of five hundred thousand" from the
autolytic system!

![](https://static.igem.wiki/teams/5136/notebook-img/nb-fig47-1.png)

![](https://static.igem.wiki/teams/5136/notebook-img/nb-fig47-2.png)

August 15, 2024

- Recording the day-to-day activities in our lab: glue making, glue running, protein purification, and
more.

![](https://static.igem.wiki/teams/5136/notebook-img/nb-fig48.png)

August 17, 2024

- Feeling awesome today! CYP199A4 253S is doing a great job with deinking—we're finally seeing some real
results after all this effort! Go, monooxygenase, go!

August 20, 2024

- This is a small portion of the lysate supernatant of deinking enzymes that came out of Chenxin Xu first
protein purification today. The lab is in full swing with all kinds of experiments, building circuits,
purifying proteins, cellular experiments and so on - it's a bustling scene.

![](https://static.igem.wiki/teams/5136/notebook-img/nb-fig49.png)

August 21, 2024

- The much-needed filter paper for the experiment finally arrived, happy to explode!!!

August 25, 2024

- BBa\_K5136023, BBa\_K5136024, BBa\_K5136025 were constructed successfully.

August 25, 2024

- We participated in an education activity at the Xiamen Science and Technology Museum, promoting waste
paper recycling and environmental awareness. Some of us organized a quiz game for children on recycling
and forest protection, while others took photos, distributed surveys, and gave presentations. Through
teamwork, we raised public awareness and deepened our understanding of the importance of environmental
education.

![](https://static.igem.wiki/teams/5136/notebook-img/nb-fig50.png)

August 25, 2024

- During spare time at the education activity at the Xiamen Science and Technology Museum, we wandered
around the science museum. A photo of Li Jia and Chenxin Xu was even displayed on the big screen.

![](https://static.igem.wiki/teams/5136/notebook-img/nb-fig51-1.png)
![](https://static.igem.wiki/teams/5136/notebook-img/nb-fig52-1.png)

August 27, 2024

- Exhibiting our protein purification heroes — the ultrasonic cell disruptor and the AKTA instrument.

August 27, 2024

- This is the first time Chenxin Xu doing it, and it made her feel truly magical. Turning knowledge into power!

![](https://static.igem.wiki/teams/5136/notebook-img/nb-fig53.png)

August 29, 2024

- The protein we purified for deinking today was in a good concentration, and it was quite something to
be happy about.

![](https://static.igem.wiki/teams/5136/notebook-img/nb-fig54.png)

August 31, 2024

- Successfully constructed a new plasmid the day before school started. If the sequencing results come
out, we can move on to the later experiments!

![](https://static.igem.wiki/teams/5136/notebook-img/nb-fig55.png)

September 1, 2024

- Finally completed the second round of deinking enzyme screening QAQ, followed by saturation mutation,
come on, 2024 XMU-China iGEMer go forward!!!

September 1, 2024

- Had a whim to show off Chenxin Xu's incomplete data to give you all a little shock haha. Every save is
the result of scrapping, a handful of bitter tears, who knows what it's like.

![](https://static.igem.wiki/teams/5136/notebook-img/nb-fig56.png)

September 2, 2024

- BBa\_K5136223 was constructed successfully.

September 4, 2024

- Finally we finished the promotion video.

- BBa\_K5136018 was constructed successfully.

September 5, 2024

- The night before, I made my debut upload of a webpage to the GitLab repository, only to mangle the
code framework like a novice chef. The pipeline crashed spectacularly. After much toil and more cups of
coffee than advisable, I finally cracked the code at the stroke of 2 AM. This reminds me of that line
from Dark Souls III, "Long may the sun shine!"

![](https://static.igem.wiki/teams/5136/notebook-img/nb-fig57.png)

September 5, 2024

- BBa\_K5136003, BBa\_K5136010, BBa\_K5136012, BBa\_K5136013, BBa\_K5136016 were constructed successfully.

September 6, 2024

- Today is a very lucky day, my conversion experiment finally succeeded. Last week's conversion
experiments were not very smooth, in some conversion experiments, no bacteria grew on the board; In
other conversion experiments, a few bacteria grew out of the board, but the bacteria were picked from
the board, the plasmid was extracted and sent for testing, and the sequencing results also told me that
the gene of the protein had been mutated, and gfpfasta could not glow. After more than a week of hard
transformation, the experiment finally succeeded, the luminous bacteria grew on the two boards, and the
colony pcr also got the correct band.

September 7, 2024

- BBa\_K5136015 was constructed successfully.

September 13, 2024

- The weather is sunny and the mood is good. I ate delicious food and petted kittens today! At this
time, I have successfully completed the current phase of my work and am ready to start working on the
next phase. To be honest, I always prefer to be alone to paint in peace and quiet.

- Yi Zhang has drawn eight schematic presentation diagrams on experimental design in the last two days,
and she even feel like a war hero herself now.

September 13, 2024

- Before and after centrifugation - what a stark contrast.

![](https://static.igem.wiki/teams/5136/notebook-img/nb-fig59-1.png)

![](https://static.igem.wiki/teams/5136/notebook-img/nb-fig59-2.png)

September 14, 2024

- These days we finished the first version of the web page, but was told that it needed to be overhauled
and that it was time to rewrite the code.

September 15, 2024

- Today, after the team meeting, we went to dinner together. We discussed something interesting in the experiment and enjoyed a variety of delicious dishes, such as big plate chicken, sliced fish, and garlic shrimp. The restaurant also offered an ice cream buffet, where I created a huge ice cream cone that was both sweet and delicious.

![](https://static.igem.wiki/teams/5136/notebook-img/nb-fig60-1.png)
![](https://static.igem.wiki/teams/5136/notebook-img/nb-fig60-2.png)

September 16, 2024

- Finally, all 20 proteins were purified! My body and mind are refreshed, and at the same time, my sense
of accomplishment is up up up!

- Accidents do happen — today, Yifei Yin hurt her leg.

September 19, 2024

- BBa\_K5136005 was constructed successfully.

September 20, 2024

- Today, we happened to work on protein-related experiments. It has to be said that we have become quite
skilled at basic operations such as preparing protein gels and running gels.

![](https://static.igem.wiki/teams/5136/notebook-img/nb-fig61-1.png)
![](https://static.igem.wiki/teams/5136/notebook-img/nb-fig61-2.png)

September 22, 2024

- The weather is rainy and Yi Zhang not in the mood at all. However, after the group meeting today, we
all got together to chat, which made me feel extraordinarily relaxed. Next, she is going to continue
working on drawings and look forward to finishing the webpage as quickly as possible these days.

September 22, 2024

- At last! The saturation mutation of CYP199A4 is complete, and the next step is to try to combine the
secretion system with the deinking enzyme. Can we really accomplish this?

September 25, 2024

Whoa, whoa, whoa! LMT-253E circuit has been built! We can finally try the effect of deinking enzyme in
combination with secretion system! Unfortunately, the circuit build of LMT-253A failed, and it may not
be possible to build it before wiki freeze.

September 27, 2024

- Today, we visited Yanwu Elementary School in Xiamen and had a wonderful time interacting with the
kids. We taught them about the importance of protecting forests and saving paper, and they were
incredibly enthusiastic and eager to learn. We even conducted some fun experiments together, and the
kids were so cooperative and engaged. It was a delightful experience, and their excitement was truly
contagious. We left feeling inspired and hopeful for the future, knowing that these young minds are so
receptive to environmental conservation!

![](https://static.igem.wiki/teams/5136/notebook-img/nb-fig62.png)

September 29, 2024

- That's great! Although 253E is not the best deinking enzyme, LMT-253E still performs well. Let us work harder to see LMT-253A performance. We look forward to showing its better deinking effect in the presentation video.

October 2, 2024

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/5136/load-new.gif)

[Overview](#section1)

[Cycle 1](#section2)

[Design](#section2-1)

[Build](#section2-2)

[Test](#section2-3)

[Learn](#section2-4)

[Cycle 2](#section3)

[Design and Build](#section3-1)

[Test](#section3-2)

[Learn](#section3-3)

[Cycle 3](#section4)

[Design and Build](#section4-1)

[Test](#section4-2)

[Learn](#section4-3)

[Conclusion](#section5)

[References](#section6)

![](https://static.igem.wiki/teams/5136/cbl-fix.png)

# . Engineering Success .

## Overview

This year, XMU-China focused on the optimization of industrial processes for
waste paper treatment and had made significant progress in this field
(See more details in our [Results](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27results%27%29%20%7D%7D) page). It is relatively easy to produce
target proteins by engineered bacteria and purify them in the laboratory, however
in industry, the additional purification process will greatly increase the cost
of paper recycling. During this season, some successful engineering iterations
for obtaining target proteins without the need of purification, particularly
using the *E. coli* autolytic system FLSA (FhuD-T7 lysozyme-SsrA mediated
autolytic system) in enzymes production and releasing, have been achieved.
All the related data are recorded below and we hope it will be helpful for
other iGEM teams and SynBio community if they meet the related issue of
obtaining recombinant proteins from microbial cell factories by means of
purification-free technology.

## Cycle 1: Verifying the function of FhuD-GGG linker-T7 Lysozyme 119V-SsrA mediated autolytic system (BBa\_K5136031)

## Design

In the autolytic system FLSA (FhuD-T7 lysozyme-SsrA mediated autolytic system), T7 lysozyme will reach the peptidoglycan layer in periplasmic space with the assistance of Sec-Tat dual pathway signal peptide FhuD to permeabilize the host inner membrane, resulting in the degradation of the peptidoglycan framework to cause cell collapse. While the degron, SsrA tag, was fused to the C-terminal of T7 lysozyme for alleviating the undesired lysis of host cells caused by leaking T7 lysozyme (1).

For determining the efficiency of FLSA autolytic system to release
intracellularly produced recombinant proteins, we chose the superfolder green
fluorescent protein (sfGFP) as the reporter (2) so that the released amount
could be easily measured as fluorescence intensity. Therefore, the **sfGFP
release efficiency** is defined as **the ratio of fluorescence intensity of
cultural supernatant to that of bacterial culture**.

## Build

First, the two parts, FhuD signal peptide and T7 lysozyme 119V
([NP\_041973.1](https://www.ncbi.nlm.nih.gov/protein/NP_041973.1)), were linked by a flexible linker (GGGGS)3
(GGG, in short) (3). Following that, the SsrA tag was fused to the
C-terminal of T7 lysozyme, generating a basic part of coding
sequence ([BBa\_K5136031](https://parts.igem.org/Part%3ABBa_K5136031)).

There were four basic parts assembled into the vector pSB1C3 (Figure 1A)
*via* Gibson assembly: *L*-arabinose inducible promoter ([BBa\_I0500](http://parts.igem.org/Part%3ABBa_I0500)),
RBS ([BBa\_B0034](http://parts.igem.org/Part%3ABBa_B0034)), FhuD-GGG linker-T7 lysozyme 119V-SsrA coding sequence
([BBa\_K5136031](https://parts.igem.org/Part%3ABBa_K5136031)) and terminator ([BBa\_B0015](http://parts.igem.org/Part%3ABBa_B0015)). Assembly product was
subsequently transformed into *E. coli* DH10β. Then the positive
transformants were selected by chloramphenicol and confirmed by colony
PCR (Figure 1B) and sequencing.

![](https://static.igem.wiki/teams/5136/engineer-success/1.png)

**Figure 1 FhuD-GGG linker-T7 lysozyme 119V-SsrA mediated autolytic system. (A)**
The gene circuit of the FhuD-GGG linker-T7 lysozyme 119V-SsrA mediated autolytic system (BBa\_K5136221). **(B)**
Agarose gel electrophoresis of the colony PCR products of BBa\_K5136221\_pSB1C3 in *E. coli* DH10β. Target bands (2332 bp) can be observed at the position between 2000 bp and 3000 bp.

