iGEM 2024 — Team pekinghsc

{% 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 %}

![notebook](https://static.igem.wiki/teams/5231/banners/notebook.png)

Wet Lab

Dry Lab

Human Practice

April

Sun.

Mon.

Tue.

Wed.

Thu

Fri.

Sat.

May

Sun.

Mon.

Tue.

Wed.

Thu

Fri.

Sat.

June

Sun.

Mon.

Tue.

Wed.

Thu

Fri.

Sat.

July

Sun.

Mon.

Tue.

Wed.

Thu

Fri.

Sat.

August

Sun.

Mon.

Tue.

Wed.

Thu

Fri.

Sat.

September

Sun.

Mon.

Tue.

Wed.

Thu

Fri.

Sat.

1st April

Human Practice

Participants

*Yunqi Xing**Tao Ma*

Items

Communicate with Professor Tian Songhai.

Participants

*Jiawei Zhao**Tao Ma*

Items

Communicate with Professor Joshi.

2nd April

Human Practice

Participants

*Yunqi Xing*

Items

Analyze stakeholders.

![](https://static.igem.wiki/teams/5231/notebook/4-2-hp.png)

3rd April

Wet Lab

Participants

*Jiawei Zhao*

Items

Design and colony PCR of homologous fragment of Csg.

4th April

Dry Lab

Participants

*Hao Zou**Han Hou*

Items

We consulted our senior for suggestions on various aspects of our project, including time management,
necessary tasks,
required learning, and strategies for building our wiki. Based on his detailed and practical advice from
previous iGEM
projects, we have broadly outlined our main tasks: conducting MD simulations and ODE computations.

![](https://static.igem.wiki/teams/5231/notebook/4-4-drylab.jpeg)

5th April

Dry Lab

Participants

*Huaizhi Wang**Shengwen Ding*

Items

After communicating with the PI of dry lab, Professor Xie Zhengwei, the general direction of the dry
experiment was
determined. It was decided to use quantum chemistry combined with artificial intelligence protein
structure prediction
to optimize the structure, solve the low availability of AlphaFold2 on NCAA-containing proteins, and
predict the NCAA
insertion efficiency through system molecular dynamics simulation. The professor also provided suggestions
on the
selection of protein insertion sites.

![](https://static.igem.wiki/teams/5231/notebook/4-5-drylab1.jpeg)
![](https://static.igem.wiki/teams/5231/notebook/4-5-drylab2.jpeg)

6th April

Human Practice

Participants

*Jiawei Zhao*

Items

Attend Professor Wei's lecture.

7th April

Wet Lab

Participants

*Jiawei Zhao*

Items

Selection of gRNA, design and construction of pgRNA-Csg.

18th April

Wet Lab

Participants

*Jiawei Zhao*

Items

Re-design and construction of pgRNA-Csg, since the previous homologous fragment went wrong.

19th April

Wet Lab

Participants

*Canpu Chen*

Items

Construct CsgA-his-BL21.

Participants

*Jiaxin He**Zheping Zhang*

Items

Construct CsgA-trucR5.

20th April

Wet Lab

Participants

*Jiawei Zhao*

Items

Confirmation of ECNΔCsg by colony PCR.

![](https://static.igem.wiki/teams/5231/notebook/4-20-wetlab-new.png)

21st April

Wet Lab

Participants

*Canpu Chen*

Items

The first time to express CsgA-histag in BL21, but failed, because the bacteria died when cultured.

22nd April

Wet Lab

Participants

*Canpu Chen*

Items

Construct CsgBtruncR5-his-BL21.

23rd April

Wet Lab

Participants

*Tao Ma*

Items

Construct ECN1917genome-konck down
\_mt.

24th April

Wet Lab

Participants

*Tao Ma*

Items

Construct pBbB8k-mutCsgA\_F97TAG\_mt.

25th April

Wet Lab

Participants

*All team members*

Items

All iGEM members underwent a 1-hour experimental safety training.

![](https://static.igem.wiki/teams/5231/notebook/4-25-wetlab.jpeg)

Participants

*Canpu Chen*

Items

The first time to express and purify the CsgBtruncR5.

26th April

Wet Lab

Participants

*Jiawei Zhao*

Items

Discard of plasmids inside ECNΔCsg and confirmed.

![](https://static.igem.wiki/teams/5231/notebook/4-26-wetlab1.png)
![](https://static.igem.wiki/teams/5231/notebook/4-26-wetlab2.png)
![](https://static.igem.wiki/teams/5231/notebook/4-26-wetlab3.png)

27th April

Dry Lab

Participants

*Hao Zou*

Items

Attending molecular training course for dynamics simulation software GROMACS.

![](https://static.igem.wiki/teams/5231/notebook/4-27-drylab.jpeg)

Human Practice

Participants

*Murong Lei*

Items

Attend the Capital Biomedical Innovation and Entrepreneurship Elite Exchange Meeting.

28th April

Wet Lab

Participants

*Jia Yuan**ZhePing Zhang**Jiaxin He*

Items

Communicate with Tao Liu and report on the progress of the experiment.

Human Practice

Participants

*Jia Yuan**Murong Lei*

Items

Engage in an exchange with Southern Medical University.

29th April

Wet Lab

Participants

*Canpu Chen*

Items

Arrival of the purchased plasmid pBbB9k-CsgA-nbGFP.

Participants

*Jiawei Zhao*

Items

The purification of CsgBtrucR5-His protein has been preliminarily completed by denaturing purification.
However the
purity and yield were relatively poor.

1st May

Wet Lab

Participants

*Canpu Chen*

Items

Purification of CsgBtruncR5-histag by non-denaturing purification from the supernant was preliminarily
completed.
However, the purity and yield were relatively poor.

Participants

*Jiawei Zhao**Jia Yuan**Canpu Chen**Tao Ma**Jiaxin He**Zheping Zhang*

Items

Construction of multiple plasmids.

3rd May

Wet Lab

Participants

*Jiaxin He**ZhePing Zhang*

Items

Construct PBbB8K-Csg opreon-F-delete.

5th May

Wet Lab

Participants

*Jiawei Zhao**Jia Yuan*

Items

Preparation of several competent cells including: ECNΔCsg, ECNΔCsg+pUltra-Tet v2.0 (ECNΔCsg+OTS),
ECNΔCsg+pBbB8k-CsgAWT.

6th May

Human Practice

Participants

*All*

Items

"Five-Team Exchange Meeting".

7th May

Human Practice

Participants

*Jiawei Zhao*

Items

Communicate with Professor Lv.

8th May

Wet Lab

Participants

*Zheping Zhang**Zifeng Qiao*

Items

Construct CsgBtrucR5-Flag tag.

9th May

Wet Lab

Participants

*Jiawei Zhao**Jia Yuan*

Items

Preparation of several competent cells including: ECNΔCsg, ECNΔCsg+pUltra-Tet v2.0 (ECNΔCsg+OTS),
ECNΔCsg+pBbB8k-CsgAWT.

Participants

*Jiaxin He**Zifeng Qiao**Zheping Zhang*

Items

Construct CsgBtrucR4R5&CsgA(-).

12th May

Wet Lab

Participants

*Jiawei Zhao**Tao Ma*

Items

Construction of plasmids: S89TAG, F97TAG.

Dry Lab

Participants

*Hao Zou*

Items

Solve the key issue for nonnatral amino acid TeT molecular dynamics simulation with GROMACS. Successfully
conducted force field topology file.

![](https://static.igem.wiki/teams/5231/notebook/5-12-drylab.png)

13th May

Wet Lab

Participants

*Zheping Zhang*

Items

Construct pBbB8k-δCsgA.

14th May

Wet Lab

Participants

*Jiaxin He**ZhePing Zhang*

Items

Literature survey on proteins that CsgA can bind.

15th May

Human Practice

Participants

*Murong Lei*

Items

Questionnaire survey.

16th May

Wet Lab

Participants

*Jiaxin He*

Items

Construct Csg-operon-δFδB(with nb His).

17th May

Wet Lab

Participants

*Jiaxin He*

Items

Literature research on protein purification conditions.

18th May

Wet Lab

Participants

*Zheping Zhang**Canpu Chen*

Items

Purification of CsgBtruncR5-histag.

19th May

Wet Lab

Participants

*Jiaxin He**Zifeng Qiao**Zheping Zhang*

Items

Construct CsgAtrucR5(PBbB8K)&Csg-MMP-nb.

23rd May

Wet Lab

Participants

*Tao Ma*

Items

Fill out the safety form.

24th May

Wet Lab

Participants

*Jiawei Zhao**Yufan Wang*

Items

Insertion of Tetv2.0 into sfGFP-151TAG, whose fluorescence was supposed to be quenced by Tet v2.0. MS of
sfGFP is sent to verify the insertion of Tet v2.0.

Participants

*Tao Ma*

Items

Check the safety form and ask for advice.

25th May

Wet Lab

Participants

*Jiaxin He*

Items

Construct pET22b-deltaFdeltaB-CsgAtrucR5-His&pET22b-deltaFdeltaB-CsgA-MMP-nb-His.

Dry Lab

Participants

*Hao Zou*

Items

Complete the molecular dynamics simulation of the csga protein with TeT. Analysis Done.

![](https://static.igem.wiki/teams/5231/notebook/5-25-drylab.png)

26th May

Dry Lab

Participants

*Shengwen Ding*

Items

Complete the first part of ODE.

28th May

Wet Lab

Participants

*Tao Ma*

Items

Construct pBbB8k-mutCsgA\_S89\_mt.

