iGEM 2019 — Team NEFU_China

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# Team:NEFU China/Notebook

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Project

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Lab

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* [Safety](https://2019.igem.org/Team%3ANEFU_China/Safety)

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* [Overview](https://2019.igem.org/Team%3ANEFU_China/Human_Practices#overview)
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Notebook

* 9/12/2018-13/12/2018

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  \*A teach-in was held about 2018 NEFU\_China.
  \*Recruitment of new members.
* 14/12/2018

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  \*Set up the 2019 NEFU\_China iGEM team.
* 15/12/2018

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  \*We held our first iGEM meeting.
* 16/12/2018-15/2/2019

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  \*We learned from excellent cases in the previous years.
  \*Brainstorm for the team project.
  \*Team registration.
* 15/2/2019- 15/3/2019

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  \*Searching for documents and references about bacterial cancer therapy.
* 16/3/2019-23/3/2019

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  \*Experimental skills training for the new team.
* 24/3/2019-29/4/2019

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  \*Perfecting our case.
  \*Adding the plasmid protection system.
* 31/4/2019-31/5/2019

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  \*We made preparation for the experiment.
  \*We ordered a strain.
* 1/6/2019

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  \*First day in the lab.
* 2/6/2019-5/6/2019

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  \*We got our first strain.
* 6/6/2019-20/6/2019

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  \*A simple device was developed for the hypoxia experiment.
* 21/6/2019-30/6/2019

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  \*Hypoxia-induced promoter worked out.
* 1/7/2019-18/7/2019

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  \*Uric acid-induced promoter worked out.
* 19/7/2019-20/7/2019

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  \*We attended the iGEM Jilin University Meetup.
  \*In-depth communication with other teams.
* 22/7/2019

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  \*Selecting members to go abroad by a public vote.
* 30/7/2019-31/7/2019

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  \*Meeting with Prof. Guangchao Sui.
* 1/8/2019-15/8/2019

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  \*We built mathematical models.
* 22/8/2019-24/8/2019

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  \*Human Practice : we used an internet-based questionnaire to quiz people about bacteria cancer therapy.
  \*Nearly 1000 people participated in the survey via questionnaires over the Internet.
* 25/8/2019

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  \*Bacterial cancer therapy was determined as the final case.
* 26/8/2019-30/8/2019

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  \*We began preparing our Wiki.
  \*We designed our posters and uniforms.
* 26/8/2019-30/9/2019

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  \*We cooperated with 2019 OUC\_China team so as to ensure the reproducibility of the experiments.
* 1/9/2019

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  \*Reorganizing tasks to accomplish them efficiently.
* 7/9/2019

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  \*We went to Harbin Pharmaceutial Group to investigate the application of our case.
* 12/9/2019

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  \*A lecture was held in Harbin No.6 Middle School to let students share the ideas about iGEM.
* 14/9/2019

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  \*Laboratory Open Day.
  \*Students in Harbin No.6 Middle School went to our laboratory
* 20/9/2019

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  \*We have constructed the plasmids pYB1a and pRB1s which work in the final pathways.
* 25/9/2019

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  \*We interviewed Professor Shuling Liu, an disease expert, in Harbin Medical University to ask some adivice on our case.
* 27/9/2019

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  \*We have knocked out the gene *alr* in *E.coli BW*.
* 7/10/2019

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  \*Another gene *dadX* has been knocked out in *E.coli BW*.
* 9/10/2019

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  \*Target and self-regulation module has been completed.
* 14/10/2019

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  \*Tumor microenvironment sensor module has been completed.
* 15/10/2019

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  \*Uric acid regulation module has been completed.

# Sponsors

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# Contact us

### iGEM-NEFU\_China2019

### E-mail:nefu\_igem2019@163.com

### No.26 Hexing Road, Xiangfang District, Harbin, Heilongjiang Province 150040

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# Team:NEFU China/Experiments

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HUMAN PRACTICES

[Human Practices](https://2019.igem.org/Team%3ANEFU_China/Human_Practices)
[Education & Engagement](https://2019.igem.org/Team%3ANEFU_China/Public_Engagement)

AWARDS

[Entrepreneurship](https://2019.igem.org/Team%3ANEFU_China/Entrepreneurship)
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# Experiments

Describe the research, experiments, and protocols you used in your iGEM project. These should be detailed enough for another team to repeat your experiments.

Please remember to put all characterization and measurement data for your parts on the corresponding Registry part pages.