Afterwards, the reporter plasmid (*sfgfp*\_pET-28a(+), laboratory stock) and
the correct autolytic plasmid ([BBa\_K5136221](https://parts.igem.org/cgi/partsdb/dna_info.cgi?part=BBa_K5136221)\_pSB1C3) were co-transformed
into *E. coli* BL21(DE3). The [BBa\_I0500](http://parts.igem.org/Part%3ABBa_I0500)\_pSB1C3 (laboratory stock) was set
as the control and co-transformed with the reporter plasmid into *E. coli*
BL21(DE3) to obtain the control strain. Correct dual-plasmid transformants
were selected by chloramphenicol and kanamycin.

## Test

For obtaining recombinant proteins (sfGFP) *via* the FLSA autolytic system,
the *E. coli* BL21(DE3) strain harboring [BBa\_K5136221](https://parts.igem.org/cgi/partsdb/dna_info.cgi?part=BBa_K5136221)\_pSB1C3 and
*sfgfp*\_pET-28a(+) was firstly induced to intracellularly produce sfGFP by
adding isopropyl-*β*-D-thiogalactopyranoside (IPTG). After **10 hours′ induction
for accumulating sfGFP**, the second inducer *L*-arabinose was added to trigger
the autolytic system. The fluorescence intensity (λex = 475 nm, λem = 515 nm)
of bacterial culture and supernatant were measured and the release efficiency
was calculated after **autolysis for 16 hours**. (See detailed protocol in our
[Experiments](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27experiments%27%29%20%7D%7D) page)

![](https://static.igem.wiki/teams/5136/engineer-success/2.png)

**Figure 2 sfGFP release efficiency of the *E. coli* FhuD-lysozyme-SsrA mediated autolytic system.**
The original FLSA system showed a 2.3 times release efficiency of the control (I0500).

The release efficiency of the original FLSA system (FhuD-GGG linker-T7 lysozyme 119V-SsrA) was higher than that of the control group, indicating that the system functioned indeed. However, the low release efficiency (~6%) might hinder the further and practical implementation of this autolytic system.

## Learn

The original FLSA autolytic system (FhuD-GGG linker-T7 lysozyme 119V-SsrA)
suffered from low release efficiency. Since the T7 lysozyme plays a key role
in degrading the peptidoglycan framework to cause cell collapse, we then
focused on the sequence of T7 lysozyme we used in the experiments. In addition
to the amidase activity, the T7 lysozyme was identified to inhibit the T7 RNA
polymerase, thus regulating the transcription level of T7 promoter (4).
Therefore, a well-known plasmid, pLysS ([GenBank: HB865373.1](https://www.ncbi.nlm.nih.gov/nuccore/HB865373.1)), which contained
the coding sequence of T7 lysozyme, was constructed to improve the inducible
T7 expression system in *E. coli* BL21(DE3), especially for some toxic proteins
due to the lower leakage resulting from the inhibition of T7 RNA polymerase
(5). We then performed sequence alignment of the T7 lysozyme encoding on the
pLysS to the sequence we used ([NP\_041973.1](https://www.ncbi.nlm.nih.gov/protein/NP_041973.1)) and found difference of a single
residue at the 119th site (Figure 3). Even though no evidence showed the
residue at this site will affect either the activity of amidase or inhibitor
of T7 RNA polymerase (6), we wonder whether the change at this site (V to G)
contribute to the improved release efficiency of FLSA autolytic system or not.

![](https://static.igem.wiki/teams/5136/engineer-success/3.png)

**Figure 3 Sequence alignment between NP\_041973.1 and the T7 lysozyme sequence encoding on pLysS, where the different residue is highlight in green.**

## Cycle 2: Changing a single residue of T7 lysozyme for FLSA system (BBa\_K5136032)

## Design and Build

We isolated the pLysS plasmid from the commercial strain BL21(DE3) pLysS.
Then the coding sequence of T7 lysozyme (119G) was amplified and cloned to
replace the original T7 lysozyme sequence (119V) on
[BBa\_K5136221](https://parts.igem.org/Part%3ABBa_K5136221)\_pSB1C3 *via*
Gibson assembly so as to generate [BBa\_K5136222](https://parts.igem.org/Part%3ABBa_K5136222)\_pSB1C3 (Figure 4A).
The assembly product was transformed into *E. coli* DH10β.
Then the positive transformants were selected by chloramphenicol and
confirmed by colony PCR (Figure 4B) and sequencing.

![](https://static.igem.wiki/teams/5136/engineer-success/4.png)

**Figure 4 FhuD-GGG linker-T7 lysozyme 119G-SsrA mediated autolytic system. (A)**
The gene circuit of the FhuD-GGG linker-T7 lysozyme 119G-SsrA mediated autolytic
system (BBa\_K5136222). **(B)** Agarose gel electrophoresis of the colony PCR
products of BBa\_K5136222\_pSB1C3 in *E. coli* DH10β. Target bands (2332 bp) can be
observed at the position between 2000 bp and 3000 bp.

Afterwards, the reporter plasmid and the correct autolytic plasmid ([BBa\_K5136222](https://parts.igem.org/Part%3ABBa_K5136222)\_pSB1C3) were co-transformed into *E. coli* BL21(DE3) and the correct dual-plasmid transformants were selected by chloramphenicol and kanamycin.

## Test

The experimental procedures are as mentioned in Cycle 1.

![](https://static.igem.wiki/teams/5136/engineer-success/5.png)

**Figure 5 sfGFP release efficiency of the *E. coli* FhuD-lysozyme-SsrA mediated autolytic system, in which the 119th residue of T7 lysozyme was changed to Glycine.** The FLSA (119G) system showed a 5.2 times release efficiency of the control (I0500).

Interestingly, changing the valine residue to glycine residue at the 119th site of T7 lysozyme does not impair the lysis effect of FLSA autolytic system but improves the sfGFP release efficiency to ~15% (Figure 5).

## Learn

The V119G variant of T7 lysozyme presented a better performance when played the role
to cause cell collapse, which implied that this change of a single residue
might promote the amidase activity of T7 lysozyme whereas the influence of
119th residue has not been reported. The effect of this site should be
further verified and the mechanism need to be explored in the future. However,
this improvement inspired us that other sequences in the FhuD-lysozyme-SsrA
fusion may also affect the efficiency of FLSA autolytic system. Given the case
of last season that the linker between signal peptide (Kpsp) and the cargo (GFP)
has an nonnegligible influence on the folding of cargo
([Engineering Success of XMU-China 2023](https://2023.igem.wiki/xmu-china/engineering)), we turned to investigate whether the change of linker between FhuD and T7 lysozyme will have a positive effect on improving the release efficiency or not.

## Cycle 3: Changing the linker between FhuD signal peptide and T7 lysozyme for FLSA system (BBa\_K5136030)

## Design and Build

We replaced the GGG linker ((GGGGS)3) with “TSSSIASSSPSSVAGS”
(TSS in short), a linker we have used in various surface display system
([Parts of XMU-China 2020](https://2020.igem.org/Team%3AXMU-China/Parts) and
[Parts of XMU-China 2022](https://2022.igem.wiki/xmu-china/parts)).
Linker change was achieved in the coding sequence of FhuD-GGG linker-T7
lysozyme 119G-SsrA *via* Gibson assembly, thus generating
[BBa\_K5136220](https://parts.igem.org/Part%3ABBa_K5136220) on
pSB1C3 vector (Figure 6A). Positive transformants were selected by
chloramphenicol and confirmed by colony PCR and sequencing (Figure 6B).
Then, the reporter plasmid and the correct autolytic plasmid
([BBa\_K5136220](https://parts.igem.org/Part%3ABBa_K5136220)\_pSB1C3) were co-transformed into *E. coli* BL21(DE3) and the correct dual-plasmid transformants were selected like before.

![](https://static.igem.wiki/teams/5136/engineer-success/6.png)

**Figure 6 FhuD-TSS linker-T7 lysozyme 119G-SsrA mediated autolytic system. (A)**
The gene circuit of the FhuD-TSS linker-T7 lysozyme 119G-SsrA mediated autolytic system (BBa\_K5136220).
**(B)** Agarose gel electrophoresis of the colony PCR products of BBa\_K5136220\_pSB1C3 in *E. coli* DH10β. Target bands (2335bp) can be observed at the position between 2000 bp and 3000 bp.

## Test

The experimental procedures are as mentioned in Cycle 1.

![](https://static.igem.wiki/teams/5136/engineer-success/7-newest.png)

**Figure 7 sfGFP release efficiency of the *E. coli* FhuD-lysozyme-SsrA mediated autolytic system, in which the GGG linker was changed to TSS linker.**
The FLSA (119G, TSS linker) system showed a 7.9 times release efficiency of the control (I0500).

The alternative FLSA system which harboring TSS linker between FhuD signal peptide and T7 lysozyme had an improved sfGFP release efficiency of ~22% (Figure 7), which again emphasizes the significance of linker sequence between different functional elements (7).

## Learn

Changing the linker sequence between the signal peptide and recombinant protein to be secreted might affect both the folding of cargo and the secretion efficiency (8). Depending on the secretion pathway, useful machinery of protein-folding enhancers in the periplasm could be accessible (9). The dual-pathway signal peptide FhuD, possessing both Sec- and Tat- pathway properties, might help solve the folding issue of lysozymes (1). Anyway, the better-performed TSS linker contributes to the improvement of release efficiency of the FLSA autolytic system, even though the mechanism is elusive and need to be further investigated.

## Conclusion

For realizing the purification-free obtainment of target proteins, the deinking enzymes in our case, we implemented an autolytic system named FLSA (FhuD-T7 lysozyme-SsrA mediated autolytic system) to lysis the microbial cell factory after the target proteins are produced and accumulated. The original FLSA (FhuD-GGG linker-T7 lysozyme 119V-SsrA) suffered from low release efficiency. For optimizing the effect of FLSA system, we changed a single residue on the sequence of T7 lysozyme (V119G) to obtain an improved release efficiency from ~6% to ~15% at first. Then we replaced the linker between FhuD and T7 lysozyme with the one we used in 2020 and 2022 seasons, thus finally achieved a **considerable** release efficiency about 22% (Figure 8). This improved FLSA system will assist the release of deinking enzymes, offering a promising method for obtaining target proteins without purification, which is critical to lower the cost of paper recycling industry.

![](https://static.igem.wiki/teams/5136/engineer-success/8-new.png)

**Figure 8 Comparison of the sfGFP release efficiency of various engineered FLSA systems.**

Engineering a biological system cannot be easy, we need to pay our all
attention to it and move towards success through experiments and
feedback step by step. Here, we have tried to demonstrate how we
**engineered the sequence of FLSA** to **improve the release efficiency of
autolytic system** through successive iterations of the **DBTL**
(**D**esign-**B**uild-**T**est-**L**earn) cycle (Figure 9).

![](https://static.igem.wiki/teams/5136/engineer-success/9-new.png)

**Figure 9 Summary of the different engineering steps completed before reaching the goal of obtaining efficient autolytic system.**

## References

1. F. Zhang et al., Development of a Bacterial FhuD-lysozyme-SsrA Mediated Autolytic (FLSA) System for Effective Release of Intracellular Products. ACS Synth. Biol. 12, 196-202 (2023).
2. J. D. Pedelacq, S. Cabantous, Development and Applications of Superfolder and Split Fluorescent Protein Detection Systems in Biology. Int. J. Mol. Sci. 20, 14 (2019).
3. D. Wen, S. F. Foley, X. L. Hronowski, S. Gu, W. Meier, Discovery and Investigation of O-xylosylation in Engineered Proteins Containing a (GGGS)n Linker. Anal. Chem. 85, 4805-4812 (2013).
4. B. A. Moffatt, F. W. Studier, T7 Lysozyme Inhibits Transcription by T7 RNA Polymerase. Cell 49, 221-227 (1987).
5. F. W. Studier, Use of Bacteriophage T7 lysozyme to Improve an Inducible T7 Expression System. J. Mol. Biol. 219, 37-44 (1991).
6. X. Cheng, X. Zhang, J. W. Pflugrath, F. W. Studier, The Structure of Bacteriophage T7 Lysozyme, a Zinc Amidase and an Inhibitor of T7 RNA Polymerase. Proc. Natl. Acad. Sci. U S A 91, 4034-4038 (1994).
7. X. Chen, J. L. Zaro, W. C. Shen, Fusion Protein Linkers: Property, Design and Functionality. Adv. Drug. Deliv. Rev. 65, 1357-1369 (2013).
8. M. J. Burggraaf et al., Optimization of Secretion and Surface Localization of Heterologous Ova Protein in Mycobacteria by Using Lipy as a Carrier. Microb. Cell. Fact. 18, 44 (2019).
9. L. A. Burdette, S. A. Leach, H. T. Wong, D. Tullman-Ercek, Developing Gram-negative Bacteria for the Secretion of Heterologous Proteins. Microb. Cell. Fact. 17, 196 (2018).