29th May

Wet Lab

Participants

*Jiaxin He**Zifeng Qiao**Zheping Zhang*

Items

Construct pET22b-deltaFdeltaB-CsgAtrucR5-His&pET22b-deltaFdeltaB-CsgA-MMP-nb-His.

1st June

Wet lab

Participants

*Zheping Zhang*

Items

Purification of CsgAtruncR5-histag.

Human Practice

Participants

*Yunqi Xing*

Items

Consult the safety literature.

![](https://static.igem.wiki/teams/5231/notebook/6-1-hp.jpeg)

5th June

Dry Lab

Participants

*Huaizhi Wang*

Items

Attending the webinar on "Modeling and Analysis of Synbio Systems" presented by Mathworks. Learned about
Simbiology, a biological system modeling tool, and determined to use it as the main method for ODE modeling
and subsequent optimization.

![](https://static.igem.wiki/teams/5231/notebook/6-5-drylab.png)

9th June

Wet Lab

Participants

*Jiawei Zhao**Yufan Wang*

Items

Congo Red and ThT assay of curli fibre including CsgAWT, S89TAG, F97TAG, finding that insertion of amber
codon at site 89 and 97 didn't end the formation of curli fibre, it can be formed with only R1 and R2.
Furthermore confirmation of curlie fibre formation needs to be completed.

Participants

*Zifeng Qiao**Zheping Zhang**Jiaxin He*

Items

Purification of pET22b-CsgAtruncR5-his.

Dry Lab

Participants

*Shengwen Ding*

Items

Complete the second part of ODE, make a presentation of past work.

10th June

Dry Lab

Participants

*Hao Zou*

Items

Second phase simulation of nucleation. Attempt to construct a mem-protein system but failed.

![](https://static.igem.wiki/teams/5231/notebook/6-10-drylab.png)

11th June

Human Practice

Participants

*Yunqi Xing*

Items

Contact the pharmaceutical company.

14th June

Wet Lab

Participants

*Jiawei Zhao**Canpu Chen*

Items

Purification of CsgA-S89TAG, Csg-F97TAG and CsgAWT in ECNΔCsg. SDS-PAGE was done to verify the protein.
However, the
purity and rate of expression of proteins with Tet v2.0 needs to be further improved. A preliminary try of
verification
of the insertion of Tetv2.0 of the protein is done using TCO-cy5 and tested by Typhoon.

29th June

Human Practice

Participants

*Jiawei Zhao**Jia Yuan*

Items

Exchange with Beijing Normal University.

1st July

Human Practice

Participants

*Jiawei Zhao**Zhichao Deng**Canpu Chen*

Items

Communicate with Professor Liu Zhuang.

3rd July

Human Practice

Participants

*Murong Lei**Yunqi Xing**Shengwen Ding**Zhichao Deng*

Items

Communicate with the public mutual fund researcher.

5th July

Wet Lab

Participants

*Jiawei Zhao*

Items

Verification of the formation of Curli fibre is done by SEM. It was confirmed that, without Tet v2.0, under
the induction of arabinose, curli fibre was still formed.

![](https://static.igem.wiki/teams/5231/notebook/7-5-wetlab.png)

Human Practice

Participants

*Murong Lei*

Items

Collaborate with Jilin University.

8th July

Wet Lab

Participants

*Jiawei Zhao**Yufan Wang*

Items

Cy5-TCO to mark Tet v2.0. S89TAG and F97TAG all displayed significant rise in signal,
while S89TAG did a much better job. It was considered that S89TAG was chosen as the better candidate.

![](https://static.igem.wiki/teams/5231/notebook/7-8-wetlab.jpg)

11th July

Wet Lab

Participants

*Zifeng Qiao**Zheping Zhang**Jiaxin He*

Items

Purification of CsgA-His& CsgBtrucR5.

12th July

Human Practice

Participants

*Tao Ma*

Items

Teaching experiments to international high school igem team students for four days.

13th July

Human Practice

Participants

*Canpu Chen*
*Hao Zou*
*Zhichao Deng*
*Murong Lei*
*Jiawei Zhao*
*Jia Yuan*
*Han Hou*
*Kelibinuer·Kuerban*
*Yong Jie Tan*
*Huaizhi Wang*
*Shengwen Ding*
*Yufan Wang*

Items

Presentation in CCiC.

![](https://static.igem.wiki/teams/5231/notebook/7-13-hp.jpeg)

Dry Lab

Participants

*Hao Zou**Shengwen Ding*
*Huaizhi Wang**Murong Lei*

Items

Communicate with iGEM team leader of Jilin University.

14th July

Wet Lab

Participants

*Zifeng Qiao**Jiaxin He*

Items

Purification of CsgB& CsgBtrucR5.

15th July

Human Practice

Participants

*Yishenghui Li**Zhenghao Liu**Yunqi Xing**Zheping Zhang*

Items

Visit the ACROBiosystems company and communicate with the company manager.

![](https://static.igem.wiki/teams/5231/notebook/7-15-hp.jpg)

16th July

Human Practice

Participants

*Canpu Chen**Murong Lei**Kelibinuer·Kuerban*

Items

Contact Kai Zheng from innoModels.

![](https://static.igem.wiki/teams/5231/notebook/7-16-hp.jpeg)

Participants

*Huaizhi Wang**Hao Zou**Shengwen Ding*

Items

Communicate with Chen Zihao from Southern University of Science and Technology.

Dry Lab

Participants

*Hao Zou**Huaizhi Wang*

Items

Exchanged the details of MD simulation with instructor Zhengwei Xie and senior Lisheng Zhang, and solved
the problem of lack of computational resources.Exchange views with senior students, and obtain guidance
on more comprehensive molecular dynamics simulation methods of protein polymerization;
Got high performance server help

![](https://static.igem.wiki/teams/5231/notebook/7-16-drylab.jpg)

18th July

Wet Lab

Participants

*Jiawei Zhao*

Items

Growth Curve of E.coli in existence of Tet. Previous experts reflected their worries about the toxity
of Tet v2.0, so we constructed experiments of E.coli growing in 2YT with 1mM Tet v2.0 inside. The problem
is that, the experiment is conducted under 25℃ rather than 37℃, but the growth curve can surely prove
the safety of Tet v2.0.

![](https://static.igem.wiki/teams/5231/notebook/7-18-wetlab1.jpg)
![](https://static.igem.wiki/teams/5231/notebook/7-18-wetlab2.jpg)
![](https://static.igem.wiki/teams/5231/notebook/7-18-wetlab3.jpg)
![](https://static.igem.wiki/teams/5231/notebook/7-18-wetlab4.jpg)

19th July

Dry Lab

Participants

*Huaizhi Wang*

Items

It was decided to build Deepro, a neural network based promoter strength prediction system. searched for
data,
constructed the dataset and completed the skeleton part of Deepro (data loading module, training module,
testing module,
plotting module, etc.).

![](https://static.igem.wiki/teams/5231/notebook/7-19-drylab1.png)

Human Practice

Participants

*Jiawei Zhao**Yunqi Xing*

Items

Communicate with Director Qi.

20th July

Dry Lab

Participants

*Huaizhi Wang*

Items

Completed construction of the MLP portion of the Deepro model for initial parameter optimization, which
performed
poorly. Next step is to try using 1dCNN model.

21th July

Dry Lab

Participants

*Huaizhi Wang*

Items

Completion of the Deepro 1dCNN model part of the construction, compared to the MLP parameter amount is
greatly
increased, in the case of training the same number of rounds in the training set of better performance
compared to the
MLP model, but in the test set of the performance of the performance of the improvement is not obvious, in
general,
unsatisfactory. The next step is to try the LSTM model and XGBoost model, and at the same time hope to carry
out the
extended dataset.

Human Practice

Participants

*Huaizhi Wang**Hao Zou**Shengwen Ding*

Items

Communicate with Zhang Lisheng.

22nd July

Wet Lab

Participants

*Jiawei Zhao*

Items

Synthesis of TCO-coumarine. It was successfully done at the third time. First failure was because
incomplete protection of water, Beijing was really rainy at that moment. The molecule was successfully
synthesized at the second time, and synthesized more at the third time.

![](https://static.igem.wiki/teams/5231/notebook/7-22-wetlab.jpg)

23rd July

Wet Lab

Participants

*Jiawei Zhao**Yufan Wang*

Items

Flow cytometry of EcN. The bacteria was labled if Tet v2.0 was inserted into the curli fibre.
The experiment was accomplished in one single attempt. The problem of the experiment, is that the
bacteria unlabled cannot be distinguished from impurity. However, the amout of labled bateria was
so outstanding, that we didn't think that it would influence the results much.

![](https://static.igem.wiki/teams/5231/notebook/7-23-wetlab1.jpg)
![](https://static.igem.wiki/teams/5231/notebook/7-23-wetlab2.jpg)

25th July

Wet Lab

Participants

*Yunqi Xing**Murong Lei*

Items

Create a stakeholder map for the commercial version.

26th July

Wet Lab

Participants

*Jiaxin He*

Items

Prepare a batch of competent cells for electroporation.

Dry Lab

Participants

*Huaizhi Wang*

Items

Try to use embedded vector instead of one hot vector as CNN model input. The new model performs weaker than
the original model under the same training conditions, which is to be expected since word embedding matrices
tend to
struggle to exhibit DNA base features initially, and thus the model requires longer training rounds.
Increasing the number of
training rounds to 300 shows a slight but insignificant improvement in the model's performance on the test
set, with
hyperparameters to be optimized. Meanwhile I think the main problem at the moment is centered on low quality
and small
datasets.

27th July

Wet Lab

Participants

*Jiaxin He**Han Hou*

Items

Purification of CsgA-His and BCA quantification.