### What should this page contain?

* Protocols
* Experiments
* Documentation of the development of your project

### Inspiration

* [2014 Colombia](https://2014.igem.org/Team%3AColombia/Protocols)
* [2014 Imperial](https://2014.igem.org/Team%3AImperial/Protocols)
* [2014 Caltech](https://2014.igem.org/Team%3ACaltech/Project/Experiments)

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# Team:NEFU China/Results

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Project

* [Description](https://2019.igem.org/Team%3ANEFU_China/Description)
* [Design](https://2019.igem.org/Team%3ANEFU_China/Design)
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Parts

* [Basic](https://2019.igem.org/Team%3ANEFU_China/Basic_Part)
* [Composite](https://2019.igem.org/Team%3ANEFU_China/Composite_Part)
* [Improvement](https://2019.igem.org/Team%3ANEFU_China/Improve)

[Results](https://2019.igem.org/Team%3ANEFU_China/Results)

Lab

* [Experiments](https://2019.igem.org/Team%3ANEFU_China/Demonstrate)
* [Protocol](https://2019.igem.org/Team%3ANEFU_China/Protocol)
* [Notebook](https://2019.igem.org/Team%3ANEFU_China/Notebook)
* [Safety](https://2019.igem.org/Team%3ANEFU_China/Safety)

[Model](https://2019.igem.org/Team%3ANEFU_China/Model)

[HP](https://2019.igem.org/Team%3ANEFU_China/Human_Practices)

* [Overview](https://2019.igem.org/Team%3ANEFU_China/Human_Practices#overview)
* [Education & Engagement](https://2019.igem.org/Team%3ANEFU_China/Human_Practices#edu)
* [Community](https://2019.igem.org/Team%3ANEFU_China/Human_Practices#community)

[Judging](https://2019.igem.org/Team%3ANEFU_China/Judging)

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Results

### [1. Tumor Microenvironment Sensor Module](https://2019.igem.org/Team%3ANEFU_China/Results#tumor_micro)

#### [Hypoxia Promoter Selection](https://2019.igem.org/Team%3ANEFU_China/Results#Hypoxia)

#### [Dual-sensing Promoter](https://2019.igem.org/Team%3ANEFU_China/Results#Dual)

### [2. Target and Self-regulation Module](https://2019.igem.org/Team%3ANEFU_China/Results#targeting)

#### [Magnetic Targeting Cassette](https://2019.igem.org/Team%3ANEFU_China/Results#targeting)

#### [Inducing Suicide Cassette](https://2019.igem.org/Team%3ANEFU_China/Results#cd)

#### [Activity of ECF20 & Anti-ECF20](https://2019.igem.org/Team%3ANEFU_China/Results#ecf)

### [3. Uric Acid Regulation Module](https://2019.igem.org/Team%3ANEFU_China/Results#uric_acid)

#### [Phuc＊ Screening](https://2019.igem.org/Team%3ANEFU_China/Results#phuc)

#### [Characterization of Uricase](https://2019.igem.org/Team%3ANEFU_China/Results#cha)

### [4. Tumor Killer Module](https://2019.igem.org/Team%3ANEFU_China/Results#tumor_killer)

### [5. Plasmid Protection Module](https://2019.igem.org/Team%3ANEFU_China/Results#plasmid)

### Tumor microenvironment sensor module

#### Description

To ensure our therapeutic system can work only when reaching tumor cells, we chose two characteristics of tumor microenvironment, hypoxia [1] (dissolved oxygen is less than 1%) and high lactic acid [2] levels as the detection indexes of our sensor module.

We selected the hypoxia-inducible promoter better respond to the tumor hypoxia environment. We use sodium sulfite to make hypoxia environment during experiment. When oxygen level is at a normal range in the internal environment of human body, the transcription factor FNR [3] will associate with oxygen, leading to its impeded binding to the hypoxia-inducible promoter, and reduce the transcription of its downstream gene. However, in hypoxic environment of tumors, FNR will disassociate with oxygen and activate its downstream gene.

The natural operon repressed by lldR consists of two operators (O1 and O2) and a promoter that is intercalated between the operators. It regulates the expression of the polycistronic lldPRD, involved in L-lactate metabolism. In the absence of lactate, two lldR dimers bind to the operator sites in the lldPRDp promoter and form a homo-tetramer, sequestering the DNA and preventing transcription of the operon. When lactate enters the bacteria via the glycolate permease (GlcA) or lldP, it interacts with the lldR regulator protein. When this interaction happens, the lldR dimer on the O2 site will dissociate, but the dimer bound to the O1 site will become a transcriptional activator to promote the transcription of the operon [4].

We replaced the promoter of the natural operon of lldR with a hypoxia responsive promoter to construct a promoter that can respond to both hypoxic and high lactate signals. In normal tissue with relatively high levels of oxygen and low levels of lactic acid, the lldR molecules will form a homo-tetramer and the binding of the transcription factor FNR to the hypoxia-inducible promoter will be impeded, leading to repressed expression of the downstream gene. In tumor microenvironment with relatively low levels of oxygen and high levels of lactic acid, lldR will be released from the O2 site and the FNR transcription factor can bind to the [PfnrF8](http://parts.igem.org/Part%3ABBa_K2913009) promoter, leading to the expression of the downstream gene.