{% 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/5136/load-new.gif)

[Commom Experiment](#section1)

[Enzymatic Deinking](#section2)

[Others](#section3)

![](https://static.igem.wiki/teams/5136/cbl-fix.png)

# . Experiments .

## Commom Experiment

### Molecular Cloning Handbook

#### [Handbook](#collapse2024)

[molecular-cloning-handbook-10-0.pdf](https://static.igem.wiki/teams/5136/molecular-cloning-handbook-10-0.pdf)

## Enzymatic Deinking

Protein Purification

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

- AKTA pure™ chromatography system
- LB or TB liquid medium
- 50 mM Tris-HCl buffer (pH 7.5)
- Elution Solution A (50 mM Tris-HCl, 0.3 M NaCl, 20 mM imidazole, pH 7.5)
- Elution Solution B (50 mM Tris-HCl, 0.3 M NaCl, 200 mM imidazole, pH 7.5)
- HisTrap HP His tag protein purification columns
- Kanamycin (50 mg/mL)

#### [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 reached at 0.6~0.8.
3. Corresponding inducer is added into bacteria culture from step 2 and kept at Suitable culture
conditions and last 16-20 h.
4. Bacteria culture form step 3 is centrifuged at 6500 rpm at 4 °C for 7 minutes to collect the cell
and resuspended with 25-30 mL Tris-HCl buffer (pH 7.5)
5. Repeat step 4 twice.
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 20 minutes to remove the cell debris and then
the
supernatant is filter through a 0.22-μm membrane filter.
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 Tris-HCl buffer (pH 7.5) overnight to
remove imidazole for subsequent uses.

Deinking Pulp Experiment

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

- Deli Ruiguan multifunctional office copy paper (A4)
- Blender
- Drying Oven
- Stirrer
- Water bath
- Gel imaging system
- 60 mm Büchner funnel
- i-Quip 7 cm fast quantitative filter paper
- Deink-enzymes
- Blotting papers

#### [Protocol](#collapse4)

1. Papers printed identical content are shredded into small strips.
2. Strips from step 1 are weighed and added into appropriate amount of purified water to prepare 1%
(w/v) pulp.
3. The complex from step 2 is grinded by a blender continuing 30 minutes.
4. 30 mL of the pulp is transferred into a flask and treated with 2 mL of 7.5 deink-enzyme solution
and kept at the optimal temperature of the enzyme for 60 minutes while being stirred by a
fixed-speed
stirrer.
5. After step 4, the pulp is added 0.6 mL limonene and shaken at 25 °C, 200 rpm for 1 minute, and
then
transferred into a beaker and stirred by a fixed-speed magnetic stirrer for 1 minute.
6. After 1 minute, a blotting paper is placed on the surface of the pulp for 30 seconds then is
removed.
7. The pulp is filtered by i-Quip 7 cm fast quantitative filter paper using a Büchner funnel to
collect paper fibers.
8. Paper fibers from step 7 are complete dried at 65 °C in a drying oven to get a recycled paper.

9. The recycled paper from step 8 is imaged by the Coomassie Brilliant Blue mode of gel imaging
system
and then its average grayscale value is analyzed by Image Lab and our
[Image Gray Read Tool](https://gitlab.igem.org/2024/software-tools/xmu-china).

In-Situ Deinking Experiment with Rhamnolipids

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

- Gel imaging system
- Fume hood
- 96-well plate
- 6-well plate
- Water-based fountain pen
- MUJI smooth oil-based blue ballpoint pen
- Deli Ruiguan multifunctional office copy paper (A4)
- Rhamnolipid samples
- rhamnolipid-limonene mixture
- SDS solution
- SDS-limonene mixture

#### [Protocol](#collapse6)

1. The paper is cut into 2.5 cm × 2.5 cm squares.
2. Square papers are written with the identical content and dried at 65 °C for 10 minutes to ensure
the ink is completely dry.
3. Each well of a 6-well plate is placed with a square paper from step 2 and added with 2.0 mL
rhamnolipid-limonene mixture.
4. The square papers are taken out per 15 minutes and air-dried until 1 h.

*Note: Take three parallel samples per treatment/time point to ensure experimental accuracy.*
5. The square papers are imaged by the Coomassie Brilliant Blue mode of gel imaging system and
analyzed by Image Lab.

Production and Adsorption Efficiency Measurement of
Cross-Linked Chitosan

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

- Chitosan powder
- 2.0% (w/w) acetic acid (HAc)
- 0.1 M NaOH
- 2.5% (w/w) glutaraldehyde
- 0.1 M HCl
- Diluted ink solution (diluted 2000 times)
- Water bath
- Tecan Infinite M200 microplate reader

#### A. Preparation of Crosslinked Chitosan

#### [Protocol](#collapse7)

1. 0.45 g of chitosan is dissolved by 15 mL of 2.0 % (w/w) HAc.40 mL of 0.1 M NaOH is added into
the
chitosan solution.
2. The solution from step 2 is centrifuged at 3600 rpm for 5 minutes to collect the precipitation.

3. The precipitation is resuspended by 50 mL of purified water to repeat step 3 until the pH of the
supernatant is neutral.
4. The precipitation is transferred to a petri dish, flatten with a spatula, dried at 80 °C to form
a
film and then weighed.
5. The dry film is treated with 2.5% (w/w) glutaraldehyde at 60 °C for 24 h to form crosslinked
Chitosan.

*Note: The ratio of glutaraldehyde to chitosan is 25 mL/g.*
6. The cross-linked Chitosan is separated from the complex from step 6 and then dried at 80 °C.
7. Discard the glutaraldehyde, and dry the sample again in the oven at 80 °C.

#### B. Chitosan Adsorption Experiment

#### [Protocol](#collapse8)

1. 0.10 g of crosslinked chitosan is added to a 1.5-mL centrifuge tube.
2. The crosslinked chitosan is soaked by 1.0 mL 0.1 M HCl for 90 minutes.
3. The crosslinked chitosan is wished by water until the water is neutral and air-dried.
4. 1.0 mL of diluted ink is mixed with the crosslinked chitosan from step 3.
5. OD400 of the supernatant is measured by Tecan Infinite M200 microplate reader
immediately and
then
measured per 1 h until 4 h.
6. Calculate the adsorption rate based on the standard curve.

Ink Coating and Deinking Method

#### [Materials & Apparatus](#collapse10)

- 2.5% (w/w) rhamnolipid solution
- 2.5% (w/w) SDS solution
- M&G MG6128 black ink refill
- 75% (v/v) alcohol
- 1 cm × 1 cm square papers- Metal bath
- Tecan Infinite M200 microplate reader

#### [Protocol](#collapse11)

1. The ink is transferred into a 2-mL centrifuge tube and diluted by 75% (v/v) alcohol 10-20 times
approximately.
2. 15 µL diluted ink is added on a square paper and spread evenly by a pipette tip.
3. The square paper is air-dried for 3 minutes and then placed into a 2-mL centrifuge tube.

*Note: Take five parallel samples per experiment to ensure experimental accuracy.*
4. 1 mL of the reaction liquid (SDS solution, rhamnolipid solution, or purified water) is added into
the centrifuge tube from step 4.
5. The centrifuge tube is shaken at 25 °C, 1000 rpm for 10 minutes by metal bath.
6. OD400 of the supernatant in the centrifuge tube is measured.
7. The ink concentration of the supernatant is calculated by using the standard curve.

## Others

Lysis Efficiency Measurement of Autolysis System

#### [Materials & Apparatus](#collapse12)

- Tecan Infinite M200 microplate reader
- Centrifuge
- Chloramphenicol (34 mg/mL)
- Kanamycin (50 mg/mL)
- LB liquid medium
- IPTG (Isopropyl β-D-1-thiogalactopyranoside) (1 mol/L)
- double sterile water
- L-arabinose stock solution (20% (w/v))

#### [Protocol](#collapse13)

1. Bacteria culture is inoculated into LB liquid mediums at 2% (v/v) and kept at 37 °C, 200 rpm
overnight.
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 0.6~0.8.
3. 0.5 mM IPTG is added into bacteria culture to induce the expression of sfGFP at 18 °C for 20
h.
4. After 10 h, 0.25% arabinose is added into bacteria culture to initiate autolysis system
expression.
5. The fluorescence intensity of the bacteria culture is measured immediately by Tecan Infinite M200
microplate reader.
6. The bacteria culture is centrifuge at 6500 rpm for 10 minutes and then measured the fluorescence
intensity of the supernatant by Tecan Infinite M200 microplate reader.
7. After 10 h, the fluorescence intensity of the bacteria culture and the supernatant are
measured
again.
8. The lysis efficiency is calculated by dividing the fluorescence intensity of the supernatant by
the
fluorescence intensity of the bacteria culture.

*Note:
To further improve the autolysis efficiency of the E. coli FLSA system, the following two
methods
can be applied:
1. When inducing the Fhud-T7Lysozyme-SsrA module with 0.25% arabinose, add 0.5% Triton X-100 to
the
culture (add Triton X-100 along with arabinose).
2. Adjust the pH of the induced culture medium to 8.0 using 2 M NaOH ( before adding arabinose),
and
then shake at 25°C for 30 minutes. Analyze the sfGFP distribution with a microplate reader and
10%
SDS-PAGE. Quantify the band intensity using ImageJ. Define the lysis or release efficiency as
the
percentage of the target protein in the supernatant divided by the total target protein in the
culture.*

Verification of the Surface Display System

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

- LB liquid medium chloramphenicol
- Chloramphenicol (34 mg/mL)
- PBS buffer (pH 7.5)
- L-arabinose stock solution (20% (w/v))
- Tecan Infinite M200 microplate reader
- Centrifuge
- HisTag-Spytag-GFP

#### [Protocol](#collapse16)

1. Bacteria culture is inoculated into LB liquid mediums at 2% (v/v) and kept at 37 °C, 200 rpm
overnight.
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 0.6~0.8.
3. 0.2% arabinose is added into bacteria culture to induce the expression of INPNC-HisTag-SpyCatcher
in *E. coli* BL21 (DE3) at 37 °C for 12 h.
4. 0.2 mg/mL HisTag-Spytag-GFP is added into bacteria culture and kept at 37 °C, 200 rpm for 12
h.

5. 1 mL of bacteria culture is centrifuged at 6500 rpm for 5 minutes, then 1 mL fresh LB liquid
medium
was used to resuspend the precipitate, repeat 2 times for centrifugation and suspension under the
same
conditions.
6. Then the fluorescence intensity of the precipitate is measured by Tecan Infinite M200 microplate
reader.

Verification for the Cytotoxicity of CcdB

#### [Materials & Apparatus](#collapse24)

- LB liquid medium
- Ampicillin (100 mg/mL)
- LB solid plates
- *L*-arabinose stock solution (20% (w/v))
- *D*-glucose stock solution (50% (w/v))

#### [Protocol](#collapse25)

1. Bacteria culture is inoculated into LB liquid medium at 2% (v/v) and kept at 37 °C, 200 rpm
overnight (12-16 h).
2. The overnight 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 (*L*-arabinose) is added *L*-arabinose stock solution to
reach the concentration of 0.02% and the other group (*D*-glucose) is added *D*-glucose stock solution
to reach the concentration of 0.05%.

*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 *L*-arabinose group)/(CFU/mL of the *D*-glucose group)].