Dry Lab

Participants

*Huaizhi Wang*

Items

The construction of the LSTM model was completed, and the training was extremely time consuming and the model
generalization ability was quite limited, and it is not intended to be used as a skeleton part of the hybrid
model in
the future.

28th July

Human Practice

Participants

*Murong Lei*

Items

Visit Jingfeng Pharmaceutical.

29th July

Wet Lab

Participants

*Jiaxin He**Han Hou*

Items

Purification of CsgA-His and BCA quantification.

Dry Lab

Participants

*Huaizhi Wang*

Items

By searching the literature, two datasets with sizes of 936 sequences and 3512 sequences were added
to the model. However, it still faces the same problem as before, i.e., data from different sources
cannot be merged with each other. The data characteristics of the two datasets are quite different,
with the smaller dataset having lower DNA sequence homology and the larger dataset having higher sequence
homology, a distribution that can have a serious impact on the generalization ability of the model.

![](https://static.igem.wiki/teams/5231/notebook/7-29-drylab1.png)
![](https://static.igem.wiki/teams/5231/notebook/7-29-drylab2.png)

31th July

Dry Lab

Participants

*Huaizhi Wang*

Items

Based on our teacher's advice, we decided to try it out using some relatively simple models.The XGBoost
model was added and tuned, which was the fastest to train and performed the best of all the current models,
which may be related to the limited size of our data. This model and the subsequent models were chosen
to be trained on the third dataset, which is the largest dataset, and of course our program supports
training the model from scratch on the first and second datasets.

1st August

Dry Lab

Participants

*Huaizhi Wang*

Items

Using the API provided by Scikit-learn, AdaBoost, SVR, RandomForest, and GBDT models are added
to Deepro, where the AdaBoost model performs poorly, GBDT and XGBoost perform close to each other,
and the rest of the models perform moderately well.

Human Practice

Participants

*Murong Lei*

Items

Visit Xinhua Medicine.

Participants

*Murong Lei*

Items

Visit HICOOL.

2nd August

Human Practice

Participants

*All*

Items

ECN Conference.

4th August

Human Practice

Participants

*Murong Lei*

Items

Visit Intco.

5th August

Dry Lab

Participants

*Huaizhi Wang*

Items

A joint CNN-LSTM model was completed, with improved performance compared to CNN and LSTM models,
but with a significant increase in training time.

10th August

Dry Lab

Participants

*Huaizhi Wang*

Items

Completed the Transformer model (Encoder only), which is the largest and most structurally complex of
all models to date, with a significant increase in training time compared to the other models, and a
final performance similar to XGBoost. Our concern is that the high homology of the dataset will limit
the generalization ability of the model, especially in the prediction of the strength of promoters from
other organisms or artificial promoters modified based on promoters from other organisms, and the model
lacks the consideration of the external environmental conditions, for example, the strength of the
promoter expression under different oxygen conditions may be significantly different.

Human Practice

Participants

*Shengwen Ding**Shengwen Ding*

Items

Dry Lab Conference.

11th August

Humam Practice

Participants

*Huaizhi Wang**Hao Zou*

Items

Communicate with Yan Zeyu.

13th August

Wet Lab

Participants

*Jiawei Zhao*

Items

Decage verification of TCO-coumarine in level of molecule. We wanted to observe the complete decaging
time. Suggested by Yanbo Jing, later decaging experiment conducted in culture medium rather than ddH2O
would be conducted.

![](https://static.igem.wiki/teams/5231/notebook/8-13-wetlab.jpg)

15th August

Dry Lab

Participants

*Huaizhi Wang*

Items

Refactor Deepro project code to increase its readability and optimize code structure.

Human Practice

Participants

*Murong Lei**Yunqi Xing**Zhichao Deng*

Items

Write two white papers.

16th August

Wet Lab

Participants

*Jiawei Zhao**Yufan Wang*

Items

Decage verification of TCO-coumarine in level of bacteria. A really tough experiment, which I have to
handle about 54 samples at the same time. Fortunately, the results came out well.

![](https://static.igem.wiki/teams/5231/notebook/8-16-wetlab.jpg)

19th August

Human Practice

Participants

*Huaizhi Wang*

Items

Communicate with You Siying.

22nd August

Human Practice

Participants

*Hao Zou*

Items

Communicate with Wang Ge.

23rd August

Dry Lab

Participants

*Huaizhi Wang**Hao Zou*

Items

Participating in the MindSpore SPONGE summer course, hoping to master MD simulation methods
based on machine learning and augmented sampling for use in projects.

![](https://static.igem.wiki/teams/5231/notebook/8-23-drylab.png)

27th August

Human Practice

Participants

*Huaizhi Wang*

Items

Communicate with Song Renxiu.

2nd September

Wet Lab

Participants

*Jiawei Zhao**Yufan Wang*

Items

Synthesis of TCO-dox. It was accomplished in 3 attempts. The first failure was because technical error,
which all the products were leaked out from Flash. The second time, I mistakenly collected the wrong
product, and it was successful for the third time.

![](https://static.igem.wiki/teams/5231/notebook/9-2-wetlab1.png)
![](https://static.igem.wiki/teams/5231/notebook/9-2-wetlab2.png)

5th September

Wet Lab

Participants

*Jiawei Zhao*

Items

Click-to-Release verification of TCO-Dox performed by HPLC.

![](https://static.igem.wiki/teams/5231/notebook/9-5-wetlab.jpg)

10th September

Wet Lab

Participants

*Jiawei Zhao*

Items

The condition of MC38 went worse, so we decided to focus on CT26

Human Practice

Participants

*Murong Lei**Zifeng Qiao*

Items

Project Exchange Meeting.

13th September

Dry Lab

Participants

*Huaizhi Wang*

Items

Participate in an online forum on the theme "Navigating the Future of AI in Synthetic Biology: A Multi Stakeholder
Dialogue".

![](https://static.igem.wiki/teams/5231/notebook/9-13-drylab.png)

15th September

Wet Lab

Participants

*Jiawei Zhao*

Items

Started to culture CT26 and MC38, and explored ways to conduct cytoxicity experiment.

20th September

Wet Lab

Participants

*Jiawei Zhao*

Items

Cell Cytoxicity of Dox and TCO-Dox on CT26 was done.

![](https://static.igem.wiki/teams/5231/notebook/9-20-wetlab.jpg)

28th September

Wet Lab

Participants

*Jiawei Zhao*

Items

Cell Cytoxicity of TCO-Dox decaging on molecular level on CT26 was done.

![](https://static.igem.wiki/teams/5231/notebook/9-28-wetlab.jpg)

Human Practice

Participants

*Murong Lei**Yunqi Xing**Zhichao Deng*

Items

Write an HP article to participate in building a wiki.

29th September

Wet Lab

Participants

*Jiawei Zhao*

Items

Cell Cytoxicity of TCO-Dox decaging on protein and bacteria level on CT26 was done.

![Cell toxicity of TCO-Dox on CT26](https://static.igem.wiki/teams/5231/results/27.jpg)

Human Practice

Participants

*Zhichao Deng**Mingyu Liang**Yunqi Xing**Shengwen Ding*

Items

Consult Mr. Hou for legal advice.

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{% block lead %}Demonstrate engineering success in a part of your project by going through at least one iteration of the engineering design cycle.{% endblock %}
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Introduction

Engineering 1: Bacteria Construction
Cycle 1: Acquisition of EcNΔCsg
Cycle 2:EcNΔCsgΔThyA

Engineering 2: Site Selection
Cycle 1: Initial Selection
Cycle 2: Continue Selection of Insertion Sites
Cycle 3: Final Selection of Insertion Sites

Engineering 3: Safety Cycle
Cycle 1: Promoter Verification

References

Introduction

**Design, Build, Test, and Learn.** This is the engineering cycle when we are designing new products. We first design new
modules that fit the function we want. Then we build, building the module, creating what we design. Next, we test the
module to see if it runs as expected. At last, we learn, that since the design could succeed or fail, learning from the
result would give us knowledge that we could use in the next round of the engineering cycle. In areas of engineering,
the engineering cycle is the most fundamental and efficient process we must go through for better products.

Synthetic biology is the subject of engineering in biology. We discover new modules, generating new genetic circuits,
aiming to design new “genetic machines” with new or better functions. **Our product is bacterial therapy, using *Escherichia coli* Nissle 1917 (EcN) as the
carrier, displaying tetrazine on the surface to release the prodrug at the tumor microenvironment (TME).** The design
includes **construction of the EcN strain; display of tetrazine on the surface of CsgA; control of click-to-release
reaction; and safety module**. Here is the engineering cycle we've done in our experiments:

Engineering 1: Bacteria Construction

## Cycle 1: Acquisition of EcNΔCsg

### Design

To guarantee the effectiveness and safety of our design, we need to change several properties of the EcN strain.

For effectiveness, the goal is to display more tetrazine on the bacterial surface. The two things that we can do
so far according to the previous works1are displaying more engineered CsgA on
the surface and displaying tetrazine on CsgA better. **So, We started our first cycle on displaying more engineered
protein: constructing a new fundamental EcN strain, EcNΔCsg.**

### Build

We have to knock out all the genes associated with Csg from the genome of EcN. We designed the homologous fragment,
guide RNA and successfully constructed pgRNA-Csg-1. After rounds of induction and selection, **we obtained bacterial
strains with the target gene deleted and the plasmid responsible for deletion removed through screening.**

![Figure 1](https://static.igem.wiki/teams/5231/engineering/1.png)
![Figure 2](https://static.igem.wiki/teams/5231/engineering/2.png)

### Test

**The test was conducted in two dimensions: Gene and Protein.** We wanted to make sure that, the gene deletion was
successful, and no curli fiber could be generated by EcNΔCsg.