![](https://static.igem.org/mediawiki/2019/8/83/T--NEFU_China--lac1.png)

Fig. 1 a. When lactic acid is not present, lldR binds the O1 and O2 sites. Depending on the affinity of these two sites to lldR (the O1 site is greater than the O2 site), lldR will bind to the O1 site first and the O1 site binding of lldR may facilitate the interaction between new lldR molecules and the O2 site. At this point, lldR proteins form homo-tetramer that causes the DNA strains to form a stem-loop structure and prevent the RNA polymerase from binding to the promoter sequence region, leading to repressed transcription [5]. b. When oxygen level is in a normal range in the internal environment of normal tissues of a human body, the transcription factor FNR will associate with oxygen, leading to its impeded binding to the hypoxia-inducible promoter and thus reduce the transcription of its downstream gene. In the hypoxic environment of tumors, FNR will disassociate with oxygen and activate its downstream gene.

#### Methods

##### Hypoxia parts

With the hope to find a hypoxia-inducible promoter which can only activate its downstream gene regularly in a hypoxic environment, we inserted the lacZ gene at downstream of the hypoxia-inducible promoter and evaluated β-galactosidase activity by measuring o-Nitrophenyl β-D-galactopyranoside (ONPG) conversion. We used a series of concentrations of sodium sulfite (0, 0.25, 0.5, 0.75 and 1 g/l Na2SO3) to create an hypoxia environment and cultured the bacteria for 6-8 h at 16 ℃ in a shaker. We recorded the absorbance at 420 nm for each well every 30 s in an automatic microplate reader at 37 ℃ 30 min. The experiments of each group were repeated for at least 3 times.

##### High lactate & Hypoxia parts

To test the effectiveness of the promoter, Phll, that responds to both hypoxia and high lactate signals created by us, we provided conditions with low oxygen and high lactic acid levels to check if the element worked. We inserted the lacZ gene at downstream of the [Phll](http://parts.igem.org/Part%3ABBa_K2913023) regulating promoter to detect β-galactosidase activity by ONPG conversion. In order to prevent the growth inhibition of bacteria by excessively low pH, we used sodium lactate instead of lactic acid for the promoter induction. We used sodium sulfite (1 g/l Na2SO3) and a series of concentrations of sodium lactate (10, 25, 50, 200 and 300 mM) to create a hypoxia and high lactic acid environment. We cultured bacteria for 8-12 h at 16 ℃ in a shaker and recorded the absorbance at 420 nm for each well every 30 s using an automatic microplate reader at 37 ℃ for 1-2 h. Each group were repeated for at least 3 times.

#### Results

##### Hypoxia parts

We used different concentrations of sodium sulfite (0, 0.25, 0.5, 0.75 and 1 g/l Na2SO3) to make an hypoxia environment (Fig. 2a). Being driven by the PfnrF8 [6] promoter, the activity of LacZ could markedly increase between 0 g/l and 1 g/l of Na2SO3 (Fig. 2c). Thus, this promoter can respond to our hypoxia environment regularly. Comparing with a control promoter FF+20 (with mutations at the hypoxia resposive element; it was reported to respond to the hypoxia condition, but it did not show this response in our experimental setting), the downstream gene of the inducible promoter showed much higher expression (Fig. 2d). We will further improve the hypoxia sensitivity of the PfnrF8 promoter in the future.

![](https://static.igem.org/mediawiki/2019/a/a3/T--NEFU_China--lac2.png)

Fig. 2a. a. Dissolved oxygen changed curve under a series of concentrations of sodium sulfite (0, 0.25, 0.5, 0.75 and 1 g/l Na2SO3). b. Hypoxic induction of the control promoter FF+20 (with mutations at the hypoxia resposive element) in *E. coli Nissle 1917*. Expression of LacZ varied in different sodium sulfite concentrations (0, 0.25, 0.75 and 1 g/l Na2SO3). β-gal activity was measured as the method described in text. Values are expressed as percentage of the promoter activity at 1 g/l Na2SO3. c. Hypoxia induction of the PfnrF8 promoter in *E. coli Nissle 1917*. Expression of LacZ shows the Na2SO3 concentration dependence in increasing sodium sulfite concentrations (0, 0.25, 0.5, 0.75 and 1g/l Na2SO3). β-gal activity was measured as described above. Values are expressed as percentage of the promoter activity of the positive control (a mutant of the PfnrF8 promoter with strong expression and irresponsive to hypoxia induction). d. The comparison of hypoxia inductions of the PfnrF8 promoter and FF+20 promoter (with mutations at the hypoxia resposive element) in *E. coli Nissle 1917*. Expression of LacZ was presented with increasing sodium sulfite concentrations (0, 0.25, 0.75 and 1 g/l Na2SO3). β-gal activity was measured as described above. Values are expressed as percentage of the promoter activity of a positive control (a mutant of the PfnrF8 promoter with strong expression and irresponsive to hypoxia induction). Error bars represent the standard deviations.

##### High Lactate & Hypoxia Parts

We compared the response of the Phll promoter to hypoxia and normal oxygen environments after adding sodium lactate of different concentrations (0, 10, 25, 50, 200, 300 mM) (Fig. 3b). We expected that β-galactosidase expression would be higher when culturing the bacteria in medium without sodium lactate and sodium sulfite, because the bacterial growth at this condition was better than that in the medium with these two chemicals. Under the conditions of hypoxia and high lactic acid, the expression of β-galactosidase was significantly increased, which indicated that the Phll promoter was effective.