Spot Assay for Kill Switch

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

- LB liquid medium
- Ampicillin (100 mg/mL)
- Self-made blue-light illumination device

#### [Protocol](#collapse18)

1. Bacteria culture is inoculated into LB liquid medium at 2% (v/v) and kept at 37 °C, 200 rpm
overnight (12-16 h).
2. The overnight 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 (ILLUMINATION) is exposed to blue light (with a relative
light intensity of 250) and the other group (DARKNESS) is kept in darkness.

*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 6 times, resulting in 7 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
ILLUMINATION group)/(CFU/mL of the DARKNESS group)].

Verification of Heavy Metal Adsorption System

#### [Materials & Apparatus](#collapse31)

-1 M MgSO4 solution
-1 M CaCl2 solution
-40% Glycerin solution
-5×M9 Salt Solution
![](https://static.igem.wiki/teams/5136/experiment/exp-zjs-1.png)
- LB liquid mediums
- L-arabinose stock solution (0.2 g/mL)
- Potassium dichromate solution (1 g/L)
- 50% H2SO4
- 50% H3PO4
- Chloramphenicol (34 mg/mL)
- Camera
- Potassium dichromate solids
- Chromogenic Agent
![](https://static.igem.wiki/teams/5136/experiment/exp-zjs-2.png)

#### A. Preparation of M9 Medium

#### [Protocol](#collapse32)

1. The 5×M9 Salt solution, 1 M CaCl2 solution and 1 M MgSO4 solution are
sterilized at 115 °C for 30
minutes.
2. The 40% Glycerin is filtered by using a 0.22-μm syringe filter.
3. These solutions are mixed according to the following formula to prepare the M9 medium:
![](https://static.igem.wiki/teams/5136/experiment/exp-zjs-3.png)

#### B. Plotting of Standard Curves of Cr2O72-

#### [Protocol](#collapse33)

1. K2Cr2O7 solution is prepared at concentrations of 1 mg/L, 2
mg/L, 3 mg/L, 4 mg/L, 5 mg/L in a 10
mL centrifuge tube.
2. 100 μL of 50% H2SO4 and 100 μL of 50% H3PO4 are added
to tubes from step 1, mixed well, and then
add 400 μL of the chromogen agent, react for 10 minutes.
3. After reaction, the pictures of tubes from step 2 are taken by using the high-saturation mode of
the camera.
4. RGB values of three points in each tube are measured, and take the average G values to draw the
standard curve.

#### C. Verification of Cr2O72- Adsorption

#### [Protocol](#collapse34)

1. Bacteria culture is inoculated into LB liquid mediums at 2% (v/v) and kept at 37 °C, 200 rpm
overnight.
2. 1 mL of bacteria culture is centrifuged at 6500 rpm for 3 minutes, then 1 mL fresh M9 medium was
used to resuspend the precipitate, repeat 2 times for centrifugation and suspension under the same
conditions.
3. The bacteria culture from step 2 is inoculated into 20 mL of M9 medium at a concentration of 2%
and kept at 37 °C, 200 rpm for 12 h.
4. Bacteria culture from step 2 is inoculated into M9 mediums at 4% (v/v) and kept at 37 °C, 200 rpm
until the OD600 of bacteria culture reached at 0.7~0.9.
5. 0.2% arabinose is added into bacteria culture to initiate the expression of
INPNC-linker-MT3-linker-MT2A at 37 °C, 200 rpm.
6. After 4 h, 1.25 mL 1 g/L K2Cr2O7 solution is added into
bacteria culture and then incubate at
200 rpm at 37°C for 24 h.
7. 4.5 mL of the bacterial cultures is collected at 0 h and 24 h.
8. 1.5 mL of the supernatant is placed into a 2.0 mL centrifuge tube and centrifuge at 6500 rpm for
20 minutes at room temperature.
9. The supernatant is filtered by using a 0.22-μm syringe filter.
10. 15 μL of 50% H2SO4 and 15 μL of 50% H3PO4 are added
to the filtrate, and then 60 μL of chromogen
agent is added after mixing, and the reaction is carried out for 10 minutes.
11. After reaction, the pictures of tubes from step 2 are taken by using the high-saturation mode of
the camera.
12. RGB values of three points in each tube are measured, take the average G values and substitute
it into the standard curve to calculate the chromium concentration in the original samples.

Characterization of the secretion system

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

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

#### [Protocol](#collapse20)

1. Bacteria culture is inoculated into LB liquid mediums at 2% (v/v) and kept at 37 °C, 200 rpm
overnight.
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 0.6~0.8.
3. 0.1 mM IPTG is added into bacteria culture to induce the expression of signal peptide-GFP at 37
°C for 10 h.
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 bacteria culture is centrifuged at 6500 rpm for 10 minutes and then measured the fluorescence
intensity of the supernatant by Tecan Infinite M200 microplate reader.
7. 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 deinking enzymes

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

- Tecan Infinite M200 microplate reader
- Centrifuge
- Kanamycin (50 mg/mL)
- LB liquid medium
- IPTG (Isopropyl β-D-1-thiogalactopyranoside) (1 mol/L)
- double sterile water
- δ-aminolevulinic acid hydrochloride (0.5 mol/L)

#### [Protocol](#collapse22)

1. Bacteria culture is inoculated into LB liquid mediums at 2% (v/v) and kept at 37 °C, 200 rpm
overnight.
2. Bacteria culture from step 1 is inoculated into LB liquid mediums at 2% (v/v) and kept at 25 °C,
150 rpm until the OD600 of bacteria culture reached at 0.6.
3. 0.2 mM IPTG and 0.5mM δ-aminolevulinic acid hydrochloride are added into bacteria culture to
induce the expression of signal peptide-GFP at 37 °C for 36 h.
4. 1 mL of the bacterial cultures is collected per 6 h until 36 h.
5. The bacteria culture is centrifuged at 6500 rpm for 10 minutes and take the supernatant for
SDS-PAGE (Gray scale value analysis was performed on the bands).
6. The supernatant from the 36th h is taken for deinking pulp experiment.

{% 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/5136/load-new.gif)

[Overview](#section1)

[Screen Enzymes](#section3)

[Screen Monooxygenase](#section4)

[Autolysis System](#section5)

[Secretion System](#section6)

[Secrete Monooxygenase](#section8)

[Display System](#section9)

[Kill Switch](#section10)

[Further Explorations](#section11)

[Conclusion](#section12)

[Future Prospects](#section13)

[References](#section15)

# . Results .

## 1 Overview

Deinking is the bottleneck for waste paper recycling, which requires a substantial amount of toxic
chemicals. It also releases poisonous organic agents and heavy metals that may cause environmental
contamination and expensive disposal costs.

Our project, REPARO, focuses on developing an unprecedented solution based on synthetic biology, which could
serve as a viable alternative to the chemical method on the industry scale. We have successfully established
a screening method for enzymes with high deinking efficiency based on the gray scale changes.
Leveraging the monooxygenase CYP199A4 and its mutants reported in the literature, we have identified a
monooxygenase with high efficiency in deinking, a significant milestone in the wastepaper recycling process.
This, combined with a rapid screening method, structure-activity analysis, and saturation mutation, has led
us to the CYP199A4 mutant T253A, which exhibits superior deinking performance. By characterizing classic
signal peptides, we proved that LMT has a high secretion efficiency. It realized the continuous secretion of
CYP199A4 T253E with significant deinking performance. Furthermore, we expanded the application in erasing
the handwriting based on the combination of crosslinked chitosan and rhamnolipid. Beyond that, displaying
the metallothioneins on the surface of engineered bacteria could successfully reduce the heavy metal ion
Cr2O72- in the wastewater. Finally, we proved that the scheme we designed
is reasonable and feasible, which
paves the way for its application at an industrial scale.

## Highlight

##### Develop a screen method and explore the enzymes mutants with the best deinking performance.

##### Continuous secretion of CYP199A4 T253E with significant deinking performance.

##### Construct surface display system to significantly absorb heavy metal ion.

##### Construct a blue light-induced kill switch to achieve biocontainment.

##### Propose multi-scale estimation (MUSES) for the secretion kinetics of signal peptides for real world engineering.

## 2 Screen Enzymes with the Best Deinking Performance

### 2.1 Deinking Blank System

The deinking blank system consisted of a 50 mL pulp with 1 g Na2SiO3, and 1.67 mL
H2O2 (1), which is the same
in each deinking method. Pulp was produced by the paper that printed the same article, ensuring the same ink
concentration. The pulp preparation referred to standard operating procedure (see [SOP](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27sop%27%29%20%7D%7D) for
more details).
Then, limonene (see [design](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27design%27%29%20%7D%7D) and [IHP](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27human-practices%27%29%20%7D%7D) for more details) was selected from many
hydrophobic solvents to extract
the ink from the mixture after the reaction. The suction filtration was carried out through the Büchner
funnel to remove the water and dry it in ovens. Take a picture of the paper cake after drying, and read the
gray scale through the written program by automatically selecting (see [SOP](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27sop%27%29%20%7D%7D) for
more details). These
processes were summarized to a standard operating procedure (see [SOP](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27sop%27%29%20%7D%7D) for
more details). It was worth noting
that the higher the gray scale, the better the deinking effect. The experimental results showed that the
deinking effect was almost invalid for **the deinking blank system, which was set as the negative
control** (Figure 1).

![](https://static.igem.wiki/teams/5136/result/1.jpg)

**Figure 1 The gray scale of the paper cake deinked by
deinking negative control system.**

### 2.2 Chemical Method

Currently, chemical deinking is the classical method used in industry (see [IHP](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27human-practices%27%29%20%7D%7D) for more details), which was
selected as a positive control. In the chemical method, 1 g NaOH was added to the deinking blank system and
reacted at 30 °C for 30 min. After the standard operating procedure (see [SOP](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27sop%27%29%20%7D%7D) for
more details), read the
gray scale automatically. The experimental results showed a high value of the gray scale (Figure 2),
demonstrating that the chemical method exhibits a remarkable effect in deinking.

![](https://static.igem.wiki/teams/5136/result/2.jpg)

**Figure 2 The gray scale of the paper cake deinked by
chemical method.**

### 2.3 Enzymatic Method

#### 2.3.1 Cellulase

The plasmid [BBa\_K5136026](http://parts.igem.org/Part%3ABBa_K5136026) was
transformed into *E. coli* BL21(DE3), then the positive transformants were
selected by kanamycin and confirmed by colony PCR (Figure 3A) and gene sequencing. The plasmid verified by
sequencing was successfully transformed into *E. coli* BL21(DE3).

![](https://static.igem.wiki/teams/5136/result/3.png)

**Figure 3** (**A**) DNA gel electrophoresis of the colony PCR products of
BBa\_K5136026\_pET-28a(+) in *E.
coli*
BL21(DE3). Target bands (1518 bp) can be observed at the position between 1000 bp and 2000 bp.
(**B**)
SDS-PAGE
analysis of cellulase EG5C protein. Target bands (56.1 kDa) can be observed at the position around 52
kDa.

After being cultivated and induced by 0.5 mM IPTG, the GE AKTA Prime Plus FPLC System was employed to
collect purified protein from the lysate supernatant. Cellulase was verified by sodium dodecyl sulfate
(SDS)-12% (wt/vol) polyacrylamide gel electrophoresis (PAGE) and Coomassie blue staining (Figure 3B).

After purification, we harvest the cellulase EG5C successfully. 1 mL cellulase (0.2 mg/mL) was added in the
deinking blank system and reacted at 60 °C for 60 min. After the standard operating procedure (see [SOP](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27sop%27%29%20%7D%7D) for
more details), read the gray scale automatically. The experimental results showed that the gray scale is
higher than that of deinking blank system (negative control), while lower than that of chemical
method (positive control). So, cellulase has the ability to remove ink, but there is still insufficient
compared with the chemical deinking (Figure 4).

