iGEM 2023 — Team syphu-china

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# Notebook

What we do

[**Overview**](#Overview)
◢
**Team work**

[Team-Work](#teamwork)
[brain-storm](#brain-storm)
[Wet lab](#Wet lab)
[Dry lab](#Dry lab)
[Human-practice](#Human-practice)
[Art design](#Art design)
[Wiki Editing](#Wiki Editing)

#### Overview

* **2022.12.25The first Team formed**
* We set up the first iGEM team in SYPHU with 28 members.
* **2022.12.6-2023.3.4**
* Experimental skills training and literature reading training
* 2023.2.4-2023.4.8 Brain storm
* We were devided into different groups, each group put forward their ideas.
* **2023.4.10 Peoject determination**
* We decided to focus on HER2 positive Tumor therapy
* **2023.4.10-2023.7.25 Background research and preliminary experiments**
* The preliminary design of the complete project system and a large number of background research and
  literature research
  were completed. All experiments were started.
* **2023.6.15-2023.10.12 Experiment process and project iteration**
* We solved out all problems appeared during experiment and improve our design.
* **2023.10.12 Finished all experiment and wiki upload**
* **2023.11.2 iGEM Jamboree**

#### Team Work

#### 2023.2.4-2023.4.8 brain-storm

* **Production of non-alcoholic beer**
* Zayn Yang--Put forward chassis design and beer production methods.
* **Seborrheic Dermatitis Treatment**
* Yi Ding and Mengting Li--Do some research about Seborrheic Dermatitis and design a plan about therapy.
* **Insecticide Production**
* Shaohan Ye-- She put forward this project but she didn't take part in our team.
* **KRAS antibody design**
* Jialong Xie -- He ran after some hot topic so put forward KRAS antibody design.

#### Wet lab

* **2023.6.10-2023.10.12 We all work hard!!!!!!**
* **6.10-7.1**
* Zayn Yang and Mengting Li--Deletion of Tsr and Tar gene sequences. Prepare for all necessary materials we
  need in later experiment.
* Culture bacteria and Extract plasmid
* **6.18-7.30**
* Yi Ding-- Design Tlpc fusion protein and construct plasmid.
* Culture bacteria and Extract plasmid.
* **6.20-9.26**
* Junjie Shi,Mengting Li and Lanzhuo Xu ---Preparation for competent cell for all experiment.
* Culture bacteria and Extract plasmid
* **7.22-9.26**
* Yi Ding--Design ADC Drug and connect all parts.
* Culture bacteria and Extract plasmid
* **8.21-9.26**
* Junjie Shi,Mengting Li and Jiarui Bai--PCR for all needed parts and quantify them.
* **8.30-10.08**
* Junjie Shi and Zayn Yang --Two ways to test Chemotaxis under the guide of Ziang Su .

#### Dry lab

* **2023.6.10-2023.7.12**
* Yi Ding,Zhihao Dong,Qi Wang and Guoqing Xu--Literature searching
* **2023.7.12-2023.7.20**
* Yi Ding,Zhihao Dong and Qi Wang--Learning method of MOE、Rosetta、Gromacs and so on
* **2023.7.20-2023.8.28**
* Yi Ding and Zhihao Dong--Molecular docking modeling
* **2023.8.28-2023.10.12**
* Yi Ding and Zhihao Dong--Molecular dynamics analysis and establishment of bacterial cell model

#### Human-practice

* **2023.6.10--2023.6.15**
* All team members carry out cultural and creative design
* **2023.7.27**
* Xiaoling Yang and Zhangyi Yang--The production of tweets from the 10th iGEMer Conference
* **2023.8.7**
* Xiaoling Yang--Went back to the high school and speck
* **2023.9.19**
* Jialong Xie and Xiaoling Yang--Lectures on biology on college campuses
* **2023.9.20**
* Shuwen Cheng--Lectures on biology on college campuses
* **2023.10.5**
* Shuwen Cheng Jiarui Bai and Xiaoling Yang--Make propaganda tweets
* **2023.10.9**
* Jiarui Bai and Zishuo Wang--Analysis of questionnaire results

#### Art design

* **All time**
* Designed all event posters during this iGEM period. (Respect to them!!!)
* **4.12-5.3**
* Shuya Hu and Yiqing Wang designed our Team emblem
* **6.1-6.26**
* They designed our original project poster for CCiC(Conference of China iGEMer Community)
* **7.1-8.2**
* They designed our team uniforms.
* **8.21-9.30**
* They and Lanzhuo Xu record all activities we hold.
* **8.21-9.13**
* They complete promotion video.