![](https://static.igem.org/mediawiki/2019/5/56/T--NEFU_China--lac3.png)

Fig. 3a. a. In an environment of normal oxygen and low lactic acid, the lldR molecules would form a homo-tetramer, and the binding of the transcription factor FNR to the hypoxia-inducible promoter would be impeded, leading to repressed expression of the downstream gene. In an environment of hypoxia and high lactic acid levels, lldR would be released from the O2 site and thus the transcription factor can bind to the PfnrF8 promoter, leading to the expression of downstream gene. b. LacZ expression in *E. coli Nissle 1917* was induced by different concentrations of sodium lactate (0, 10, 25, 50, 200 and 300 mM) with (+) or without (-) 1 g/l Na2SO3. β-gal activity was measured as described above.

### Targeting and Self-regulation Module

#### Magnet targeting cassette:

#### Description

In order to enhance the tumor-targeting specificity of our engineered bacteria, we used a mutant form of ferritin, ftnA-M, to construct its expression cassette and integrate it into the genome of bacteria. Theoretically, bacteria expressing ftnA-M should respond to a magnetic field and aggregate to the tumor site if it is exposed to a magnet. In this case, we can reduce the number of bacteria when it is possibly used to treat cancer patients and minimize the potential adverse effects of bacteria on humans.

Actually, endogenous ferritin of *E. coli* creates an inner cavity to store iron in a hydrated amorphous form of iron oxide that is biocompatible and magnet responsive depending on its crystal structure. However, the mineralized iron stored inside natural ferritin exhibits poor crystallinity that facilitates iron release when needed but shows very modest inherent magnetic moment.

Based on previous literature, ftnA-M is a mutant form of ferritin harboring amino acid substitutions of H34L and T64I, which located at the “B-channel” (Fig. 4, marked by a red circle) responsible for transporting iron. These mutations could likely enlarge the B-type channel and could enhance the ferromagnetism. [7]

![](https://static.igem.org/mediawiki/2019/5/50/T--NEFU_China--ma1.png)

Fig. 4. The three-dimensional structure of the ftnA-M protein[7]

To verify the increased magnetotactic ability of the *E. coli* expressing ftnA-M, we carried the following experiments:

We constructed an expression cassette with the P-BAD promoter driving ftnA-M expression as shown in Fig. 5.

![](https://static.igem.org/mediawiki/2019/1/1a/T--NEFU_China--ma2.png)

Fig. 5. Diagram of the expression cassette for ftnA-M in PYB1a plasmid.

#### Methods

1. We used the sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) to analyze protein expression levels of ftnA-M in the engineered *E. coli BW2 5113*:

(1) We transformed the PYB1a (with or without ftnA) into *E. coli BW 25113*

(2) To induce the expression of ftnA-M in bacteria, we added arabinose to a final concentration of 0.2 % in LB medium at the log-phase growth of the bacteria (OD600 about 0.4), and simultaneously supplied the medium with 100 μM of Fe2SO4.

(3) We then continuously cultured the bacteria for another 6 h at 16 ℃ in a shaker.

(4) We collected the bacteria by centrifugation and lyzed the bacteria using sonication. We mixed an equal volume of the bacterial lysates and 2xSDS loading buffer, and heated the sample at 100 ℃ for 5 min. The samples were then analyzed by 10 % acrylamide of SDS-PAGE.

The result of SDS-PAGE for ftnA-M expression is shown in Fig. 7.

2. A neodymium magnet (also known as NdFeB, NIB or Neo magnet) was used because it is the most widely used rare-earth magnet with a permanent magnet made from an alloy of neodymium, iron and boron to form the Nd2Fe14B tetragonal crystalline structure). We employed to verify the increased magnetotactic ability of the engineered *E. coli* BW25113 expressing ftnA-M:

(1) We induced expression of ftnA-M protein as described above (the steps of (1) to (3) in 1).

(2) After the induction, the bacteria were centrifuged and resuspended in the M9 liquid medium to a density of OD600 at about 0.4.

(3) For further bacterial culture, 0.2 % arabinose and 100 μM Fe2SO4 were still present in the medium to maintain the induction. We then set the RbFeB magnet between the two 50 ml tubes containing the experimental bacteria (expressing ftnA-M) and the control bacteria (without the ftnA-M cassette) (Fig. 6). The tubes and the magnet were left at 37 °C for 24 h.

![](https://static.igem.org/mediawiki/2019/0/0d/T--NEFU_China--ma3.png)

Fig. 6. The experiment setting to determine the magnetotactic ability of engineered bacteria. The neodymium magnet was set between the two 50 ml tubes containing the bacteria expressing ftnA-M (right) and the bacteria without ftnA-M expression (left). We set the white area of the tube facing to the neodymium magnet.

The result verifying the magnetism of bacteria is shown in Fig. 8.

#### Results

1.The result of SDS-PAGE indicated that the ftnA-M protein was expressed upon the induction of arabinose.