![](https://static.igem.wiki/teams/5136/result/4.jpg)

**Figure 4 The gray scale of paper cake deinked with cellulase.**

#### 2.3.2 Laccase

The plasmid [BBa\_K5136027](http://parts.igem.org/Part%3ABBa_K5136027) was
transformed into *E. coli* BL21(DE3), then the positive transformants were
selected by kanamycin and confirmed by colony PCR and gene sequencing. The plasmid verified by sequencing
was successfully transformed into *E. coli* BL21(DE3).

![](https://static.igem.wiki/teams/5136/result/5.png)

**Figure 5** (**A**) DNA gel electrophoresis of the colony PCR products of
BBa\_K5136027\_pET-28a(+) in *E.
coli*
BL21(DE3). Target bands (1659 bp) can be observed at the position between 1000 bp and 2000 bp.
(**B**)
SDS-PAGE analysis of laccase-his protein. Target bands (62.6 kDa) can be observed at the position around
66 kDa.

After being cultivated and induced by 0.03 mM IPTG and 0.5 mM CuSO4, the GE AKTA Prime Plus FPLC
System was
employed to collect purified protein from the lysate supernatant. Laccase was verified by sodium dodecyl
sulfate (SDS)-12% (wt/vol) polyacrylamide gel electrophoresis (PAGE) and Coomassie blue staining.

After purification, we harvest the laccase successfully. 1 mL laccase (0.2 mg/mL) was added to the deinking
blank system and reacted at 70 °C for 60 min. After the standard operating procedure (see [SOP](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27sop%27%29%20%7D%7D) for
more
details), read the gray scale automatically. The experimental results show that the waste paper
treated with cellulase alone exhibits a similar gray scale to cellulase. So, laccase also has the
ability to remove ink, but is still insufficient compared with chemical deinking.

![](https://static.igem.wiki/teams/5136/result/6.jpg)

**Figure 6 The gray scale of paper cake deinked with laccase.**

#### 2.3.3 Monooxygenase (P450)

The engineering bacteria were cultivated and induced by 0.2 mM IPTG, the GE AKTA Prime Plus FPLC System was
employed to collect purified protein from the lysate supernatant. P450 was verified by sodium dodecyl
sulfate (SDS)-12% (wt/vol) polyacrylamide gel electrophoresis (PAGE) and Coomassie blue staining.

![](https://static.igem.wiki/teams/5136/result/7.png)

**Figure 7 SDS-PAGE analysis of P450 CYP102A1 protein. Target bands (117.8 kDa) can be observed at the
position around 95 kDa.**

After purification, we harvest P450 successfully. 1 mL P450 (0.35 mg/mL) and 0.012 mg NADPH were added in the
deinking blank system and reacted at 30 °C for 60 min. After standard operating procedure (see [SOP](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27sop%27%29%20%7D%7D) for
more
details), read the gray scale automatically. The gray scale treated by P450 was slightly higher
than that of cellulase and laccase (Figure 8).

![](https://static.igem.wiki/teams/5136/result/8-p450.jpg)

**Figure 8 The gray scale of paper cake deinked with P450 CYP102A1.**

#### 2.3.4 Multienzymes

We try to investigate the deinking using multienzymes, coupling the function of two enzymes with different
functions. For one combination, 1 mL cellulase (0.2 mg/mL) and 1 mL laccase (0.2 mg/mL) were added in the
deinking blank system and reacted at 65 °C (the average of the two optimal temperatures) for 60 min. Because
the optimal temperature of cellulase (60 °C) varies from that of P450 (30 °C), for the other combination, we
first added 1 mL cellulase (0.2 mg/mL) to the deinking blank system at 60 °C for 60 min. After
the reaction, 1 mL P450 (0.35 mg/mL) was added and reacted at 30 °C for 60 min. After the standard operating
procedure (see [SOP](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27sop%27%29%20%7D%7D) for more details), read the gray scale
automatically. The experimental results
showed that there was no obvious improvement (Figure 9 and 10) in the gray scale in the two multienzymes
compared with
that of a single enzyme. We found that gray scale of waste paper treated by monooxygenase was the
highest one among the enzymes. Therefore, we hope to conduct further research on monooxygenase to obtain the
enzymes with better deinking performance.

![](https://static.igem.wiki/teams/5136/result/9.jpg)

**Figure 9 The gray scale of paper cake deinked with cellulase and laccase.**

![](https://static.igem.wiki/teams/5136/result/10-p450.jpg)

**Figure 10 The gray scale of paper cake deinked with cellulase and P450.**

## 3 Screen the Monooxygenase with Higher Deinking Efficacy

NADPH is an expensive coenzyme (428.7 $/g), which propels us to give up the way to use the NADPH-dependent
monooxygenases in the waste paper deinking. After reviewing the literature, we chose three kinds of
monooxygenases,
CYP199A4, OleTJE and SfmD and their mutants, which use H2O2 as an electron
donor. The targeted sequence was
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 11), and sequencing.
After being
cultivated and induced by 0.5 mM IPTG at 20 °C, the induction of CYP199A4 requires the addition of
δ-aminolevulinic acid hydrochloride (0.5 mM), the GE AKTA Prime Plus FPLC System was employed to get
purified protein from the lysate supernatant. SDS-PAGE and Coomassie blue staining were used to verify the
expression of the target protein (Figure 12).

**Table 1 The part information of CYP199A4, OleTJE and SfmD and their mutants.**

| Name | Type | Description |
| --- | --- | --- |
| [BBa\_K5136020](http://parts.igem.org/Part%3ABBa_K5136020) | coding | *SfmD WT* |
| [BBa\_K5136021](http://parts.igem.org/Part%3ABBa_K5136021) | coding | *SfmD 277F* |
||  |  |  |
| --- | --- | --- |
| [BBa\_K5136023](http://parts.igem.org/Part%3ABBa_K5136023) | coding | *OleTJE* |
| [BBa\_K5136024](http://parts.igem.org/Part%3ABBa_K5136024) | coding | *OleTJE 236S 100K 166K* |
| [BBa\_K5136025](http://parts.igem.org/Part%3ABBa_K5136025) | coding | *OleTJE 236S 360K 167K* |
| [BBa\_K5136000](http://parts.igem.org/Part%3ABBa_K5136000) | coding | *CYP199A4 WT*-His tag |
| [BBa\_K5136001](http://parts.igem.org/Part%3ABBa_K5136001) | coding | *CYP199A4 T253E*-His tag |
| [BBa\_K5136002](http://parts.igem.org/Part%3ABBa_K5136002) | coding | *CYP199A4 T253S*-His tag |

![](https://static.igem.wiki/teams/5136/result/11.png)

**Figure 11 DNA gel electrophoresis of the colony PCR products of CYP199A4 (A), SfmD (B) and OleTJE (C)
and
their mutants\_ pET-28a(+) in *E. coli* BL21(DE3)**. Target bands (1412 bp) can be observed
between 1000 bp and
2000 bp.

![](https://static.igem.wiki/teams/5136/part/047/result-figure12.png)

**Figure 12 SDS-PAGE analysis of CYP199A4 (A), SfmD (B), and
OleTJE (C, D, and E) and their mutants**. Target bands (about 48.6 kDa) can be observed
between 42 kDa and
52 kDa.

1 mL monooxygenase (0.2 mg/mL) was added in the deinking blank system and reacted at 30 °C for 60 min. After the standard operating procedure (see [SOP](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27sop%27%29%20%7D%7D) for
more details),
read the gray scale automatically. As shown in Figure 13, the pulp treated by SfmD-277F showed a
slight increase in ΔGray scale value compared to that of wild type. However, the value decreased
significantly in the mutant of OleTJE than that of the wild type. The pulp treated by
CYP199A4 T253E exhibited
the **highest ΔGray scale value**, which was increased by more than ten-fold compared to the wild-type
enzyme.
As shown in the picture from the microscope (Figure 14), the paper treated with CYP199A4 T253E (Figure 14)
has minimal ink residue among these enzymes, demonstrating the **highest deinking efficiency**.

![](https://static.igem.wiki/teams/5136/result/formula1.png)

![](https://static.igem.wiki/teams/5136/result/13.png)

**Figure 13 Comparison of the Relative Gray Scale of
different monooxygenases and their mutants.**

![](https://static.igem.wiki/teams/5136/result/14.png)

**Figure 14 Pulp Recycled Paper under a High-resolution
Microscope, the percentage is the value obtained by dividing by the Gray of negative.**

The experimental results showed that CYP199A4 and its mutants exhibited the best deinking efficiency.
Therefore, we carried out saturation mutagenesis at position T253 of CYP199A4, aiming to screen the enzyme
with the best deinking effect. The steps for circuit construction, induced expression, and protein
purification were conducted with the same conditions for CYP199A4 WT and mutants, which were detailed
described in the part page below (Table 2).

**Table 2 The part information of CYP199A4 and its mutants.**

| Name | Type | Description |
| --- | --- | --- |
| [BBa\_K5136000](http://parts.igem.org/Part%3ABBa_K5136000) | coding | *CYP199A4 WT*-His tag |
| [BBa\_K5136001](http://parts.igem.org/Part%3ABBa_K5136001) | coding | *CYP199A4 T253E*-His tag |
| [BBa\_K5136002](http://parts.igem.org/Part%3ABBa_K5136002) | coding | *CYP199A4 T253S*-His tag |
| [BBa\_K5136003](http://parts.igem.org/Part%3ABBa_K5136003) | coding | *CYP199A4 T253A*-His tag |
| [BBa\_K5136004](http://parts.igem.org/Part%3ABBa_K5136004) | coding | *CYP199A4 T253R*-His tag |
| [BBa\_K5136005](http://parts.igem.org/Part%3ABBa_K5136005) | coding | *CYP199A4 T253N*-His tag |
| [BBa\_K5136006](http://parts.igem.org/Part%3ABBa_K5136006) | coding | *CYP199A4 T253D*-His tag |
| [BBa\_K5136007](http://parts.igem.org/Part%3ABBa_K5136007) | coding | *CYP199A4 T253C*-His tag |
| [BBa\_K5136008](http://parts.igem.org/Part%3ABBa_K5136008) | coding | *CYP199A4 T253Q*-His tag |
| [BBa\_K5136009](http://parts.igem.org/Part%3ABBa_K5136009) | coding | *CYP199A4 T253W*-His tag |
| [BBa\_K5136010](http://parts.igem.org/Part%3ABBa_K5136010) | coding | *CYP199A4 T253G*-His tag |
| [BBa\_K5136011](http://parts.igem.org/Part%3ABBa_K5136011) | coding | *CYP199A4 T253H*-His tag |
| [BBa\_K5136012](http://parts.igem.org/Part%3ABBa_K5136012) | coding | *CYP199A4 T253I*-His tag |
| [BBa\_K5136013](http://parts.igem.org/Part%3ABBa_K5136013) | coding | *CYP199A4 T253L*-His tag |
| [BBa\_K5136014](http://parts.igem.org/Part%3ABBa_K5136014) | coding | *CYP199A4 T253K*-His tag |
| [BBa\_K5136015](http://parts.igem.org/Part%3ABBa_K5136015) | coding | *CYP199A4 T253M*-His tag |
| [BBa\_K5136016](http://parts.igem.org/Part%3ABBa_K5136016) | coding | *CYP199A4 T253F*-His tag |
| [BBa\_K5136017](http://parts.igem.org/Part%3ABBa_K5136017) | coding | *CYP199A4 T253P*-His tag |
| [BBa\_K5136018](http://parts.igem.org/Part%3ABBa_K5136018) | coding | *CYP199A4 T253V*-His tag |
| [BBa\_K5136019](http://parts.igem.org/Part%3ABBa_K5136019) | coding | *CYP199A4 T253Y*-His tag |

The targeted sequences were inserted in pET-28a(+) to construct circuits, which were transformed into *E.
coli* BL21(DE3) to express each enzyme. The positive transformants were confirmed by kanamycin, colony
PCR,
and sequencing. After being cultivated and induced by the addition of IPTG (0.2 mM) and δ-aminolevulinic
acid hydrochloride (0.5 mM) at 20 °C, the GE AKTA Prime Plus FPLC System was employed to get purified
protein from the lysate supernatant. SDS-PAGE and Coomassie blue staining were used to verify the expression
of the target protein.