#### 1. Gene dimension

We tested in gene dimension by colony PCR. By designing two primers before and after the homologous fragments, we
could see the length of homologous fragments in between, which should be 1312 bp if the deletion was successful.
**The Agarose Gel Electrophoresis image validated the result.**

![Figure 3](https://static.igem.wiki/teams/5231/engineering/3.png)

#### 2. Protein dimension

On dimension of Protein, we performed Scanning Electron Microscope (SEM). By directly looking at the surface of the
bacteria, we could check if there were curli fiber. Comparing the pictures from EcNΔCsg (Left) and EcNΔCsg +
pBbB8k-CsgAWT (Right), which served as positive control, no fibers were generated on EcNΔCsg.
**The result validated that Csg gene was deleted successfully.**

![Figure 4-1](https://static.igem.wiki/teams/5231/engineering/4-1.png)
![Figure 4-2](https://static.igem.wiki/teams/5231/engineering/4-2.png)

### Learn

In this cycle of engineering, we learnt:

* Techniques of gene editing and Scanning Electron Microscope. They could be later used for other cycle of design.
* The displaying of Curli Fiber on the surface of EcN was abundant. If all of these fibers could carry a tetrazine per protein, the concentration should be enough for click reaction and
  click-to-release reaction.

## Cycle 2: EcNΔCsgΔThyA

### Design

To guarantee the safety of EcN, we tend to engineer upon the basis of EcNΔCsg. One design that we found might be
effective is the deletion of ThyA, which was part of safety design of bacterial therapy2 that had passed Phase I trials.
ThyA is responsible for the synthesis of thymidine. **Knocking out ThyA is thought to enhance the bacteria's safety**,
proliferating in thymidine-rich area such as TME or other places we expected.

### Build

It was another round of gene editing, which we had to design new homologous fragments and guide RNA. However, according
to the reference3, we couldn't knock out the complete gene as what we did in Csg, since umpA also used some of the
genes, knocking out both would cause death of the bacteria. So, we chose to conserve the first 149 bp of ThyA to
construct pgRNA-ThyA. After rounds of induction and selection, we obtained some bacteria to be tested.

![Figure 5](https://static.igem.wiki/teams/5231/engineering/5.png)
![Figure 6](https://static.igem.wiki/teams/5231/engineering/6.png)

### Test

We tested the bacteria in dimension of gene. As we did in last cycle, we performed colony PCR on the bacteria on plates.
However, **we did not find bacteria whose ThyA gene was successfully deleted**.

### Learn

We estimated that our failure was **because inappropriate site selection of gene deletion**, maybe still we delete base pair
in umpA. As the experiment was conducted in September, time was very limited and we failed to get our expected results; but later
we'll continue our research on ThyA deletion.

Cycle 2: Site Selection

## Cycle 1: Initial selection

### Design

To maximum the effectiveness of tetrazine, we have to find a better site to insert Tet v2.0 into the protein CsgA.
Previous work reported S89TAG (means inserting the unnatural amino acid at site 89 of the protein, which is
originally the place for serine) for the insertion of pAzF4. Whether this site is still the best for Tet v2.0 needs to
be tested. Also, after simulation and selection of site from the Dry Lab, we find another site F97TAG might have a
better displaying property. So, **we decide to have a initial selection and investigation between the two site, S89TAG
and F97TAG**.

### Build

Based on the plasmid we bought from addgene, we successfully constructed pBbB8k-mutCsgA-F97TAG and pBbB8k-mutCsgA-S89TAG,
which both the protein mutCsgA had the correct insertion site and His-tag at C-terminal. Then, we transferred each of
the plasmid with pUltra-Ambrx, which was responsible for the expression of RS-tRNA pair of Tet v2.0, into EcNΔCsg. As
the result, we successfully constructed the bacteria: EcNΔCsg-S89TAG and EcNΔCsg-F97TAG.

![Figure 7-1](https://static.igem.wiki/teams/5231/engineering/7-1.png)
![Figure 7-2](https://static.igem.wiki/teams/5231/engineering/7-2-new.png)

### Test

We had abundant test on these two sites. However, for this cycle, we began our testing on the generation of Curli Fiber.
**We wanted to make sure that the insertion of Tet v2.0 wouldn't affect too much on the expression of the protein.** The verification was conducted by two-steps.

#### 1. Congo Red quantitative binding assay

Congo Red has the property of characterizing the generation of Curli Fiber. It will be absorbed to the fiber, so
calculating the Congo Red left on supernatant after centrifugation could partially reflect the generation of Curli
Fiber. We set EcNΔCsg with only pUltra-Ambrx and with pBbB8k-CsgAWT as control.

![Figure 8](https://static.igem.wiki/teams/5231/engineering/8.jpg)

#### 2. Scanning Electron Microscope (SEM)

Another test we performed was SEM, to directly find the fiber on the bacterial surface. It could also serve as
supporting evidence to the results of CR quantitative binding assay.

![Figure 9](https://static.igem.wiki/teams/5231/engineering/9-5.png)

### Learn

For this cycle, we validated the formation of almost same amount of curli fiber of S89TAG and F97TAG. However, some
interesting results drove to conduct the next cycle of engineering, adding new sites into our
consideration. **We found that, after induction, even without Tet v2.0, the protein could still be expressed as the same
level of expressing with Tet v2.0.** The result was validated by the pictures of SEM. We thought that **it might be because
truncated CsgA expression with only 2 repeats of β sheets could still be piled and formed curli fibre**. Thus, we decided
to **select two more sites which was at the beginning of CsgA** to see if we could tackle the problem.

## Cycle 2: Continue selection of insertion sites

### Design

After the last cycle of engineering, we decided to find two more sites at the beginning of CsgA. **We selected two sites
Y48TAG and Y50TAG** which expressed tyrosine naturally, since the structure of Tet v2.0 is similar to tyrosine, the
insertion might cause the least side effect to the folding of CsgA. For this cycle of selection, we didn't consider the
display of tetrazine. We just wanted to make clear the function of truncated CsgA that we thought we observed in the
last engineering cycle.

### Build

As we did in the last cycle, we constructed the plasmids pBbB8k-mutCsgA-Y48TAG and obtained the bacteria EcNΔCsg-Y48TAG and EcNΔCsg-Y50TAG.

![Figure 10-1](https://static.igem.wiki/teams/5231/engineering/10-1.png)
![Figure 10-2](https://static.igem.wiki/teams/5231/engineering/10-2.png)

### Test

In this cycle, we tested the generation of curli fiber primarily on CR quantitative binding assay. Altogether, we compared the
results of the previous cycle and this cycle to verify our hypothesis.

![Figure 11](https://static.igem.wiki/teams/5231/engineering/9.jpg)

After the experiment, we simulated the ability of each insertion site to display tetrazines. According to the simulation
results from the dry lab, the F97TAG site has the strongest hydrophobicity in tetrazine display. **This indicates that
this site is more inclined to react rather than be exposed in the aqueous phase, and it has stronger reactivity.**

### Learn

According to the result, sites Y48TAG and Y50TAG didn't affect the formation of curli fiber with or without Tet v2.0,
which meant our previous hypothesis was wrong. The formation of curli fiber even decreased for Y50TAG. After discussing
with our PI, **we thought another reason might be the false insertion of natural amino acids** such as phenylalanine or
tyrosine since the RS we used would have such a false positive effect. **We planned to test such a hypothesis after the
competition using the GMML culture medium which lacks nutrition and natural amino acids.** As a result of this engineering
cycle, **we decided to keep our selection range within S89TAG and F97TAG**, since they were simulated to display tetrazine
better, and the expression of CsgA was better than Y48TAG and Y50TAG.

## Cycle 3: Final selection of insertion sites

### Design

After the last engineering cycle, we realized that the CR quantitative binding assay couldn't help us decide on the
final site, so we continued our experiment for the next cycle. We still chose **S89TAG and F97TAG** to investigate. **This
cycle of engineering mainly continued our test in cycle 3.**

### Build

The plasmids and bacteria have been already built in cycle 3.TCO-Coumarin was synthesized through a two-step reaction and verified by MS.

### Test

We tested S89TAG and F97TAG this engineering cycle mainly on performance on displaying tetrazine. In total, we conducted
3 sets of experiments:

#### 1. TCO-Cy5 assay

Cy5 is a type of Cyanine dye which would generate fluorescence after excitement. TCO-Cy5 would react with tetrazine, the
more tetrazine displayed on the surface, the more fluorescence signal would be generated. Using TCO-Cy5, we tested the 2
sites from 3 dimensions:

##### a. Bacterial dimension

In this dimension, we tend to compare the 2 sites overall. We first directly incubated TCO-Cy5 with the bacteria which were
overnight induced the expression of CsgA. After rounds of washing, S89TAG displayed a better Cy5 fluorescence signal
compared to F97TAG, indicating that S89TAG might express more CsgA with Tet v2.0 inserted.