#### Wiki Editing

* **7.10-10.12**
* Fushun Xu and Xuan Guo--Painting all materials we needed on the web.
* **7.10-7.31**
* Zayn Yang--Learn principle of iGEM wiki Editing and learn other team's design
* **7.20-9.26**
* Zayn Yang--Learning HTML,CSS and JavaScript.(Show respect to Pink teacher in Bilibili).Began to know VUE
  structure.
* **8.21--9.20**
* Zayn Yang build the wiki framework for SYPHU-China 2023.Collect some icons.
* **9.20-10.12**
* Zayn Yang-- Complete our wiki design. It's our first wiki

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# Engineering

Content

◢
**Construction of the Chemotaxis modules**

[Construction of the Chemotaxis modules](#title1-0)
[Construction of fusion proteins](#title1-1)
[To form better combine](#title1-2)
[Genome insertation replaced Plasmid](#title1-3)

◢
**Construction of ADC Drugs**

[Construction of ADC Drugs](#title2-0)
[Iteration of lactate-regulated promoters](#title2-1)

◢
**Future application**

[Future vision](#title3-0)
[Development of sensor proteins](#title3-1)
[Development of lactate-regulated promoters](#title3-2)
[Double AND gate logic-regulation of hypoxia promoter](#title3-3)

[**References**](#title4)

Our project mainly consists of two parts, namely the detection of chemotaxis module and ADC killing drug module.
The
chemotaxis module causes the bacteria to swim to the high lactic acid area of the tumor. The ADC drug module
expresses
ADC drugs under the control of different lactic acid concentrations to achieve the tumor killing effect.

#### Construction of the Chemotaxis modules

We proposed two ideas for the lactate chemotaxis module, one is to retain the extramembrane sensor part of the
*H. pylori*
lactate sensor, and the other is to retain the extramembrane and transmembrane parts of the *H. pylori*
lactate sensor protein.

#### Construction of fusion proteins

In the initial design of the experiment, we planned to directly introduce the fused sensor protein into
*Nissle1917*,
express this protein through a plasmid, and combine it with the corresponding signaling protein to induce
flagellar
movement. One of our fusion proteins is a fusion of the extramembrane sensor part of the lactic acid sensor
protein of
Helicobacter pylori and the transmembrane and intramembrane parts of the serine sensor protein of
*Nissle1917*.
The second
solution is to retain only the intramembrane part of *Nissle1917*, and the rest are from *Helicobacter
pylori*.

#### To form better combine

However, we noticed that the native sensor protein of *Nissle1917* may bind itself better, which will cause
our
fusion
protein to compete for ligands, which makes it difficult for our fusion protein to operate normally. Our initial
idea
was to let the bacteria overexpress these signal transduction proteins again, but this would cause greater
pressure on
the bacteria. Therefore, after many opinions, we decided to knock out the gene for the serine sensor protein of
*Nissle1917*.

When knocking out the serine sensor protein, we found that *Nissle1917* also has a homologous protein of
the
serine sensor
protein. Because it is not clear which type of biological membrane this protein is on, we knocked it out again.
The whole bacteria after knocking out the two sensor proteins did not show any difference in the growth status
of the
wild type.

![img](https://static.igem.wiki/teams/4617/wiki/engineering/deletion.png)

Figure 1: Deletion of Tsr and Tar

#### Genome insertation replaced Plasmid

*Nissle1917*, which contains a plasmid containing lactate sensor protein, has obvious lactate chemotaxis,
but the
wild-type strain does not show this property. However, we noticed that the plasmid would be lost during the
passage, and
the offspring *Nissle1917* would lose its ability to be deoxidized. Therefore, we decided to knock the
fusion
protein
directly into the genome of *Nissle1917*, which can ensure the stability of passage.

![img](https://static.igem.wiki/teams/4617/wiki/engineering/partinsert.png)

Figure 2: Plasmid expression

![img](https://static.igem.wiki/teams/4617/wiki/engineering/fusion.jpg)

Figure 3: Genome insertation

Finally, we preliminarily proved the role of lactate chemotactic protein through the disk method and capillary
method.

#### Construction of ADC Drugs

At first we decided to directly use a complete antibody for drug design, but we noticed that the complete
antibody did
not express well in the prokaryotic expression system, and its penetration ability was also poor, so we decided
to use a
single-chain antibody. The reduced molecular weight ensures tissue penetration and immunogenicity.