![](https://static.igem.wiki/teams/5136/result/15.png)

**Figure 15 DNA gel electrophoresis of the colony PCR
products of CYP199A4 and its mutants\_ pET-28a(+) in *E. coli* BL21(DE3).** Target bands (1412 bp) can
be
observed between 1000 bp and 2000 bp.

![](https://static.igem.wiki/teams/5136/result/16.png)

**Figure 16 SDS-PAGE analysis of CYP199A4, its mutants.**
Target bands (about 48.8 kDa) can be observed between 42 kDa and 52 kDa.

Based on the results from the preliminary experiment, many enzymes exhibit excellent deinking performance,
resulting in the saturation of the gray scale. Thus, each enzyme was diluted from 0.2 mg/mL to 0.05
mg/mL. 1 mL of each monooxygenase (0.05 mg/mL) was added to the deinking blank system and reacted at 30 °C for 60 min. After the standard operating procedure (see [SOP](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27sop%27%29%20%7D%7D)
for more details), read the gray scale automatically. As shown in Figure 17, the gray scale of
some mutants increased by 50% at least, in which CYP199A4 T253A shows the best performance in deinking. As
shown in
the picture from the microscope (Figure 18), the paper treated with CYP199A4 T253A (Figure 18) has minimal
ink residue among these enzymes. The paper is relatively white, and the observed effect has reached 1.06
times higher than chemical deinking (Figure 18 chemical method).

![](https://static.igem.wiki/teams/5136/result/17.png)

**Figure 17 Comparison of the Relative Gray Scale of
CYP199A4 and its mutants.**

![](https://static.igem.wiki/teams/5136/result/18.png)

**Figure 18 Pulp Recycled Paper under a High-resolution
Microscope, the percentage is the value obtained by dividing by the Gray of A.**

## 4 The Effectiveness of An Autolysis System in Lysing Bacteria

Protein purification was a tedious procedure that also costs a lot of money. We attempted to construct
engineered bacteria based on synthetic biology to address the release of target proteins, which could skip
the protein purification step. Therefore, we obtained a modified LLSA system (please see [Design](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27design%27%29%20%7D%7D)
and
[Engineering Success](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27engineering%27%29%20%7D%7D) for details) from the *E.
coli* autolytic system FLSA (FhuD-T7 lysozyme-SsrA
mediated
autolytic system) (2). The system is designed to ensure that the target proteins can be efficiently produced
and smoothly released outside the cell, thus simplifying the process of obtaining the target proteins and
decreasing the treatment process cost (FLSA) (2). For the functional validation of both autolytic modules,
it is of utmost importance to assess the protein release capacity.

In order to verify whether the FLSA/LLSA autolytic system can release the target proteins to the
extracellular environment, we tried to optimize the gene sequences within the *E. coli* autolytic
system.
Using different signal peptides, linker and T7 lysozyme sequences, we assembled and constructed four
composite parts [BBa\_K5136221](http://parts.igem.org/Part%3ABBa_K5136221), [BBa\_K5136222](http://parts.igem.org/Part%3ABBa_K5136222), [BBa\_K5136220](http://parts.igem.org/Part%3ABBa_K5136220), [BBa\_K5136223](http://parts.igem.org/Part%3ABBa_K5136223), which were assembled into the
expression vector pSB1C3 by standard assembly serving as the correct autolytic plasmids. Besides, we
constructed a basic part [BBa\_K5136028](http://parts.igem.org/Part%3ABBa_K5136028)
as a reporter gene and then
assembled it into the expression vector
pET-28a(+) by standard assembly, serving as the reporter plasmid (*sfgfp*\_pET-28a(+), laboratory
stock).

Afterward, we co-transformed the reporter plasmid and the correct autolytic plasmid into *E. coli*
BL21(DE3),
and the cultures were grown overnight in the LB medium containing corresponding antibiotics. When the
OD600
value reaches 0.6-0.8, added 0.5 mM IPTG to induce sfGFP expression at 18 °C. After 10 h, 0.25%
*L*-arabinose was added to activate the autolytic system. The total fluorescence intensity was measured after
16 h of expression of the induced autolytic system, and the fluorescence intensity of the supernatant was
measured too. The ratio of the fluorescence intensity of the culture and supernatant was used to assess the
lysis efficiency of the FLSA system.

As shown in Figure 19, the ratio of supernatant fluorescence intensity to total fluorescence intensity in
the experiment group ([BBa\_K5136221](http://parts.igem.org/Part%3ABBa_K5136221), [BBa\_K5136222](http://parts.igem.org/Part%3ABBa_K5136222), [BBa\_K5136220](http://parts.igem.org/Part%3ABBa_K5136220),
and [BBa\_K5136223](http://parts.igem.org/Part%3ABBa_K5136223)) was significantly
higher
than that of the control group, demonstrating that the autolytic system FLSA/LLSA can release target
proteins into the extracellular environment efficiently. Moreover, we achieved effective protein release by
optimizing the structural combinations of our autolytic system FLSA/LLSA. (please see [Engineering Success](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27engineering%27%29%20%7D%7D)
for details).

![](https://static.igem.wiki/teams/5136/result/19-new.png)

**Figure 19 Comparison of the sfGFP release efficiency of
various engineered FLSA systems.**

## 5 The Secretion Efficiency of the Extracellular Secretion System

**Performance comparison of multiple signal peptides.**

The LMT sequence has been identified as a signal peptide capable of directing recombinant proteins out of
bacterial cells ([XMU-China 2021](https://2021.igem.org/Team%3AXMU-China)). Given the variety of engineered signal
peptides and corresponding
translocation systems developed for secretory production of heterologous proteins in *E. coli* (3), we
sought
to compare the performance of the LMT sequence against other commonly used signal peptides. Specifically,
several signal peptides or translocation systems associated with the Type II Secretion System
(T2SS)—including both the Sec-dependent system and the Twin-Arginine Translocation (TAT) system—as well as
the Type I Secretion System (T1SS), were selected and fused to the reporter protein sfGFP. The sequences of
the various signal peptides are provided in Table 1. Most of the secretion systems utilized in this
comparative analysis contain critical N-terminal regions necessary for recognition and function within the
translocation machinery (4), which resulted in N-terminal fusions with sfGFP. However, the HlyABD secretion
system, which belongs to T1SS, requires the C-terminal sequence of HlyA, as well as the heterologous
expression of HlyB and HlyD as assistants (5, 6). Therefore, a C-terminal fusion of sfGFP with HlyA was
constructed.

All signal peptide-sfGFP fusions were expressed under the control of the T7 promoter on the pET-28a(+)
vector in *E. coli* BL21(DE3) for parallel testing. Following induction, we monitored the fluorescence
intensity of the culture, the supernatant after centrifugation, and the OD600 of each group over
time.
Secretion efficiency was calculated at each sampling point as the ratio of fluorescence intensity of
supernatant to culture.

![](https://static.igem.wiki/teams/5136/result/20.png)

**Figure 20 Comparative tests of performance of multiple
signal peptides.** (**A**) Secretion efficiencies of different groups were calculated as time
progressed. (**B**)
The OD600, fluorescence intensity of supernatant and normalized culture fluorescence intensity
of
different groups after induction for 10 h.

Our results revealed that both OsmY- and HlyA-mediated secretion, with the assistance of HlyB and HlyD,
exhibited higher secretion efficiencies than any other signal peptides, including the newly discovered LMT
sequence, which ranked third. YebF also performed well in the comparison (Figure 20A). Most of the other
signal peptides exhibited secretion efficiencies below 2.5%. These findings demonstrate the variability in
secretion efficiencies among different signal peptides, despite the long-term usage of some of them (3).

Interestingly, certain groups showed lower calculated secretion efficiencies than the sfGFP control group
(which lacked a signal peptide, No SP). This may be due to the rapid growth and high levels of cytoplasmic
sfGFP expression driven by the strong T7 promoter in *E. coli* BL21(DE3) (7). The normalized
fluorescence
intensity of the sfGFP-only (No SP) culture was much higher than that of sfGFP fusions with signal peptides,
suggesting that even minor leakage due to cell growth or lysis contributed significantly to the fluorescence
intensity in the supernatant (Figure 20B). In the case of the LMT signal peptide, despite producing only
half the total sfGFP as the no-signal-peptide group (No SP), the fluorescence intensity in the supernatant
was comparable to that of the control group and significantly higher than that of any other signal peptide
after 10 h of induction. This highlights the LMT sequence's efficiency in exporting recombinant proteins
out of the bacterial cells.

While OsmY- and HlyA-mediated secretion showed the highest secretion efficiencies, they also impaired cell
growth (reflected by the lowest OD600 values) and reduced the normalized fluorescence intensity
in the
culture (Figure 20B). This indicates that overexpression of these systems places a metabolic burden on the
host cells, possibly explaining the larger error bars associated with their calculated secretion
efficiencies (Figure 20A). Additionally, the normalized fluorescence intensities in all groups with signal
peptides were reduced (Figure 20B), potentially due to adverse effects on sfGFP folding caused by the fusion
with signal peptides (8).

Classic signal peptides such as PelB and TorA, which direct recombinant proteins to the periplasmic space,
exhibited relatively low efficiency for extracellular translocation (Figure 20A) (9). Similarly, OmpA,
another well-known signal peptide, showed poor efficiency for recombinant protein secretion, unlike YebF and
OsmY, which also mediate extracellular expression (10). Notably, the artificial intelligence-generated
signal
peptide AIgen, which functions efficiently in *Bacillus subtilis*, performed poorly in exporting recombinant
proteins from *E. coli* (Figure 20A) (11). However, the OD600 values for the AIgen group
suggested no
significant growth burden (Figure 20B).

In summary, our self-discovered LMT signal peptide showed a slight growth defect but achieved considerable
secretion efficiency for the extracellular expression of recombinant proteins, making it a competitive
alternative compared to other commonly used signal peptides in *E. coli*.

**Highest Secretion Efficiency and Proper Metabolic Burden of LMT Signal Peptide**

The secretion kinetics of various signal peptides from both the Sec pathway (LMT, PelB, OmpA, OsmY, YebF, and AIgen) and the Tat pathway (TorA) were analyzed with our multi-scale model (see [model](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27Model%27%29%20%7D%7D) for more details). The two secretion kinetic parameters, α and γ, describe translocation rate from the cytoplasm to the periplasm and secretion efficiency (Figure 21A-B), respectively. LMT, a newly discovered signal peptide by Team XMU-China 2021 with excellent intrinsic secretion kinetic parameters, had a γ value that is 6.55-45.44 times higher than the others, indicating the highest secretion efficiency. With a considerable α value and proper metabolic burden, LMT achieved the highest supernatant fluorescence (Figure 20B). While the percentages of secretion, leakiness, and lysis, which are three primary contributors to supernatant fluorescence, changed dynamically with cell growth. This dynamic resulted in varying proportions of supernatant fluorescence among different signal peptides by the end of the experiment (Figure 21C).

![](https://static.igem.wiki/teams/5136/result/21.png)

**Figure 21 The model fitting results.**  (**A**) Estimated α for different signal peptides. (**B**) Estimated γ for different signal peptides. (**C**) The percentage of different sources contributes to the supernatant fluorescence.

**LMT-mediated Secretion under Constitutive Expression for Optimal Economic Cost**

We constructed circuits driven by promoters of different strengths (J23100, J23103, J23104, J23106, J23110, J23114), each containing RiboJ-B0034-LMT-linker-*sfgfp*-B0010.

By measuring the fluorescence intensity in the supernatant of each circuit, we can quantitatively analyze the secretion of the LMT signal peptide under different promoter strengths and thus evaluate its performance (Figure 22A).

We find that this follows exponential kinetics, which is consistent with the T7-mediated secretion. As shown in Figure 3A, secretion efficiency is affected by many factors, but not positively related to promoter strength (Figure 22B).