![](https://static.igem.org/mediawiki/2019/8/8c/T--NEFU_China--ma4.png)

Fig. 7. SDS-PAGE analysis to evaluate protein expression of ftnA-M induced by arabinose. Lane 1: *E. coli BW 25113*, Lane 2: ftna-M/*E. coli BW 25113*. Lane 3: protein molecular weight markers.

2. The engineered *E. coli* showed the aggregation at the side of the magnet at the slope bottom of the tube while the control group of *E. coli BW 25113* did not show obvious aggregation.

![](https://static.igem.org/mediawiki/2019/2/20/T--NEFU_China--ma5.png)

Fig. 8. Image of cells cultured in the M9 liquid medium for 24 h. 0.2 % arabinose, 100 μM Fe2SO4 as supplements were added and the neodymium magnet was set between the 50 ml tubes of experimental group (right) and control group (left). We set the white area of the tube facing to the neodymium magnet.

#### Inducing Suicide Cassette

#### Description

The target and self-regulation part helps the bacteria survive in the microenvironment of tumors, and clear them in normal tissues. We designated the functions above as a suicide system.

Our plasmid can express the cytosine deaminase (CD) in the normal tissue, but stop the expression when sensing the signals of hypoxia and high lactic acid levels in the tumor microenvironment. Here, we control the survival of bacteria by adding 5-Fluorocytosine (5-FC), a chemical without noticeable adverse effects to human.

![](https://static.igem.org/mediawiki/2019/a/a3/T--NEFU_China--t1.png)

Fig. 9. CD transforms 5-Fluorocytosine into 5-fluorouracil.

The bacteria were engineered to express CD that can convert 5-FC, a nontoxic nucleoside analog with antifungal activities, into a highly cytotoxic chemical, 5-fluorouracil (5-FU), which is a common anticancer drug. As a result, bacteria transformed with the CD gene expression cassette can be adversely affected by 5-FC, leading to their growth arrest or death [8].

#### Experiments

##### Experimental design

![](https://static.igem.org/mediawiki/2019/f/ff/T--NEFU_China--t2_2.png)

Fig. 10. The schematic diagram showing the cassette to express CD. PYB1a and PRB1s are the plasmids with different selection gene. ECF20 is the factor σ for the promoter Pecf20.

We tested the engineered bacteria (BW 25113) carrying a cassette of constitutive CD expression.

We cultured the engineered bacteria with a concentration gradient of 5-FC in the LB-medium containing ampicillin and streptomycin. This experimental setting imitated the situation of normal tissues. For comparison, another set of experiment was designed with a concentration gradient of 5-FU (5- Fluorouracil) in the LB-medium containing ampicillin and streptomycin. We monitored them in 96-well plates in order to measure growth of bacteria continuously and generated their growth curves.

##### Pre-experiments

We conducted preliminary experiments to choose the best constitutive promoter. We tested 3 different types of promoters available to us: the P-Anderson promoter, T7 promoter and CPA1 promoter. We cultured the engineered bacteria with gradient concentrations of 5-FC and 5-FU (0, 100 and 500 mg/l) to determine the promoter that could express the highest level of CD.

![](https://static.igem.org/mediawiki/2019/9/92/T--NEFU_China--t3.png)

Fig. 11. Black curves represent the growth of bacteria in response to different concentrations of 5-FC. Culture the engineered bacteria after inoculating bacteria in LB medium with a concentration gradient of 5-FC (0, 100 and 500 mg/l) in the LB medium containing ampicillin and streptomycin. In a, b and c, the growth curves of bacteria using the Anderson promoter, the T7 promoter and the CPA1 promoter, respectively, to drive CD expression in different 5-FC concentrations. d. The growth curve of original bacteria without the capability of expressing CD in different 5-FC concentrations. Red curves represent the growth of bacteria in response to different 5-FU concentrations. Culture the engineered inoculating bacteria in LB medium with a concentration gradient of 5-FC (0, 100 and 500 mg/l) and ampicillin/streptomycin. In a, b and c, the growth curves of the bacteria with the Anderson promoter, the T7 promoter and the CPA1 promoter, respectively, to drive CD expression in different 5-FU concentrations. d. Growth curves of the original bacteria without CD expressing capability in different 5-FU concentrations.

There was not significant difference among these promoters based on the growth curves. We can know from the graphs above that adding 5-FU can clearly inhibit the growth of the bacteria.

##### Selection of the promoters

We also checked the protein expression of CD using SDS-PAGE.

![](https://static.igem.org/mediawiki/2019/6/61/T--NEFU_China--t4.png)

Fig. 12. SDS-PAGE analysis to evaluate protein expression of CD. Lane 1: the original bacteria without CD expression plasmid. Lanes 2, 3 and 4: The bacteria with CD expression driven by the Anderson promoter (2), the T7 promoter (3) and the CPA1 promoter(4). Lane 5: protein molecular weight standard.

The results indicated that the T7 promoter could express the highest CD level among the three promoters. Thus, we chose the T7 promoter to drive CD expression in this part.

##### Test and verification

We cultured the engineered bacteria with the T7 promoter driving CD expression.