At the same time, we noticed that the single-chain antibody and PE38 of Pseudomonas aeruginosa chlorotoxin can
achieve
complementary advantages and offset each other's disadvantages.

#### Iteration of lactate-regulated promoters

Considering that there is no T7RNA polymerase in *Nissle1917*, we knocked in the T7RNA polymerase gene in
the
genome and
directly expressed it through the Trc strong promoter. At first, we wanted to directly use the T7 promoter to
control
drug expression, but the T7 promoter is a strong promoter, which would cause overexpression and risk of leaky
expression. Therefore, we hope to have sufficient expression intensity but also sufficient induction
sensitivity, so
that T7 RNA polymerase is also expressed under the induction of PlldR.
And finally achieve the induction effect of plldR-T7 promoter.

![img](https://static.igem.wiki/teams/4617/wiki/engineering/characterization-of-promoter-effects.png)

Figure 4: Characterization of promoter effects

#### Future vision

#### Development of sensor proteins

We plan to develop a complete set of sensor protein components that can be used in different scenarios. And it
can be
expanded from therapy to more areas of life.

![img](https://static.igem.wiki/teams/4617/wiki/engineering/chemotaxis-signal.png)

Figure 5: A complete of Biosensors[1]

#### Development of lactate-regulated promoters

During the experiment, we discovered that the promoter used was not ideal. Initially, we used the default T7
promoter,
but leaky expression was severe. For convenient control, we redesigned the promoter PlldR-T7 lactate-regulated
promoter.
The LldR protein belongs to the GntR regulatory family and is a gene involved in L-lactate metabolism in
*Escherichia
coli*.

There are two operating sites O1 and O2 at 25-54 bases upstream and downstream of the promoter. Complete
inhibition of
gntT is achieved through DNA circularization through the interaction between two GntR molecules.The upstream O1
is a key regulatory site. Its mutation will eliminate the induction effect of L-lactate, but the
mutation of the O2 site will not.

The PlldR-T7 promoter is a hybrid promoter of lldR protein and T7 promoter. The inducer is lactic acid, which is
universal and its mechanism is relatively clear. However, due to insufficient experimental time, our web page
has not
been able to display specific experimental data. We hope to show the results live in a subsequent presentation.

![img](https://static.igem.wiki/teams/4617/wiki/engineering/whole.jpg)

Figure 6: Exact data of promoter

![img](https://static.igem.wiki/teams/4617/wiki/engineering/whole1.png)

Figure 7: A series of Promoters [2]

#### Double AND gate logic-regulation of hypoxia promoter

This project focuses on lactate tropism, and the drug is also expressed under the regulation of lactate. At the
same
time, we noticed that high lactate may also be caused by strenuous anaerobic exercise.Therefore, the regulation
of future drugs can be expressed under a double AND gate logic, and another regulatory factor
can be hypoxia, which is also a characteristic of the tumor microenvironment.In other words, under the
expression regulated by lactate, whether there are other inhibitory factors that can prevent
its expression in other high-lactate environments, this is also something that needs to be explored in the
future.

![img](https://static.igem.wiki/teams/4617/wiki/engineering/future-induced.png)

Figure 8: Future And Gate induced Express

#### Reference

[1]Stock AM. A nonlinear stimulus-response relation in bacterial chemotaxis. Proc Natl Acad Sci U S A. 1999 Sep
28;96(20):10945-7. doi: 10.1073/pnas.96.20.10945. PMID: 10500102; PMCID: PMC34220.

[2]Meyer AJ, Segall-Shapiro TH, Glassey E, Zhang J, Voigt CA. Escherichia coli "Marionette" strains with 12
highly
optimized small-molecule sensors. Nat Chem Biol. 2019 Feb;15(2):196-204. doi: 10.1038/s41589-018-0168-3. Epub
2018 Nov
26. PMID: 30478458.