To ensure that these results reflect actual secretion rather than differences in cell growth, we measured fluorescence intensity data in 30 h of culture (Figure 22C) and supernatant (Figure 22D), and calculated the secretion efficiency (supernatant/culture) (Figure 22E).

![](https://static.igem.wiki/teams/5136/result/22.png)

**Figure 22 Characterization of LMT secretion performance with different Anderson promoters.** (**A**) Secretion Kinetic. (**B**) Fitting Result. (**C**) Secretion efficiencies. (**D**) Fluorescence intensity of supernatant. (**E**) Culture fluorescence intensity.

Interestingly, when we compared the secretion efficiency at 30 h, we found that high secretion efficiency does not mean high protein content in supernatant and culture. Although the secretion efficiency of weak promoter and the strong promoter were both higher, the weak promoter could fully realize extracellular secretion due to its low efficiency and less protein production by the exosystem, but the amount of protein in supernatant was less. For strong promoters, although the amount of protein secreted is large, the total fluorescence is low, indicating that the metabolic burden is heavy, which affects the survival rate. If the detection continues, very few bacteria will survive. , we use multi-scale model to deconstruct the dynamic secretion process of signal peptides accurately. The experimental results aligned well with the model's predictions (see [model](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27Model%27%29%20%7D%7D) for more details). By trading off, BBa\_K5136202 with promoter J23104 has the most appropriate secretion efficiency.

## 6 The deinking efficiency of deinking enzymes secreted by signal peptide

We have proved that some CYP199A4 mutants showed stronger deinking performance, and LMT showed a good secretion effect.
So, we try to verify the deinking efficiency of CYP199A4 mutants secreted to the supernatant by the LMT. The
engineered bacteria were cultured at 25 °C, and the supernatant culture was taken at 12 h, 18 h, 24 h, and
36
h, respectively, using SDS-PAGE to demonstrate that the fusion protein could be successfully secreted into
the supernatant. Gray scale analysis was performed on the bands, proving that the concentration of
LMT-CYP199A4 T253E in the culture supernatant gradually increased with time (Figure 23A). At the same time,
the supernatant from the culture in 36 h was used for the pulp deinking experiment (see [SOP](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27sop%27%29%20%7D%7D) for more
details), and the results are shown in Figure 23B. As shown in the picture from the microscope, LMT-CYP199A4
T253E in the supernatant showed a perfect deinking effect. **The above results showed that the LMT signal
peptide could secrete CYP199A4 T253E to the extracellular environment continuously, which further exhibits
the perfect performance in removing the ink from the pulp**.

![](https://static.igem.wiki/teams/5136/result/re-23.png)

**Figure 23 Characterization of His tag-LMT-CYP199A4 T253E.** (**A**) SDS-PAGE analysis (left) and gray scale value analysis (right) of the supernatant at different times. (**B**) Deinking characterization of His tag-LMT-CYP199A4 T253E (BBa\_K5136047)..

## 7 Display the Metallothioneins on the Surface of the Cell to Adsorb Heavy Metal Ions

### 7.1 The Surface Display Efficiency of INPNC

To verify whether INPNC can display target protein on the surface of bacteria, we introduced the Spy system,
which consists of SpyCatcher and SpyTag. SpyCatcher is an engineered split fragment of the
fibronectin-binding protein (FbaB) in *Streptococcus pyogenes*, and SpyTag is a peptide tag that could
steadily bind to SpyCatcher by forming an isopeptide bond. In our project, SpyCatcher was fused with INPNC
and SpyTag was fused with GFP. Then, His tag-SpyTag-GFP was added to the medium containing the engineered
bacteria, which express the fused protein INPNC-His tag-SpyCatcher. By monitoring the fluorescence intensity
of bacteria, we can verify if INPNC could anchor target proteins on the cell surface.

The His tag-SpyTag-GFP coding sequence ([BBa\_K5136034](http://parts.igem.org/Part%3ABBa_K5136034)) was assembled into the expression vector
pET-28a(+) by
Gibson assembly (Figure 24A). The constructed plasmid was transformed into *E. coli* BL21(DE3), then
the
positive transformants were selected by kanamycin and confirmed by colony PCR and sequencing. Target
bands (953 bp) can be observed at the position around 1000 bp (Figure 24B). After that, the bacteria were
cultivated and induced by 0.5 mM IPTG at 25 °C, and then the GE AKTA Prime Plus FPLC System was employed to
get purified protein His tag-SpyTag-GFP from the lysate supernatant. Purified protein was verified by sodium
dodecyl sulfate (SDS)-10% (wt/vol) polyacrylamide gel electrophoresis (PAGE) and Coomassie blue staining. As
shown in the gel image (Figure 24C), the target protein (29 kDa) can be observed at the position around 30
kDa on the purified protein lanes (FR).

![](https://static.igem.wiki/teams/5136/result/re-24.png)

**Figure 24 Constructing and purifying His tag-SpyTag-GFP
fused protein.**
(**A**) the expression gene circuits for expressing SpyTag-GFP.
(**B**) DNA gel electrophoresis of the colony PCR products of BBa\_K5136034\_pET-28a(+) in *E.
coli* BL21(DE3).
(**C**) SDS-PAGE analysis of His tag-SpyTag-GFP.

The promoter ([BBa\_I0500](http://parts.igem.org/Part%3ABBa_I0500)),
RBS ([BBa\_B0034](http://parts.igem.org/Part%3ABBa_B0034)), INPNC-His
tag-SpyCatcher coding sequence ([BBa\_K5136035](http://parts.igem.org/Part%3ABBa_K5136035)), and
terminator ([BBa\_B0015](http://parts.igem.org/Part%3ABBa_B0015))
were used to construct a composite part [BBa\_K5136224](http://parts.igem.org/Part%3ABBa_K5136224), which was then assembled into
the expression vector pSB1C3 by standard assembly. The constructed plasmid was transformed into *E.
coli* BL21(DE3), then the positive transformants were selected by chloramphenicol and confirmed by
colony PCR
(Figure 25B) and sequencing. Target bands (2974 bp) can be observed at the position around 3000 bp.

![](https://static.igem.wiki/teams/5136/result/re-25.png)

**Figure 25 Constructing INPNC-His tag-SpyCatcher fused
protein.**
(**A**) the expression gene circuits for INPNC displaying SpyCatcher on the surface of *E. coli*
BL21(DE3).
(**B**) DNA gel electrophoresis of the colony PCR products of Bba\_K5136224\_pSB1C3 in *E. coli*
BL21(DE3).

After being induced with 0.2% (w/v) *L*-arabinose for 12 h,
the *E. coli* BL21(DE3) culture solution (express INPNC-His
tag-SpyCatcher) was incubated with His tag-SpyTag-GFP at 37 °C in
the shaker. After cultivation for 12 h, the culture was
centrifuged to get the precipitate. Then, the precipitate was
washed and resuspended in the PBS buffer (see [Experiment](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27experiments%27%29%20%7D%7D) for more details). As shown in Figure 26,
the fluorescence intensity of the resuspension sample in the experimental group (INPNC-His
tag-SpyCatcher\_pSB1C3) was significantly higher than that of the control group (I0500\_pSB1C3). In addition,
we can also observe significant fluorescence from precipitation in the experiment group, while the control
group was contrary (Figure 27). These results indicated the combination between His Tag-SpyTag-GFP and the
INPNC-HisTag-SpyCatcher displayed on the surface of *E. coli* BL21(DE3), demonstrating that INPNC
exhibits
excellent capacity to anchor target proteins on the cell membrane.

![](https://static.igem.wiki/teams/5136/result/re-26.png)

**Figure 26 Fluorescence intensity of samples after culturing
12 h.**

![](https://static.igem.wiki/teams/5136/result/re-27.png)

**Figure 27 Samples placed under blue light after culturing
12 h.**

### **7.2 Surface-Displayed Metallothioneins Adsorb Cr2O72-**

In order to adsorb heavy metal ions in the wastewater after deinking, we chose metallothioneins (MTs) MT2A
and MT3 to treat the wastewater. In addition, to increase the contact area between MTs and heavy metal ions,
we utilized the anchor protein INPNC to display MTs on the surface of bacteria. The composite part
([BBa\_K5136226](https://parts.igem.org/Part%3ABBa_K5136226)), which expresses the fused protein INPNC-linker-MT3-linker-MT2A with the induction of
*L*-arabinose was introduced into the backbone plasmid (pSB1C3) through standard assembly. The
constructed
plasmid was transformed into *E. coli* BL21(DE3), then the positive transformants were selected by
chloramphenicol and confirmed by colony PCR (Figure 28) and sequencing. Target bands (4464 bp) can be
observed at the position around 5000 bp (Figure 28).

![](https://static.igem.wiki/teams/5136/result/re-28.png)

**Figure 28 Colony PCR of BBa\_K5136230\_pSB1C3 in *E.
coli* BL21(DE3).**

After being cultivated and induced by 0.2% *L*-arabinose at 37 °C
for 4 h, the engineered bacteria were cultured in the M9 medium
containing K2Cr2O7 (5 mg/L) for 24 h. A bit of the supernatant
of the bacterial cultures was collected at 0 h and 24 h, and was
pre-treated with 1,5-diphenylcarbazide (DPC). Then, the residual
amount of Cr2O72- in the supernatant was determined by the RGB value
measured,
which can be calculated based on a standard curve of Cr2O72- in Figure 29
(please see [Experiment](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27experiments%27%29%20%7D%7D) for details). As shown in Figure 29, the
Cr2O72- in
the culture medium at the 24 h was significantly lower than the initial value.
It indicated that the engineered bacteria could successfully reduce the heavy
metal ion Cr2O72- in the wastewater.

![](https://static.igem.wiki/teams/5136/result/re-20-7.png)

**Figure 29 Standard curve of
Cr2O72- (R2=0.987)**.

![](https://static.igem.wiki/teams/5136/result/re-30.png)

**Figure 30 Concentration of
Cr2O72- in the culture medium after 0 h and 24 h.**

## 8 Verification of the Functionality and Effectiveness of the Kill Switch

Facing the threat that the unwanted survival and accumulation of engineered bacteria might happen once they
escape to opening environment (12), we designed a light-triggered kill switch for biocontainment of the
engineered bacteria. Rather than responding to some chemical inducers, the light-triggered kill switch will
be turned to ON state after the engineered bacteria is exposed to the light illumination of specific
wavelength. We chose a blue light-inducible optogenetic system, LexRO/pColE408 (13), to control the
expression of CcdB toxin, in which an additional expression module of CcdA antitoxin was incorporated as
well to neutralize the leaky toxin when the kill switch is in OFF state. (See more details in our [Design](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27design%27%29%20%7D%7D)
page)

Here, we firstly characterized the cytotoxicity of CcdB toxin and the blue light-inducible performance of
LexRO/pColE408 system respectively, and then tested the killing effect of the blue light-induced kill
switch. Further optimization for improving the killing effect of the switch was also tried primarily.

### 8.1 Cytotoxicity of CcdB

![](https://static.igem.wiki/teams/5136/result/31.png)

**Figure 31 Cytotoxicity verification of CcdB toxin**. (**A**) The gene circuit to characterize the
cytotoxicity
of CcdB (BBa\_K5136236) on pSB4A5 vector. (**B**) Agarose gel electrophoresis of the colony PCR products
of
BBa\_K5136236\_pSB4A5 and BBa\_I0500\_pSB4A5 in *E. coli* BL21(DE3) Δ*araBAD*. (**C**) Cell
viability was measured by
CFU count and is displayed as a ratio of cells with *L*-arabinose to cells with *D*-glucose.
*p*-value: 0.0007
(\*\*\*).