![](https://static.igem.org/mediawiki/2019/8/83/T--NEFU_China--t5.png)

Fig. 13. Growth of E.coli at different concentration of 5-FC. Culture the bacteria after adding bacteria quantified to LB medium with a concentration gradient of 5-FC (0, 25, 50, 100, 250 and 500 mg/l) in the LB medium containing ampicillin and streptomycin . A. The bacteria (*E.coli*) using T7 to express CD. B. The bacteria (*E.coli*) can’t express CD.

The presence of 5-FC showed an obvious suppression to the growth of the bacteria in a concentration dependent manner. With 500 mg/l of 5-FC, the bacteria hardly proliferate. With a 5-FC concentration of 50 mg/l, an effective medicine level in the human body, 70 % bacteria could be killed.

We also observed the growth of the bacteria with a concentration gradient of 5-FU in the LB-medium. The results are presented in Fig. 14.

![](https://static.igem.org/mediawiki/2019/8/80/T--NEFU_China--t6.png)

Fig. 14. Growth of bacteria at different concentrations of 5-FU. Culture the bacteria after adding bacteria quantified to LB medium with a concentration gradient of 5-FC (0,25,50,100,250,500mg/l) in the LB medium containing ampicillin and streptomycin. A. The bacteria using T7 express CD. B. The original bacteria that without the CD expression plasmid.

We can see that the addition of 5-FU can almost suppress the growth of the bacteria at all.

##### Conclusion

Based on the experimental data presented above, the growth of our engineered bacteria can be repressed by 5-FC effectively. We can get the conclusion that target and self-regulation part can assistant bacteria to survive in the tumor environment, but eliminate them if they are transported to normal tissues.

### Activity of ECF20 & Anti-ECF20

#### Description

![](https://static.igem.org/mediawiki/2019/1/12/T--NEFU_China--ecf20_1.png)

Fig. 15 ECF20 is a kind of Sigma factors that are the subunits of the bacterial RNA polymerase (RNAP) and play critical roles in transcription initiation, including recognizing and opening the promoters, and synthesizing RNA at the initial steps a. We used EGFP as a report gene to test the activity of the ECF20 sigma factors and its corresponding promoter Pecf20. Anti-ECF20 is a repressor of ECF20, which can inhibit ECF20 b. We also tested its function using this cassette.

![](https://static.igem.org/mediawiki/2019/f/f6/T--NEFU_China--ecf20_2.png)

Fig. 16a. Plasmids pYBIa-ECf20 & pRB1s-Pecf20-EGFP were transformed into *E. coli Nissle 1917*. When arabinose (Ara) was added into the medium, the promoter P-BAD could be activated to express ECF20 that would activate the promoter Pecf20 to drive EGFP expression. b. Plasmids pYB1a-ECf20, pRB1s-Pecf20-EGFP and pSB1c-Plac-AS20 were transformed into *E. coli Nissle 1917*. The promoter Plac could be activated by isopropyl β-D-1-thiogalactopyranoside (IPTG) to express Anti-EC20 that would bind to ECF20 to suppress the expression of EGFP.

#### Methods

##### ECF20 activity

(1) Transform the plasmids into *E. coli Nissle 1917*.

(2) Bacteria were inoculated in LB medium containing 2 g/l of glucose and cultured for 12 h at 37℃.

(3) Inoculate the cultured bacteria into 5 ml fresh LB medium containing 0.5 mM Ara and 0.4 g/l glucose.

(4) Culture the bacteria for 8 h and detect the expression of GFP under a fluorescence microscope.

(5) Culture the bacteria at 37 ℃, detect OD600 and measure relative florescent signal every 10 minutes using an automatic microplate reader.

##### Anti-ECF20 activity

(1) Transform the plasmids into *E. coli Nissle 1917*.

(2) Inoculate bacteria in LB medium containing 2 g/l glucose and culture the bacteria for 12h at 37 ℃.

(3) Inoculate the cultured bacteria by 1/100 dilution into 5 tubes, each of which held 5 ml fresh LB medium containing 0.5 mM Ara and 0.4 g/l glucose. Add IPTG to these tubes with final concentrations of 0, 1, 5, 10, and 20 mM.

(4) Culture the bacteria in these tubes at 37 ℃ and measure OD600 and relative florescence unit (RFU) every 10 min using an automatic microplate reader.

#### Results

![](https://static.igem.org/mediawiki/2019/d/de/T--NEFU_China--ecf20_3.png)

Fig. 17a. Green fluorescence intensity of produced GFP driven by the Pecf20 promoter. Plasmids pYBIa-ECf20 & pRB1s-Pecf20-EGFP were transformed in to *E. coli Nissle 1917*, and the bacteria were cultured at 37 °C in medium containing 0.5 mM Ara and 0.4 g/l glucose. OD600 and green fluorescent signal were measured every 10 min using an automatic microplate reader.
b. Green fluorescence intensity of produced GFP to determine the antagonism of Anti-ECF20 to the Pecf20 promoter. Plasmids pYB1a-ECf20, pRB1s-Pecf20-EGFP and pSB1c-Plac-AS20 were individually transformed into *E. coli Nissle 1917*, and the bacteria were cultured in LB medium containing 0.5 mM Ara and 0.4 g/l glucose. IPTG was added to different final concentrations of 0, 1, 5, 10 and 20 mM. The bacteria were then cultured at 37℃ with the measurement of OD600 and green fluorescent signal every 10 min using an automatic microplate reader.