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# Experiments

Content

◢
**Chemotaxis Module**

[Chemoreceptor deletion](#title1-1)
[Expression of fusion proteins](#title1-2)
[Detection of Chemotaxis](#title1-3)

◢
**ADC Drugs**

[ADC Drugs](#title2-0)
[Lactate induced promoter](#title2-1)
[Construction of ADC Drugs](#title2-2)
[Testing of antibody drug conjugates](#title2-3)

[**References**](#title3)

#### Chemotaxis Module

#### Chemoreceptor deletion

We knocked out the gene sequences of Tsr and its homologous proteins that would cause competition, reducing the
competitiveness of binding. We measured their growth curves and found that there was no difference in the growth
curves
between the knockout strain and the wild-type strain.
![img](https://static.igem.wiki/teams/4617/wiki/experiment/sgrna.png)![img](https://static.igem.wiki/teams/4617/wiki/experiment/growth-curve.jpg)

Deletion of Tsr and growth-curve

#### Expression of fusion proteins

In the detection module, we expressed the fusion proteins eTlpC-A and eTlpC-B. This fusion protein has a gene
sequence
of approximately 1899 bp. The pET expression vector system is a widely used system for recombinant protein
expression in
*E. coli*. The system is equipped with a phage T7 promoter, which can regulate the transcription and
translation
of the
target gene. However, *EcN* lacks the gene for T7 RNA polymerase, which makes the original pET-21a system
unusable
in EcN.

Therefore, we used the Trc promoter that can be expressed in *EcN* to replace the T7 promoter on the
original
pET-21a
system to initiate the expression of downstream eTlpC. At the same time, we deleted the lactose operon repressor
protein
site on pET-21a, thereby achieving constitutive expression of eTlpC within the *EcN*.[1]

![img](https://static.igem.wiki/teams/4617/wiki/experiment/3dstructure.png)

3D structure of TlpC

![img](https://static.igem.wiki/teams/4617/wiki/experiment/tlpca.png)

The plasmid of eTlpc

#### Detection of Chemotaxis

When we introduced the constructed fusion protein into the gene-edited Nissle1917, Nissle1917 became
chemotactic. After
it grows to a certain stage, we inoculate the bacterial liquid into a new glycerol salt medium. After it grows
to
od600=0.4, take 10ml of the bacterial liquid and centrifuge it at 4°C for 10 minutes.

Pour the supernatant and add 5 ml of washing medium and gently resuspend. Centrifuge again, resuspend in 5 ml
of washing
medium, and centrifuge again. After pouring off, resuspend gently in remaining liquid. Observed with a
microscope, you
can see that more than 90% of bacteria can flow. Add 10 ml of chemotaxis medium to the resuspended culture
medium to
bring the bacterial concentration to 6\*107/ml.

![img](https://static.igem.wiki/teams/4617/wiki/experiment/capii.png)

Capillary method[2]

![img](https://static.igem.wiki/teams/4617/wiki/experiment/disc-test-method.png)

Disc test method

**(Preparation of chamber)**
Melt and bend a 5cm long capillary tube to form a U-shape. Fix the U-shaped tube on the microscope slide and
fill the
chamber with 200ul of bacterial solution. Insert the capillary containing the configured attractants of
different
concentrations, incubate for one hour, and directly move the slide to the microscope for observation and
recording.

#### ADC Drugs

In the ADC module, we use lactate-regulated promoter to express ADC drugs to kill tumor cells.

#### Lactate induced promoter

The lactate-regulated promoter LldR protein belongs to the GntR regulatory family and is a gene involved in
L-lactate
metabolism in Escherichia coli. There are two control sites, O1 and O2, at 25-54 bases upstream and
downstream of the
promoter. Complete inhibition of gntT is achieved through DNA circularization through the interaction
between two GntR
molecules, of which the upstream O1 is the key. Mutation of the regulatory site will eliminate the induction
effect of
L-lactate, but mutation of the O2 site will not.

When the LldR protein is not bound to lactate, the LldR protein will bind to the operator site and prevent
transcription
from proceeding. When LldR binds to a lactic acid molecule, it disengages from the binding site, allowing
transcription
to proceed smoothly.[3]

![img](https://static.igem.wiki/teams/4617/wiki/experiment/plldr.png)

The sequence of PlldR promoter

#### Construction of ADC Drugs

Amino acid position 106 of the light chain of trastuzumab is the light chain variable region, and amino acid
position
118 of the heavy chain is the heavy chain variable region, which are retained. Different attempts were made
to connect
single-chain antibodies to PE38 using (EAAAK)2 and (GGGGS)3. (EAAAK)2 is a rigid connecting peptide that
will form a
stable α-helical structure, while (GGGGS)3 is a flexible connection Peptides.