![Figure 12](https://static.igem.wiki/teams/5231/engineering/12-new.jpg)
![Figure 13](https://static.igem.wiki/teams/5231/engineering/13.png)

##### b. Protein dimension

In the dimension of protein, we wanted to have a deeper insight into the expression of CsgA and the insertion of Tet
v2.0. We successfully purified the Tetv2.0-incorporated CsgA variants, which we applied TCO-Cy5 for labeling, observing
that only the Tet v2.0-incorporated CsgA proteins exhibited a strong fluorescent signal. Furthermore, CsgAS89TAG and
CsgAF97TAG were validated by protein molecular weight mass spectrometry. Collectively, **these results confirm the
successful expression and purification of Tetv2.0-incorporated CsgA proteins. S89TAG exhibited a better amount of
protein expression with Tet v2.0 inserted.**

![Figure 14](https://static.igem.wiki/teams/5231/engineering/14-new.png)

The figure illustrates the fluorescent labeling of CsgAS89Tetv2.0-His and CsgAF97Tetv2.0-His (lanes 1 and 2), which
undergo reaction with TCO-Cy5 via the inverse electron-demand Diels-Alder (IEDDA) reaction, resulting in detectable
fluorescent bands. In contrast, CsgAWT-His (lane 3), lacking the tetrazine moiety, does not react with TCO-Cy5, and thus
no fluorescent labeling is observed.

![Figure 14-1](https://static.igem.wiki/teams/5231/engineering/14-1.jpg)
![Figure 14-2](https://static.igem.wiki/teams/5231/engineering/14-2.jpg)

##### c. Single bacterial dimension

Though we tested in both dimensions of bacterial and protein, we still wanted to see the performance of Tet v2.0
insertion per bacteria. We tested this through Flow Cytometry and Confocal.

For confocal experiment, we also used TCO-Cy5 to stain Tetv2.0-incorporated CsgA on the bacterial surface; moreover, to
visualize the bacteria, we chose DMAO and PI to distinguish between the live(only DMAO could stain) and the dead(both
DMAO and PI could stain) bacteria. After appropriate adjustment of excitation and emission fluorescence channel under
the CsgAS89TAG expressing with Tetv2.0, we successfully obtained the fluorescent co-localization signal of DMAO and
TCO-Cy5 in CsgAS89TAG and CsgAF97TAG, while there was no Cy5 signal in CsgAWT either under the same expression condition
or without induction, which confirmed that **Tetv2.0 was site-specifically inserted into CsgA and well displayed on
bacterial surface**. Moreover, from our fluorescence co-localization results, **CsgAF97TAG performed better than CsgAS89TAG**,
no matter considering corresponding overall fluorescence intensity or the co-localization performance.

![Figure 15](https://static.igem.wiki/teams/5231/engineering/15.png)

Adjustment of excitation and emission fluorescence channel under the single staining of
CsgAS89TAG expressing with
Tetv2.0. DMAO staining was observed through FITC channel, PI staining was observed through Cy3 staining, and TCO-Cy5
staining was observed through Cy5 channel.

![Figure 16](https://static.igem.wiki/teams/5231/engineering/16.png)
![Figure 17](https://static.igem.wiki/teams/5231/engineering/17-new.png)

Fluorescent co-localization of CsgAvariants before expression and after expression with Tetv2.0. Without induction,
there was no co-localization between DMAO and Cy5; however, after induction with 1mM IPTG, 0.2% Arabinose and 1mM
Tetv2.0, obvious fluorescent co-localization was observed in both CsgAS89TAG and CsgAF97TAG but not in CsgAWT,
confirming Tetv2.0-incorporated CsgA displayed on EcN surface.

For flow cytometry, S89TAG and F97TAG displayed a similar ratio of about 80%, much higher than that of CsgAWT which is
about 10%. After analyzing 10,000 living cells, about 65% of F97TAG were detected to be labeled by TCO-Cy5, and was slightly better than the 63% of S89TAG, indicating
that **tetrazine on F97TAG may have a better displaying**.

![Figure 18](https://static.igem.wiki/teams/5231/engineering/18-new.png)
![Figure 19](https://static.igem.wiki/teams/5231/engineering/19-new.png)

#### 2. Growth Curve

While analyzing with Confocal and Flow Cytometry, we found that a great number of bacteria were dead after induction. We
wanted to check if it was the problem of Tet v2.0's toxicity. So, we experimented with the growth curve, letting them
grow in different conditions. After induction, S89TAG and F97TAG both with or without Tet v2.0 grew in a much worse
condition.

![Figure 20-1](https://static.igem.wiki/teams/5231/engineering/20-1.jpg)
![Figure 20-2](https://static.igem.wiki/teams/5231/engineering/20-2.jpg)
![Figure 20-3](https://static.igem.wiki/teams/5231/engineering/20-3.jpg)
![Figure 20-4](https://static.igem.wiki/teams/5231/engineering/20-4.jpg)

#### 3. TCO-Coumarin assay

Testing only with TCO-Cy5 isn't adequate, since reacting with TCO for ligation and click-to-release have slight
differences. So before reacting with the prodrug, we decided to test the de-caging property by TCO-Coumarin.

TCO-Coumarin is the Coumarin whose fluorescence was partially caged by TCO. After the click-to-release reaction with
tetrazine, the fluorescence intensity would increase. So, we incubated TCO-Coumarin with the bacteria and normalized by
it OD600. S89TAG and F97TAG all displayed a similar de-caging effect, while S89TAG performed a faster reaction rate, **and F97TAG
exhibited a better reaction percentage at 30 minutes**.

![Figure 21](https://static.igem.wiki/teams/5231/engineering/21.jpg)

### Learn

With all the tests above, several conclusions can help us make the decision:

* Both S89TAG and F97TAG wouldn't affect the generation of curli fiber.
* S89TAG expressed more CsgA with Tet v2.0 inserted.
* F97TAG displayed tetrazine better than S89TAG.

Because of the need of capability of reaction, **we finally selected F97TAG as our ultimate site to incorporate Tet v2.0**.

Engineering 3: Safety cycle

## Cycle 1: Promoter Verification

### Design

In order to improve the safety and targeting of our engineered bacteria,
as well as to meet the actual needs of subsequent animal experiments and clinical
applications, we replaced the promoter of the expression system with the hypoxic promoter.

To determine the expression efficiency of the hypoxic promoter and to qualitatively and
quantitatively analyze the difference in expression efficiency between it and the arabinose
promoter, we carried out the verification and characterization of promoter expression efficiency.

### Build

To verify and analyze the difference in promoter expression efficiency,
we successfully constructed three plasmids—pBbB8k araBAD+GFP, pBbB8k JWW+GFP, pBbB8k CsgA.
The three plasmids were used as positive control, experimental group, and negative control,
respectively. They were transferred into EcN to complete the construction of the engineered bacteria.

![Figure 22-1](https://static.igem.wiki/teams/5231/engineering/22-1.jpg)
![Figure 22-2](https://static.igem.wiki/teams/5231/engineering/22-2.png)

### Test

We performed the same treatment under arabinose induction and hypoxic environment induction,
and used PBS to wash the culture medium, finally obtaining a PBS solution of bacteria.
In the enzyme marker, the OD600 and fluorescence value were measured, and the average
was taken from three repetitions of each group. The results were plotted as a bar graph
with the ratio of fluorescence value to OD600 as the Y-axis.

![Figure 23](https://static.igem.wiki/teams/5231/engineering/23-new.jpg)

Using the same culture and treatment methods, but employing different induction conditions:
arabinose induction or hypoxic environment induction, and reflecting promoter efficiency
by measuring the ratio of fluorescence value to OD600.

We found that: compared to the arabinose promoter, the expression efficiency of the hypoxic
promoter is very low. And throughout the verification process, it is difficult to control a
single variable.

### Learn

In response to the above situation, we proposed a new idea to improve the expression efficiency
of the hypoxic promoter, introducing the T7 Promoter expression system into the EcN strain.
The hypoxic promoter is used to express T7 polymerase, and then the T7 promoter is used to express
the target protein, thereby achieving an apparent increase in the expression efficiency of the
hypoxic promoter through a "cascade reaction".

![Figure 24](https://static.igem.wiki/teams/5231/engineering/24.png)

References

1. Praveschotinunt, Pichet et al. “Tracking of Engineered Bacteria In Vivo Using Nonstandard Amino Acid Incorporation.” ACS
synthetic biology vol. 7,6 (2018): 1640-1650.

2. Leventhal, Daniel S et al. “Immunotherapy with engineered bacteria by targeting the STING pathway for anti-tumor
immunity.” Nature communications vol. 11,1 2739. 1 Jun. 2020,

3. 黄维,钟辉,李平,等.大肠杆菌胸腺嘧啶合成酶thyA缺失突变菌株的构建[J].微生物学报,2004,(01):62-66.

4. Marcus, Alan et al. “Fluorescence microscopy is superior to polarized microscopy for detecting amyloid deposits in Congo
red-stained trephine bone marrow biopsy specimens.” American journal of clinical pathology vol. 138,4 (2012): 590-3.

![engineering](https://static.igem.wiki/teams/5231/banners/engineering.png)

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{% block lead %}Describe the research, experiments, and protocols you used in your iGEM project.{% endblock %}
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Experiment Record

![experiments](https://static.igem.wiki/teams/5231/banners/experiments.png)

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![results](https://static.igem.wiki/teams/5231/banners/results.png)

Acquisition of EcNΔCsg

Synthesize of Tet v2.0

Selection of Tet v2.0 Insertion Sites and Characterization
Pre-selection of the Insertion Site
Generation of Curli Fiber
Display of Tetrazine
Influence of Tet v2.0 to Bacterial Growth
Final Selection of Insertion Site

Characterization of De-cage

Cytotoxicity Assay

Promoter Verification

Conclusions

References

Acquisition of EcNΔCsg

Box 1: Why *Escherichia coli* Nissle 1917 (EcN) and not BL21?