In Fig. 17a, when pYB1a-p-BAD-EGFP (triangle), pYB1a-ECF20 & pRB1s-Pecf20-EGFP (square) was respectively transformed into *E. coli Nissle 1917*, green fluorescence intensity increased in a time dependent manner. When pRB1s-Pecf20-EGFP (circle) was transformed into the bacteria, green fluorescence intensity did not show obvious increase with time. We confirmed that ECF20 is a necessary factor for the activation of the Pecf20 promoter. In Fig. 17b, the addition of IPTG led to dramatic difference of detected green florescence signal. Without IPTG, we detected the continuously increased green fluorescence signal with the time. On the other hand, with the IPTG addition, the fluorescence signal remained at the baseline level. The data confirmed that anti-ECF20 could suppress the activity of Pecf20 promoter. Overall, our experiments indicated that ECF20 is necessary for the activation of the Pecf20 promoter, while anti-ECF20 can repress the Pecf 20 promoter through binding to ECF20 and suppressing its function.

### Uric Acid Regulation Module

#### Description

The uric acid regulation module includes a uricase coding sequence under the control of the uric acid sensor part HucR-Phuc and a constitutively expressed uric acid transporter YgfU [9]. The promoter Phuc [10] has HucR binding sites. In the absence of uric acid, the [HucR](http://parts.igem.org/Part%3ABBa_K2752003) protein binds to the Phuc promoter and prevents its association with the RNA polymerase. When uric acid reduces the affinity of HucR to the Phuc promoter, the RNA polymerase will bind to the Phuc promoter and initiate the transcription of the uricase gene. A series of the Phuc promoter variants were screened to determine which of them has the highest sensibility and intensity. YgfU is a proton-gradient dependent uric acid transporter that helps uric acid enter cells to be metabolized. With the accumulation of tumor cell lysates during chemotherapy, uric acid levels will rapidly increase and trigger the uric acid regulation module.

![](https://static.igem.org/mediawiki/2019/7/71/T--NEFU_China--uric_1.png)

Fig. 18. DNA sequences of the promoters [Phuc1](http://parts.igem.org/Part%3ABBa_K2913005), [Phuc2](http://parts.igem.org/Part%3ABBa_K2913006), and [Phuc3](http://parts.igem.org/Part%3ABBa_K2913007). The −35 and −10 sites in the promoter are indicated by underlined texts. The regulatory protein HucR binding sequences are indicated by shaded letters.

![](https://static.igem.org/mediawiki/2019/6/6e/T--NEFU_China--uric_2.png)

Fig. 19. Regulatory units of the YgfU and GFP expression cassettes. Phuc\* represents one of the Phuc1, Phuc2 and Phuc3.

![](https://static.igem.org/mediawiki/2019/3/33/T--NEFU_China--uric_3.png)

Fig. 20a. In the absence of uric acid, the HucR protein binds to the Phuc promoter and prevents its association with the RNA polymerase. b. When uric acid reduces the affinity of HucR to the Phuc promoter, the RNA polymerase will bind to the Phuc promoter and initiate the transcription of the uricase gene. c. Being catalyzed by uricase, uric acid is oxidized to allantoin until the HucR protein binds to the Phuc promoter again due to reduced levels of uric acid and prevents its association with the RNA polymerase.

#### Methods

1. Transform the plasmids into *E. coli DH5α*.

2. Prepare the uric acid solution:

• Prepare the boric acid buffer stock solution (20 mM EDTA, 0.02 % Triton-X100, pH 8.5, 1 M boric acid buffer): weigh 6.185 g of boric acid, 9.535 g of borax, and 1.169 g of EDTA, take 400 μl of 10 % Triton X-100, and then mix them in water with a final volume of 200 ml.

• Prepare the uric acid dilution (1 mM EDTA, 0.001 % Triton X-100, pH 8.5, 50 mM boric acid buffer): take 8 ml of the boric acid buffer stock solution into 152 ml water and adjust pH to 8.5.

• Prepare the uric acid stock solution (0.02 M uric acid, 1 mM EDTA, 0.001 % Triton X-100, pH 8.5, 50 mM boric acid buffer, kept in brown tubes at -20 ℃): weigh 0.336 g of uric acid and dissolve it in water to a final volume of 100 ml. This solution was stored as a 10\*stock solution at 4 ℃, and should be diluted by 10 times when used in measuring the activity.

• Prepare the uric acid solution (0.001 % uric acid, 1 M EDTA, 0.001 % Triton X-100, pH 8.5, 50 mM boric acid buffer, kept in brown tubes at -20 ℃): This uric acid solution should be diluted by 10 times when used in the experiments.

3. Cultured bacteria (150 μl) was inoculated into 15 ml of LB liquid medium containing 50 µg/ml Ampicillin (Amp) followed by culturing in a shaker (37 ℃, 160 r/min) overnight.