Various toxin-antitoxin (TA) systems have been widely utilized and engineered to construct kill switch for
biocontainment. CcdB toxin of the CcdB-CcdA TA system, interferes with the activity of DNA gyrase and thus
causes cell death (14), which will play the critical role of killing engineered bacteria. To verify the
cytotoxicity of CcdB toxin used in the kill switch, we firstly constructed a gene circuit that the toxin
encoding gene *ccdB* was placed downstream the *L*-arabinose inducible promoter (*araC*/pBAD, [BBa\_I0500](https://parts.igem.org/Part%3ABBa_I0500)) on the
pSB4A5 vector. For convenience, the expression module of CcdA controlled by a weak constitutive engineering
promoter p2)-114v was integrated into the circuit as well (Figure 31A), generating the composite part
[BBa\_K5136236](https://parts.igem.org/Part%3ABBa_K5136236). While BBa\_I0500 only on the pSB4A5 was set as control.

Cytotoxicity tests were implemented in a BL21(DE3) strain in which the *araBAD* genes were knocked out
(Δ*araBAD*) in our lab before for minimizing the influence of *L*-arabinose metabolism. After transformation,
positive transformants were selected and confirmed by colony PCR (Figure 31B) and sequencing. Spot assay (15)
was performed for characterizing the killing effect (See more details in our [Experiments](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27experiments%27%29%20%7D%7D) page), while cell
viability was measured by colony forming unit (CFU) count and is displayed as **a ratio of cells with
*L*-arabinose to cells with *D*-glucose (survival ratio)**, in which the *D*-glucose could suppress the *L*-arabinose
inducible promoter. Upon adding the inducer *L*-arabinose, the CcdB toxin (*ccdBA*) produced ~6 logs of killing
for 6 hours' culture (Figure 31C), which indicated the cytotoxicity of CcdB.

### 8.2 LexRO activates promoter pColE408 upon blue light illumination

![](https://static.igem.wiki/teams/5136/result/32.png)

**Figure 32 Characterization of blue light-induced LexRO/pColE408**. (**A**) The gene circuit to
characterize blue
light-responding performance of LexRO/pColE408 system (BBa\_K5136237) on pSB4A5 vector. (**B**) Agarose
gel
electrophoresis of the colony PCR products of BBa\_K5136237\_pSB4A5 in *E. coli* BL21(DE3). (**C**)
The relative
fluorescence units (RFU) of bacterial culture subtracted the background fluorescence of growth media,
resulting in the RFUmCherry. *p*-value: 0.0029 (\*\*).

We turned to characterize the blue light-responding performance of LexRO/pColE408 optogenetic system used in
the kill switch. The photosensor LexRO was controlled by a medium constitutive promoter J23106 and a medium
RBS SD7 (13) ([BBa\_K5136045](http://parts.igem.org/Part%3ABBa_K5136045) ), while the mCherry fluorescent protein ([BBa\_J06504](http://parts.igem.org/Part%3ABBa_J06504) ) was chosen as the reporter
under the control of promoter pColE408 ([BBa\_K5136044](http://parts.igem.org/Part%3ABBa_K5136044) ) (Figure 32A), thus generating the composite part
[BBa\_K5136237](http://parts.igem.org/Part%3ABBa_K5136237) on the pSB4A5 vector. BL21(DE3) was used to characterize this optogenetic system, and positive
transformants were selected and confirmed by colony PCR (Figure 32B) and sequencing.

Characterization was carried out in a self-made blue light (460 nm) illumination device. After cultured for
about 17 hours upon blue light illumination (with a relative light intensity of 250) or kept in dark
condition, red fluorescence intensity (*λ*ex = 585 nm, *λ*em = 615 nm) and OD600 were measured. The normalized
fluorescence intensity of “Light” group showed a significant higher value than that of “Dark” group (about 2
times), indicating that this **optogenetic system could be induced by blue light** (Figure 32C) indeed.

### 8.3 Blue Light-induced Kill Switch Characterization

![](https://static.igem.wiki/teams/5136/result/33.png)

**Figure 33 Characterization of blue light-induced kill switch.** (**A**) The gene circuit of blue light-induced
kill switch (BBa\_K5136231) on pSB4A5 vector. (**B**) Agarose gel electrophoresis of the colony PCR products of
BBa\_K5136231\_pSB4A5 (Kill Switch) and BBa\_K5136234\_pSB4A5 (Control) in *E. coli* BL21(DE3). (**C**) Cell
viability was measured by CFU count and is displayed as a ratio of cells exposed to blue light to cells
kept in dark condition. *p*-value: 0.0085 (\*\*) for D/L, 0.0077 (\*\*) for L.

After verifying the cytotoxicity of CcdB and blue light-inducible performance of LexRO/pColE408 system, we
built the blue light-induced kill switch ([BBa\_K5136231](https://parts.igem.org/wiki/index.php?title=Part:BBa_K5136231)), in which the toxin-antitoxin module is controlled
by promoter pColE408 and LexRO is constitutively expressed as in [BBa\_K5136237](https://parts.igem.org/wiki/index.php?title=Part:BBa_K5136237) (Figure 33A). While the LexRO
expression module only ([BBa\_K5136234](https://parts.igem.org/wiki/index.php?title=Part:BBa_K5136234)) on the pSB4A5 was set as the control. Positive transformants were
selected and confirmed by colony PCR (Figure 33B) and sequencing after transformed to BL21(DE3).

Spot assay was also performed after cultured upon blue light illumination or kept in dark condition. A **blue
light illumination-dependent killing effect** was observed, which indicates that this blue light-induced kill
switch functioned to kill engineered bacteria when exposed to blue light (Figure 33C). Besides, when exposed
to blue light for whole period (6 hours, “L”), the kill switch exhibited a slightly stronger killing effect
than exposed to blue light for a shorter time (kept in dark for 2 hours 25 min first then switched on the
blue light for 3 hours 35 min, “D/L”), which implied that the killing of engineered bacteria might be
illuminating time-dependent.

![](https://static.igem.wiki/teams/5136/result/34.png)

**Figure 34 Optimization of blue light-induced kill switch.** (**A**) Optimized blue light-induced kill switch
(BBa\_K5136235) on pSB4A5 vector. The RBS of LexRO was changed to SD17. (**B**) Agarose gel electrophoresis of
the colony PCR products of BBa\_K5136235\_pSB4A5 in *E. coli* BL21(DE3). (**C**) Cell viability was measured by
CFU count and is displayed as a ratio of cells exposed to blue light to cells kept in dark condition.

Although we have verified the blue light-dependent killing effect of the kill switch, we still tried to
optimize the gene circuit for further improving the killing effect. Since lower LexRO content were more
sensitive to light illumination (13), we then changed the RBS of LexRO in the gene circuit to a weaker one
(SD17, [BBa\_K5136049](http://parts.igem.org/Part%3ABBa_K5136049)) to see whether this would improve the effect of killing or not, resulting in the
generation of [BBa\_K5136235](http://parts.igem.org/Part%3ABBa_K5136235) composite part on pSB4A5 vector (Figure 34A). Colony PCR (Figure 34B) and
sequencing were performed again to confirm the positive transformants of BL21(DE3). Similar test was done to
the alternative kill switch. When lower the expression of LexRO, a slight decrease on survival ratio was
obtained for the kill switch (Figure 34C), indicating that the strategy for optimizing the kill switch might
be available and feasible.

### 8.4 Conclusions

Developing a biocontainment system to response physical factors (16) (such as light or temperature) takes the
advantage over chemical factors (such as various inducers) of no need to add any substances into the culture
or reaction systems, which is adapted to our deinking system. It is reported that the LexRO/pColE408
optogenetic system showed a better performance over the other existing single-component bacterial
light-activated expression systems (13). Even though the design of LexRO/pColE408-regulated kill switch has
been proposed before ([Tsinghua iGEM 2020](https://2020.igem.org/Team%3ATsinghua)), however, it is the first time for this optogenetic system to be
utilized and tested in constructing kill switch in actual. We achieved ~2 logs of killing within 6 hours
when exposing the engineered bacteria to blue light illumination with a light intensity that would not
impair the cell growth.

Besides, we tried to optimize the kill switch by altering the RBS of LexRO to lower its intracellular
content and obtained a decreased survival ratio. Compared to present kill switches controlled by some other
blue light-responding systems such as pDawn (17,18) or EL222/pBLind (19), further engineering of our
LexRO/pColE408-regulated kill switch should be implemented to achieve a better killing effect. As the
induction characteristics of this system is highly related to the expression level of LexRO (13), a feasible
way to optimize has been proposed according to our experimental results.

In summary, our modular verifications and engineering iterations of constructing the blue light-induced kill
switch contributed to the biosafety of our REPARO project (See more information in our [Proposed
Implementation](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27implementation%27%29%20%7D%7D) page), and we hope this design of biocontainment system will provide valuable experience to
other iGEM teams and SynBio community.

## 9 Further Explorations

In life and work, wrong words and misprints are often present in precious and important paper documents,
such as contracts, archives, and examination papers. Reconstructing these documents will take a lot of time,
cost and manpower. So, we want to expand the application of deinking for these papers with wrong words,
which may save paper, time and money.

### 9.1 Erasure Performance of Diluted Ink

We test the effect of some surfactants on removing ink. On each 1 cm×1 cm sheet of paper, 15 μL of diluted
MG6128 gel pen ink was painted uniformly. After drying in the air for 3 minutes, the papers were placed in 2-mL centrifuge tubes containing 1 mL reaction solution. The tubes were then placed in a metal bath (at 25 °C,
1000 rpm) and shaken for 10 minutes. The absorbance (at OD400 nm) of the post-reaction solution was measured
and calculated on standard curves to obtain the ink content, and then the ink removal rate was calculated.
The experimental results indicated that
**both SDS and rhamnolipid exhibit significant performance** in removing ink compared to the control
group (Figure 35).

![](https://static.igem.wiki/teams/5136/result/formula2.png)

![](https://static.igem.wiki/teams/5136/result/35.png)

**Figure 35 Ink removal rates of 2.5% (w/w) SDS, 2.5% (w/w)
rhamnolipid
and water.**

### 9.2 Erasure Performance of Handwriting

We conduct a series of erasure experiments on the ink marks of ballpoint pens. A machine wrote the same Chinese
characters on the paper, then cut them into strips and placed them in a 12-well plate with the reactant
solution. The total measurement lasted for 1 h, and we observed the erasure performance every 15 minutes.
The fade of the Chinese characters was obvious when 2.5% (w/w) SDS and 0.4 (v/v)% limonene were used together,
and erasure performance increased with the reaction time.

![](https://static.igem.wiki/teams/5136/result/36.png)

**Figure 36 Comparison of elimination effect between blank
and experimental group.**

### 9.3 Adsorption Efficiency of Crosslinked Chitosan

We also tried to remove the ink by adsorbing crosslinked chitosan. 0.1 g of crosslinked chitosan was added
to 1 mL of diluted ink solution. The adsorption efficiency was characterized by the decrease in the
solution's absorbance. The experimental results indicated that crosslinked chitosan exhibits a significant
adsorption function on both ballpoint and gel pen ink.

![](https://static.igem.wiki/teams/5136/result/formula3.png)

![](https://static.igem.wiki/teams/5136/result/37.png)

**Figure 37 The adsorption rates of chitosan to ballpoint pen
ink were calculated as time progressed.**

## 10 Conclusion

In our REPARO project, we designed and developed an unprecedented deinking method based on synthetic
biology, which could serve as a viable alternative to the chemical method on the industry scale. The
implementation of our REPARO project holds immense potential to propel technological advancement and
industrial upgrading within the waste paper recycling industry. Our project aims to introduce advanced
biotechnology and environmental protection concepts to enhance the entire industry's production efficiency,
product quality, and environmental protection level, thereby strengthening the industry's competitiveness
and promoting the development of the circular economy.

## 11 Future Prospects

In the foreseeable future, we aim to comprehensively characterize the deinking efficacy achieved through the
synergistic use of diverse deinking enzymes and explore the optimal conditions that maximize the performance
of each enzyme. Concurrently, we aim to validate the deinking capabilities further when our autolytic system
is integrated with deinking enzymes, and verify the deinking capabilities of the exogenous secretion system,
activated by the Anderson promoter, in conjunction with these enzymes. Importantly, we are committed to
continuously optimizing the design and verification processes of our erasing product, demonstrating our
dedication to the creation of a convenient, efficient, and practical erasing product for everyday use.

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