BL21 is commonly used for protein expression and characterization. Typically, when we engineer an unfamiliar protein, the initial procedure is conducted in BL21, followed by transferring the results to the final microbe we intend to use, such as EcN in our design. However, for the following reasons, we decided to engineer within EcN directly:

* **Limited time:** BL21 and EcN have the Csg gene in their genomes. Knocking out both genes would consume too much time for us.
* **Human Practices from Neel Joshi:** At the onset of our project, we contacted Professor Joshi via email, seeking advice on engineering CsgA. It was suggested that, as a protein, CsgA is difficult to purify and characterize. We should focus more on the design rather than the protein itself.
* **The Design of our project:** Our project aims to display tetrazine on the surface of EcN. Therefore, characterizing CsgA on EcN is more straightforward and relevant to our goals.

We used the way of CRISPR-Cas9 to delete the gene. Firstly, we transfer plasmid pHCY-25A, and then newly constructed
plasmid pgRNA-Csg-1 (Figure 2), which we inserted the selected gRNA (Figure 1) and N20 sequence.(Reference: [https://chopchop.cbu.uib.no/#](https://chopchop.cbu.uib.no/)).

![Selection of gRNA](https://static.igem.wiki/teams/5231/results/1-new.png)

Figure 1: Selection of gRNA

![Construction of plasmids in gene knock-out](https://static.igem.wiki/teams/5231/results/2-1-new.png)
![Construction of plasmids in gene knock-out](https://static.igem.wiki/teams/5231/results/2-2-new.png)

Figure 2: Construction of plasmids in gene knock-out

By inducting and controlling of culturing temperature, we successfully acquired EcNΔCsg with no plasmid inside. (Figure
3) The results were verified by colony PCR and growth on multiple plates with different antibiotics.

![Verification of discarding plasmids](https://static.igem.wiki/teams/5231/results/3-1.jpg)
![Verification of discarding plasmids](https://static.igem.wiki/teams/5231/results/3-2.jpg)
![Verification of discarding plasmids](https://static.igem.wiki/teams/5231/results/3-3.jpg)

Figure 3: Verification of discarding plasmids

Kanamycin(K) is the antibiotic of pgRNA; Ampicillin(A) is the antibiotic of pHCY-25A
Each monoclonal was divided into 3 portion and streaked on 3 plates (no antibiotics,
Kanamycin and Ampicillin). Ones that only grew on plate with no antibiotics were ones with no plasmids.

![Verification of knocking out gene Csg successfully](https://static.igem.wiki/teams/5231/results/4.png)

Figure 4: Verification of knocking out gene Csg successfully

The length between homologous fragments before the gene knocks out is
5444bp. After knocking out, only homologous fragments are left in the genome of EcN, which is about 1000bp.

Synthesize of Tet v2.0

Curli fibers serve as an excellent bacterial surface display platform. Praveschotinunt, Pichet, et al.1
inserted p-azido-l-phenylalanine (pAzF) into CsgA, leveraging a copper-free click reaction between the azide group
and dibenzocyclooctyl (DBCO) to enable the tracking of engineered bacteria in vivo. However, **such click
reaction isn't strictly bioorthogonal**. DBCO would react with the sulfhydryl group, which means off-target.

Compared to the former reaction, the click reaction between tetrazine and trans-cyclooctenes (TCO)
displays a faster reaction rate and has less problem in off-target. Group Mehl developed several
unnatural amino acids with tetrazine, including Tet v1.0 to 4.0. **Among the four,
Tet v2.02 was the most used and developed amino acid,
so we chose it as our unnatural amino acid**. We synthesized Tet v2.0 through a 2-step route (Figure 5)
and verified through mass spectrometry (Figure 6).

![Route of Tet v2.0 synthesize](https://static.igem.wiki/teams/5231/results/5.png)

Figure 5: Route of Tet v2.0 synthesize

![Mass spectrometry of Tet v2.0 (MW:259)](https://static.igem.wiki/teams/5231/results/6-new-new.png)

Figure 6: Mass spectrometry of Tet v2.0 (MW:259)

We tested the expression of protein with Tet v2.0 insertion with GFP-151TAG (which is, changing site
151 of GFP into TAG so that we can insert unnatural amino acids in it). We transferred pUltra-Ambrx
(Figure 7), which has an RS-tRNA pair to insert Tet v2.0, and a plasmid carrying GFP151TAG to BL21, and
purified the protein. The tetrazine would quench the fluorescence of GFP, thus making it yellow rather
than green (Figure 8). **This could verify that our system of Tet v2.0 could insert the amino acid into a protein.**

![pUltra-Ambrx](https://static.igem.wiki/teams/5231/results/7-new.png)

Figure 7: pUltra-Ambrx

![Quenching of GFP fluorescence](https://static.igem.wiki/teams/5231/results/8.jpg)

Figure 8: Quenching effect of Tet v2.0 on the fluorescence of purified GFP

Originally, the fluorescence of GFP should be green, but after insertion of Tet v2.0, the fluorescence
of GFP would be quenched and showed yellow.

Selection of Tet v2.0 Insertion Sites and Characterization

## 1. Pre-selection of the Insertion Site

One of the most important issues of our design is choosing the insertion site of Tet v2.0. Better
insertion efficiency and displaying of tetrazine could improve the effectiveness of our design.

According to previous work, S89TAG (which is, changing Serine at site 89 into amber codon where unnatural
amino acid inserts) has been reported as one possible option. However, according to our investigation,
S89TAG is at the end of the second β sheet, which may affect the structure of CsgA, and it's not the
perfect place to display tetrazine for reaction.

Box 2: Selection of insertion site

The insertion site selection may largely contribute to the effectiveness of our design. The wrong site may lead to misfolding of protein, and reduced reaction rate. On such issues, we communicate with both our PI and Dry lab, hoping to get a better site. Here's some of the advice that we got:

Dry lab: they simulate the insertion by displaying Tet v2.0 at different sites of the protein. As a result, F97TAG is recommended to be a better option, since firstly, it's on the loop of CsgA; secondly, the structure of phenylalanine is similar to Tet v2.0, so Tet v2.0 wouldn't affect the protein structure; Thirdly, F97TAG performs better in reaction rate simulation.

[For more information, please refer to dry lab.](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27md%27%29%20%7D%7D)

As the result, we chose S89TAG and F97TAG as our initial selection of Tet v2.0 insertion.

## 2. Generation of Curli Fiber

The first question we meet is,**will the insertion of Tet v2.0 affect the expression rate of CsgA**, therefore affecting the
formation of Curli Fiber. We investigate such a problem by using **Congo Red**. It's what people usually
use to quantify the formation of the curli fiber. By adding Congo Red after induction and expression of EcN,
we found that **both S89TAG and F97TAG can express the fiber with high efficiency, even higher than WT**. (Figure 9)

![CR assay of curli fiber formation](https://static.igem.wiki/teams/5231/results/9.jpg)

Figure 9: CR quantitative binding assay

S89TAG and F97TAG all displayed a much higher CR binding property,
with or without Tet v2.0. WT: pBbB8k-CsgAWT; OTS: EcNΔCsg+pUltra-Ambrx RS

The problem of the experiment is that the CR binding of CsgAWT didn't display a positive control,
after several rounds of experiment. Referring to previous works, they didn't either have a significant
positive control result.

Another strange thing that bothers us is that, without adding Tet v2.0, after induction, the bacteria can **still exhibit a high efficiency in curli fiber formation**. It was later verified by scanning electron
microscope (Figure 10), which we did the same treatment to the bacteria, with or without adding Tet
v2.0 into the expressing system.

![Scanning electron microscope results](https://static.igem.wiki/teams/5231/results/10-mew.png)

Figure 10: Scanning electron microscope results

Treatments of each group are shown in the figure. The white fibers are curli fiber
Ara:0.2%; IPTG: 0.5mM; CsgAWT: pBbB8k-CsgAWT

After discussion, we thought it might be because of the expression of truncated protein. Although the
expression stops at the amber codon, there are still two β sheets left, which might form the structure
of amyloid protein, and generate curli fiber. We inquired our PI about our guesses, and it was recommended
that the insertion site be placed at the beginning of the protein. Under such conditions, the protein
stops its initial expression and won't form curli fiber. So, we add Y48TAG and Y50TAG into our Congo Red
assay (Figure 11).

![CR quantitative binding assay](https://static.igem.wiki/teams/5231/results/11.jpg)

Figure 11: CR quantitative binding assay

WT: pBbB8k-CsgAWT; OTS: EcNΔCsg+pUltra-Ambrx RS

The change of insertion site doesn't significantly change the result. This proves that our previous hypothesis
might be wrong, and the formation of curli Fiber without Tet v2.0 might be **due to the insertion of other
natural amino acids like Tyrosine or Phenylalanine**. This is because the RS we used won't just insert
Tet v2.0, it may also have a false positive which inserts amino acids with similar structures. However,
we stopped our investigation here and paid more attention to other aspects of our design. We assumed
that CR assay was a reference rather than a concrete characterization of curli generation.
**In the aspect of curli fiber generation, S89TAG and F97TAG perform equally.**

## 3. Display of Tetrazine

The next question we want to find out is the display efficiency of tetrazine on the surface of CsgA.
We verify such display efficiency through several aspects:

### 3.1. Protein Level

To verify the insertion of Tetv2.0 into CsgA at the protein level, we attempted to purify
CsgAS89TetV2.0-His and CsgAF97Tetv2.0-His with previously reported methods. Indeed, we successfully
purified the Tetv2.0-incorporated CsgA variants, which we applied TCO-Cy5 for labeling, observing that
only the Tetv2.0-incorporated CsgA proteins exhibited a strong fluorescent signal (Figure 12).
Furthermore, CsgAS89TetV2.0-His and CsgAF97TetV2.0-His were validated by protein molecular weight mass
spectrometry (Figure 13). Collectively,  **these results confirm the successful expression and purification
of Tetv2.0-incorporated CsgA proteins.**

![TCO-Cy5 fluorescent labeling to confirm incorporation of Tetv2.0 into CsgA](https://static.igem.wiki/teams/5231/results/13-new.png)

Figure12: TCO-Cy5 fluorescent labeling to confirm incorporation of Tetv2.0 into CsgA.