In order for the ADC drug to be secreted out of the cell smoothly and play its role, an *E. coli*
signal
peptide is used
at the N-terminus of the protein to mediate the drug. Using a signal peptide that mediates secretion from
the Sec/SPI
pathway, we used an established signal peptide, Signal Peptide Website ID: 240.[4]

![img](https://static.igem.wiki/teams/4617/wiki/experiment/scfveak2.png)

alphaFold2 predicted structure of scFv-EAK2-PE38

![img](https://static.igem.wiki/teams/4617/wiki/experiment/scfvgs3.png)

alphaFold2 predicted structure of scFv-GS3-PE3

Since the N-terminal REDLK sequence of PE38 is related to protein transport, in order to avoid damaging the
C-terminal
structure of the PE38 domain, a signal peptide was inserted into the N-terminal of the fusion protein. The
inserted
protein was analyzed by Signal P4.1 and its D score was 0.862 greater than 0.5, so it can be considered to
have
secretory activity. And when it is secreted out of the cell, it is hydrolyzed by leader peptidase (Lep)
between
positions 21 and 22.

#### Testing of antibody drug conjugates

Use Nco I and Hind III restriction sites to connect the ADC drug to the expression vector pET-28a, and
transform it into
E.coli BL21 strain for expression. IPTG was used to induce its expression, and SDS-PAGE was used to extract
the strain
fermentation supernatant and cells for testing. It was purified using ion exchange chromatography and tested
using MTT
staining. HER2-positive cell lines and HER2-negative cell lines were used for control tests.[5]

![img](https://static.igem.wiki/teams/4617/wiki/experiment/example.png)

We cultured these strains and detected the expression of fluorescent protein with fluorescence intensity to
judge the actual effect of adding function modules (leghemoglobin, laccase or both) on the intracellular
oxygen concentration. We judge the concentration of bacteria in the liquid culture medium by the data of
bacterial liquid absorbance (i.e. OD value) to reflect the growth of bacteria. In order to reduce the chance
of
the experiment, we set up three parallel experiments for each group of data, and finally took the average
value
of the three groups as the experimental data of the sampling time of this group.([Click here to know about result](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27results%27%29%20%7D%7D))

#### References

[1] Callahan AM, Parkinson JS. Genetics of methyl-accepting chemotaxis proteins in
Escherichia coli: cheD mutations affect the structure and function of the Tsr transducer.
[J] J Bacteriol. 1985 Jan;161(1):96-104.

[2] Adler J. A method for measuring chemotaxis and use of the method to determine optimum conditions for
chemotaxis by
Escherichia coli. J Gen Microbiol. 1973 Jan;74(1):77-91. doi: 10.1099/00221287-74-1-77. PMID: 4632978.

[3] Chien T, Harimoto T, Kepecs B, Gray K, Coker C, Hou N, Pu K, Azad T, Nolasco A, Pavlicova M, Danino T.
Enhancing the
tropism of bacteria via genetically programmed biosensors. Nat Biomed Eng. 2022 Jan;6(1):94-104. doi:
10.1038/s41551-021-00772-3. Epub 2021 Jul 29. PMID: 34326488; PMCID: PMC8956018.

[4] Rogers LM, Veeramani S, Weiner GJ.Complement in monoclonal antibody therapy of cancer[J]. Immunol Res.
2014
Aug;59(1-3):203-10.

[5] https://bio-protocol.org/e374

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# Results

Content

[**Overview**](#title1)
◢
**Chemotaxis Module**

[Two ways of Chemotaxis evaluation](#title2-0)

[**ADC Drugs module**](#title3)

#### Two ways of Chemotaxis evaluation

Lactic acid chemotaxis experiments were performed using semi-solid medium, and wild-type EcN and EcN-SY were
used for
experiments.
In control experiments, wild-type EcN did not produce chemotactic circles on paper sheets infiltrated with
lactic acid
or 0.9% NaCl. However, EcN-SY produces a white chemotactic circle on the lactic acid-infiltrated paper piece,
and EcN-SY
does not produce a chemotactic circle on the 0.9% NaCl-infiltrated paper piece. The influence of the paper piece
itself
can be eliminated, and the test is identified as lactic acid chemotaxis positive.

![img](https://static.igem.wiki/teams/4617/wiki/etlpc10.jpg)
![img](https://static.igem.wiki/teams/4617/wiki/etlpc11.jpg)

Figure 1: Test of Chemotaxis in medium

![img](https://static.igem.wiki/teams/4617/wiki/results/results2.jpg)

Figure 2: Test of Chemotaxis in capillary tube

#### ADC Drug Module

Waiting for update results on the spots.

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