The figure illustrates the fluorescent labeling of CsgAS89Tetv2.0-His and
CsgAF97Tetv2.0-His (lanes 1 and 2), which undergo reaction with TCO-Cy5 via the inverse electron-demand
Diels-Alder (IEDDA) reaction, resulting in detectable fluorescent bands. In contrast, CsgAWT-His (lane 3),
lacking the tetrazine moiety, does not react with TCO-Cy5, and thus no fluorescent labeling is observed.

![TCO-Cy5 reacting assay WT](https://static.igem.wiki/teams/5231/results/14-1.jpg)
![Effect of TCO-Cy5 binding of different bacteria](https://static.igem.wiki/teams/5231/results/14-2.jpg)

Figure 13: Molecular weight mass spectrometry verification of CsgA variants.

### 3.2. Bacteria Level

On the Bacteria Level, we verify the displaying of tetrazine through three aspects: TCO-Cy5 assay, Flow Cytometry, and Confocal Microscopy Through Focusing.

For the TCO-Cy5 assay, we wanted to preliminarily find out the capacity of the overall tetrazine display. We labeled the bacteria with TCO-Cy5 for 5 hours after overnight induction and then washed them with PBS through centrifuging.
**S89TAG demonstrates a higher display of tetrazine on its surface after OD600 normalization.**
(Figure 14) Precisely, after comparison with the fluorescence generated by Cy5 itself, for OD600=1 of the bacteria,
S89TAG displayed about 0.13μM Cy5 in average, while F97TAG displayed about 0.11μM Cy5 in average.
It could also be directly observed through the color of centrifuged labeled bacteria.
(Figure 15) However, this is the general effect of protein expression and the insertion and display
of Tet v2.0. We couldn't identify which was the main factor, therefore we continued our experiment on
single cell dimension.

![TCO-Cy5 reacting assay WT](https://static.igem.wiki/teams/5231/results/15-new.jpg)

Figure 14: TCO-Cy5 reacting assay

WT: pBbB8k-CsgAWT

![Effect of TCO-Cy5 binding of different bacteria](https://static.igem.wiki/teams/5231/results/16-new.png)

Figure 15: Effect of TCO-Cy5 binding of different bacteria

WT: pBbB8k-CsgAWT

The next experiment we conducted was Flow Cytometry so that we could compare S89TAG and F97TAG in single-cell dimensions. We first compared the percentage of TCO-Cy5 labeled cells. (Figure 16)
**S89TAG and F97TAG displayed a similar ratio of about 80%, much higher than that of CsgAWT which is about 10%.**

![FCM results of Cy5-labeled bacteria](https://static.igem.wiki/teams/5231/results/17-new.png)

Figure 16: FCM results of Cy5-labeled bacteria

It does not distinguish between live/dead bacteria; WT: pBbB8k-CsgAWT

However, it's the overall labeled ratio of bacteria, ignoring the fact that some bacteria are dead.
Thus, we continued the experiment, identifying live/dead through DMAO/PI staining. In brief, DMAO could
pass all cell membranes, while PI could only pass damaged cell membranes from dead bacteria, so live
cells were only labeled by DMAO. Among all, about 36% of bacteria are alive. After analyzing 10,000 living cells, about 65% of F97TAG were detected to be
labeled by TCO-Cy5, and was slightly better than the 63% of S89TAG (Figure 18), indicating that **tetrazine on F97TAG may
have a better displaying**.

![FCM analysis of S89TAG and F97TAG alive](https://static.igem.wiki/teams/5231/results/18-new.png)

Figure 17: FCM analysis of S89TAG and F97TAG alive

Bacteria with DMAO green fluorescence and without PI red fluorescence are alive. Among living bacteria, labeled populations are measured.

For confocal experiment, we also used TCO-Cy5, DMAO and PI to stain our engineered bacteria. We successfully obtained
the fluorescent co-localization signal of DMAO and TCO-Cy5 in CsgAS89TAG and CsgAF97TAG, while there was no Cy5 signal
in CsgAWT either under the same expression condition or without induction(Figure 18), which confirmed that **Tetv2.0 was
site-specifically inserted into CsgA and well displayed on bacterial surface**. Moreover, from our fluorescence
co-localization results, **CsgAF97TAG performed better than CsgAS89TAG**, no matter considering corresponding overall
fluorescence intensity or the co-localization performance.

![Confocal experiment](https://static.igem.wiki/teams/5231/results/26-1.png)
![Confocal experiment](https://static.igem.wiki/teams/5231/results/26-2.png)
![Confocal experiment](https://static.igem.wiki/teams/5231/results/26-3.png)
![Confocal experiment](https://static.igem.wiki/teams/5231/results/26-4.png)

Figure 18: Confocal experiment

Fluorescent co-localization of EcN without or with induction under 1mM IPTG, 20% Arabinose and 1mM Tetv2.0. In (A),
without induction, only DMAO staining was visible; however, in (B), after induction, CsgAS89TAG and CsgAF97TAG could be
further stained with TCO-Cy5, which could be co-localized with DMAO and stained yellow. More details of CsgAS89TAG
fluorescent co-localization were shown in (C) and CsgAF97TAG in (D), where CsgAF97TAG seemed to perform better than
CsgAS89TAG, no matter considering overall fluorescent intensity or the co-localization situation.

## 4. Influence of Tet v2.0 to Bacterial Growth

Box 3: Safety issue

Safety is one of the most important issues when associated with humans. It's especially important in
bacterial therapy, since the proliferation of bacteria in vivo may affect human health.

In our project, **a total of 3 things may have safety issue: EcN, Tet v2.0, and prodrug (TCO-DOX).**
We initially thought it wasn't a big deal until experts and other iGEMers from Human Practices continued
to express their worries about safety. For example, Professor Zhuang Liu expressed his worries about the
toxicity of Tet v2.0, both to bacteria and cells. Thus, **we decided to pay a bit of attention to safety
issues**, even though we still focus more on the effectiveness of our design.

[For more information, please refer to Human Practice for the conversation with Zhuang Liu, and what we design to guarantee safety.](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27human-practices%27%29%20%7D%7D)

As experiments progressed, we found that **the growth of S89TAG and F97TAG was much worse than CsgAWT**, though the expression of CsgA was all under the control of the Arabinose promotor. Three reasons might explain such phenomenon: the toxicity of Tet v2.0; the expression pressure of protein; and the toxicity of truncated CsgA expression.

To find out the main cause, we allowed bacteria to grow under various conditions at 37°C within a microplate reader. (Figure 18) The result is that Tet v2.0 wouldn't affect the growth; after induction, S89TAG and F97TAG both with or without Tet v2.0 grew in a much worse condition, while CsgAWT didn't have such a phenomenon. It might be concluded that
**the truncated CsgA expression might largely contribute to the reduced growth of EcN**. Such a result may partially explain the safety issue of Tet v2.0, and it also gave us a hint about the purification of CsgA.

![Growth curve of bacteria and their comparison while expressing CsgA](https://static.igem.wiki/teams/5231/results/19-1.jpg)
![Growth curve of bacteria and their comparison while expressing CsgA](https://static.igem.wiki/teams/5231/results/19-2.jpg)
![Growth curve of bacteria and their comparison while expressing CsgA](https://static.igem.wiki/teams/5231/results/19-3.jpg)
![Growth curve of bacteria and their comparison while expressing CsgA](https://static.igem.wiki/teams/5231/results/19-4.jpg)

Figure 19: Growth curve of bacteria and their comparison while expressing CsgA

Bacteria are cultured in 37°C within a microplate reader. After induction by Arabinose and IPTG, the population of S89TAG and F97TAG significantly decreased compared to other groups. Toxicity of Tet v2.0 was little. WT: pBbB8k-CsgAWT

## 5. Final Selection of Insertion Site

According to previous experiments (and the next experiment of characterization of De-cage), we had three conclusions between the two sites:

* Both S89TAG and F97TAG wouldn't affect the generation of curli fiber.
* S89TAG expressed more CsgA with Tet v2.0 inserted.
* F97 displayed tetrazine better than S89TAG

Because of the need of capability of reaction, **we finally selected F97TAG as our ultimate site to incorporate Tet v2.0**.

Characterization of De-cage

Before TCO-DOX, we wanted to check the de-caging capabilities and properties of the bacteria.
The detection of DOX was comparatively difficult, so after investigation, we caged the fluorescence of
coumarin with TCO3, though later experiments showed the fluorescence wouldn't be caged
completely. We synthesized TCO-Coumarin through a two-step reaction (Figure 19), and was verified
through mass spectrometry (Figure 20):

![2-step reaction of TCO-Coumarin Synthesis](https://static.igem.wiki/teams/5231/results/20.png)

Figure 20: 2-step reaction of TCO-Coumarin Synthesis

![Mass spectrometry of TCO-Coumarin (MW: 327)](https://static.igem.wiki/teams/5231/results/21-new.png)

Figure 21: Mass spectrometry of TCO-Coumarin (MW: 327)

We tested TCO-Coumarin first on molecular dimension, which is, de-caging through tetrazine rather than
protein or bacteria. By adding the Tet v2.0 and TCO-Coumarin (Figure 21), we preliminarily assumed **the adequate de-caging time was about 30 minutes.**.
Later, the result was collected and analyzed by dry lab, to model the process of reaction so that we
could better control the reaction. (See more: please refer to dry lab) However, some problems
still occurred in such experiments because of the limited time:

* A.We didn't precisely weigh the TCO-Coumarin we got, since it's only several milligrams, so we didn't get the precise de-caging percentage. If time's available, we will re-do the experiment to get the percentage, so that we can decide the best dosage of our design.
* When communicating with the dry lab, they expected a much more complex system to simulate the effect of the environment on the de-caging reaction. For example, we are recommended to experiment with culture medium such as DMEM or RPMI 1640. However, the TCO-Coumarin was placed at -20°C for too long, and such fluorescence detection could be disturbed by the culture medium, we might think of a better way to design the experiment.

![Time-dependent Fluorescence intensity of TCO-Coumarin De-cage](https://static.igem.wiki/teams/5231/results/22.jpg)

Figure 22: Time-dependent Fluorescence intensity of TCO-Coumarin De-cage

Experiment was conducted in ddH2O. TCO-Coumarin itself has fluorescence. After adding Tet v2.0, the fluorescence increased by time.

After verifying the effect in molecular dimension, we continued our experiment on bacterial dimension.
By adding TCO-Coumarin to PBS-washed bacteria and culturing for a certain time, we tested the fluorescence
from the supernatant after centrifugation and OD600 normalization. (Figure 23) S89TAG and F97TAG all displayed a similar de-caging effect, while S89TAG performed a faster reaction rate, **and F97TAG
exhibited a better reaction percentage at 30 minutes**.The problem with such an experiment is that Coumarin might also be centrifuged under the bacteria.
This result partially supported our selection of F97TAG rather than S89TAG.

![TCO-Coumarin Click-to-Release Effect Validation at Bacterial Level](https://static.igem.wiki/teams/5231/results/23.jpg)

Figure 23: TCO-Coumarin Click-to-Release Effect Validation at Bacterial Level

WT: pBbB8k-CsgAWT

Cytotoxicity Assay

After verification of the de-caging effect of TCO-Coumarin, we came to the final stage of testing our design in the dimension of cells. In the selection of prodrug and cell line, we decided on both investigations and Human Practices.
**Finally, we set doxorubicin4 (DOX) as our prodrug to examine cell
cytotoxicity.**

Box 4: Prodrug selection

After selecting colorectal cancer as our target, we reached out to Doctor Qi as a Human Practices, to find out the problems of our design.

**Initially, we set MMAE as our drug**, since it's a widely used chemotherapeutic that has already been used
in fields of ADCs. However, Doctor Qi stated that for colorectal cancer, **MMAE might not be a preferred
option**, since its effectiveness is relatively poor to colorectal cancer, he suggested **doxorubicin and
Irinotecan** might be a better option. Doxorubicin already has references to be caged by TCO, **thus we set
DOX as our first candidate**. It is also recommended to find a site to cage Irinotecan by TCO. We told our
findings to the dry lab. However, they haven't yet found a place to cage by TCO.

[For more information, please refer to Human Practices](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27human-practices%27%29%20%7D%7D)
[Or take a look at other details about safety.](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27safety%27%29%20%7D%7D)

We synthesized TCO-DOX through a two-step reaction (Figure 24) and verified the molecular through mass spectrometry (Figure 25).

![two-step synthesize of TCO-DOX](https://static.igem.wiki/teams/5231/results/24.png)

Figure 24: two-step synthesize of TCO-DOX

![Mass spectrometry of TCO-DOX (MW: 695)](https://static.igem.wiki/teams/5231/results/25-new.png)

Figure 25: Mass spectrometry of TCO-DOX (MW: 695)

After obtaining TCO-DOX, we first tested it on the molecular dimension. After mixing TCO-DOX and overdose
Tet v2.0, we analyzed the de-caging properties by using HPLC (Figure 26) **TCO-DOX rapidly de-cage within
30 minutes**, which is compatible with the result we had before on TCO-Coumarin. However, the experiment still had several drawbacks. The standard of doxorubicin hydrochloride we used could not produce a peak with a specific retention time in HPLC. So, we couldn't identify which was the de-caged DOX and how much we got. Thus, we set one peak as DOX, and set the peak area at 125 minutes as 100%, to show its de-caging property. This could not show either the de-caging percentage.

![De-cage of TCO-DOX examined by HPLC](https://static.igem.wiki/teams/5231/results/26-new.jpg)

Figure 26: De-cage of TCO-DOX examined by HPLC

We first tested the cell toxicity in dimension of molecular. We chose CT26 as our model of colorectal cancer. It was
murine colorectal cancer cell line that we bought from Pricella. We didn't select human colorectal cancer cell line such
as 4T1, because if possible, it was more convenient for us to later test our design in mouse model.

![Cell toxicity of TCO-DOX on CT26](https://static.igem.wiki/teams/5231/results/27.jpg)

Figure 27: Cytotoxicity assay of CT26 in molecular dimension

For each of the group, we added different concentrations of DOX, TCO-DOX and TCO-DOX de-cage with Tet v2.0 (Figure 28).
**DOX exhibited an IC50 of 0.26μM, while TCO-DOX exhibited an IC50 of 5.4μM**, which was compatible with the fact that TCO
caging the toxicity of DOX. After adding Tet v2.0 to each system, the cell toxicity significantly increased, **the IC50
rose to concentration of 1.2μM, verifying the de-caging effect caused by Tet v2.0**. We previously proved that our
bacteria could display a large amount of tetrazine on the curli fiber, **we are quite confident that our design could
release prodrug efficiently and kill tumor cells.**

![Cytotoxicity assay of CT26 in protein dimension](https://static.igem.wiki/teams/5231/results/28-new.jpg)

Figure 28: Cytotoxicity assay of CT26 in protein dimension

We previously proved that our bacteria could display a large amount of tetrazine on the protein. So, we incubated 1:1
F97TAG with 12μM TCO-DOX and compared the cytotoxicity assay with F97TAG and the prodrug TCO-DOX itself (Figure 28).
Group F97TAG with 12μM TCO-DOX showed the lowest survival rate. **This indicates the successful de-caging effect by
F97TAG.** This gives us confidence that our design will work in vivo.

Promoter Verification

Due to the needs of molecular biology experiments in the laboratory, we require a promoter with high expression
efficiency, such as the arabinose promoter. However, for subsequent animal experiments or real clinical use, we will
choose a more suitable promoter for the hypoxic environment of the middle and low rectum, that is, the hypoxia-sensitive
promoter—the JWW promoter.

![Figure 22-1](https://static.igem.wiki/teams/5231/engineering/22-1.jpg)
![Figure 22-2](https://static.igem.wiki/teams/5231/engineering/22-2.png)

Figure 29: pBbB8k araBAD+GFP and pBbB8k JWW+GFP

To qualitatively and quantitatively analyze the differences between the two promoters, we designed two plasmids and
introduced them into the EcN strain, using the GFP expression of bacteria under induced conditions to characterize the
efficiency of the promoters.

The results are shown in the figure below, with the ratio of fluorescence value to OD600 as the Y-axis for bar graph
analysis and comparison.

![Figure 23](https://static.igem.wiki/teams/5231/engineering/23-new.jpg)

Figure 30: Promoter Expression Efficiency Verification Experiment

Using the same culture and treatment methods, but employing different induction conditions: arabinose induction or
hypoxic environment induction, and reflecting promoter efficiency by measuring the ratio of fluorescence value to OD600.

We found that: compared to the arabinose promoter, the hypoxic promoter has a very low expression efficiency, which is
difficult to meet our expectations for the expression quantity of the target protein by the engineered bacteria. In
response to this, we have designed a new expression system, introducing the T7 promoter system, using the hypoxic
promoter to express T7 polymerase, and then using the "cascade reaction" to induce GFP production. The plasmid design is
as follows.

![Figure 24](https://static.igem.wiki/teams/5231/engineering/24.png)

Figure 31: pBbB8k JWW+T7+GFP

Conclusions

In six months of experiments, we started our experiments from constructing the bacteria, to examine the generation of
curli fiber, and then the insertion of Tet v2.0 into CsgA, and finally examining the effect of our design in tumor
cells. **We found the comparatively better insertion site, S89TAG, and proved its effectiveness.** TCO-DOX is the prodrug we
use, and we examined its efficacy and properties in tumor cell line. For the next step, we plan to furthermore improve
our design, finding better prodrugs, and conduct animal experiments. **For now, we are quite confident that we can stand
out from other bacterial therapies in treating rectal cancer.**

References

1. Praveschotinunt, Pichet et al. “Tracking of Engineered Bacteria In Vivo Using Nonstandard Amino Acid Incorporation.” ACS synthetic biology vol. 7,6 (2018): 1640-1650.

2. Blizzard, Robert J et al. “Ideal Bioorthogonal Reactions Using A Site-Specifically Encoded Tetrazine Amino Acid.” Journal of the American Chemical Society vol. 137,32 (2015): 10044-7.

3. Fan, Xinyuan et al. “Optimized Tetrazine Derivatives for Rapid Bioorthogonal Decaging in Living Cells.” Angewandte Chemie (International ed. in English) vol. 55,45 (2016): 14046-14050.

4. Versteegen, Ron M et al. “Click to release: instantaneous DOXorubicin elimination upon tetrazine ligation.” Angewandte Chemie (International ed. in English) vol. 52,52 (2013): 14112-6.

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