4. Cultured bacteria (150 µl) was inoculated into 15 ml of LB liquid medium containing 50 µg/ml Amp followed by culturing in a shaker (37 ℃, 160 r/min) until the absorbance of 600 nm reached 0.3 - 0.4.

5. Add 100 µl of bacteria culture medium into a 96-well plate and then add uric acid to different final concentrations (0, 0.01, 0.1 and 1 mM). Fresh LB medium served as a blank control. Place the 96-well plate into an automatic microplate reader, incubate it at 37℃ overnight and record the fluorescence values at 550 and 600 nm for each well every 60 min.

6. The experiments of each group were repeated for at least 3 times.

#### Results

The promoter should be fully activated at the highest uric acid concentration safe to the human body (0.465 mM). The activity of the promoters was determined by measuring the fluorescence intensity of the GFP. The results showed that the Phuc2 promoter had the highest sensitivity and activity at the conditions of 0.1 mM and 0.01 mM of uric acid. Then, the EGFP coding sequence was replaced by the uricase gene to check whether the module could sense and metabolize uric acid. As shown in Fig. 19, the control group kept constant uric acid levels, but the experimental group displayed reduced uric acid concentrations in a time-dependent manner in the first 3 h of incubation. After that, the uric acid levels kept constant. The data suggested that the expression of uricase was induced at high concentrations of uric acid and repressed at its relatively low concentrations. The result indicated that the expression of uricase was under the control of the Phuc2 promoter responsive to the concentration of uric acid.

![](https://static.igem.org/mediawiki/2019/f/fc/T--NEFU_China--uric_4.png)

Fig. 21. Comparison of induction strength of the promoter Phuc1 (circle), Phuc2 (square), and Phuc3 (triangle) by measuring green fluorescence of EGFP at 0.1 mM of the inducer uric acid (with 1:100 dilution of culture inoculation). Error bars represent standard deviations.

![](https://static.igem.org/mediawiki/2019/e/ef/T--NEFU_China--uric_5.png)

Fig. 22. Comparison of induction strength of the promoter Phuc1 (circle), Phuc2 (square), and Phuc3 (triangle) by measuring fluorescence of EGFP at 0.01 mM of the inducer uric acid (with 1:100 dilution of culture inoculation). Error bars represent standard deviations.

![](https://static.igem.org/mediawiki/2019/1/14/T--NEFU_China--uric_6.png)

Fig. 23. The cultured bacteria were transferred into minimum medium containing 8 mM uric acid. Samples were collected every 30 min to measure the concentrations of uric acid. The control group was the Nissle bacteria while the experimental group was the Nissle bacteria with the uric acid regulation module. Error bars represent standard deviations.

### Tumor Killer Module

#### Description

Tumor necrosis factor (TNF) is a naturally occurring cytokine product of activated macrophages. The anti-tumor properties of TNF and its unique efficacy in selective destruction of tumor-associated vessels are well known[11, 12, 13]. Subsequent pre-clinical studies have demonstrated in vivo anti-tumor effects in both syngeneic murine tumor models as well as human tumor xenografts in nude mice. So we choose TNF-α as a good candidate to kill the cancer cells.

#### Methods

After the construction of whole circuit, we induce the *E. coli Nissle 1917* transformed with the whole gene circuit by 1 g/l Na2SO3 and 100 mM lactic acid in LB-medium for 20 h. Logically, the medium will contain the TNF-α, so we call it “TNF-α group”. And the control group is not induced but cultured in LB-medium for 20 h. Then we extract the supernatant of the centrifuged medium to make the “TNF-α group” and the “Control group”.

Afterwards dilute the supernatant with fresh LB-medium in 100%, 75%, 50%, 20%, 10%, 0 for supernatant volume as “TNF-α” samples and “Control” samples. We add 50 μl samples into 50 μl RPMI 1640 Medium (containing 20% FBS, 1\*105 U/l Penicillin and 0.1 g/l Streptomycin) to culture the HeLa and HepG2 cells for 24 h, then use the Cell Counting Kit-8 to measure the cell viability.

#### Results

As we can see that the TNF-α is expressed after the induction, and TNF-α sample for 50% volume can kill 82.5% HepG2 cells, the TNF-α sample for 20% can kill 70.8% HeLa cells. Eventually, our system is able to induced by hypoxia and high lactic acid to secrete cytotoxic TNF-α, causing the evident death of HeLa and HepG2 cells.

![](https://static.igem.org/mediawiki/2019/9/9b/T--NEFU_China--tnf1.png)

Fig. 24 OD450 value can represent the cell number. “TNF-α” is the very group containing TNF-α theoretically. a. Different volume of the supernatant treated the HeLa cells for 24 h. b. Different volume of the supernatant treated the HepG2 cells for 24 h.

### Plasmid Protection Module:

#### Description:

Normally, we use antibiotic resistant genes as selecting markers to ensure that the transformed bacteria carry the plasmids. However, when our engineered bacteria enter the blood vessel, the plasmid carried by the bacteria will be lost as the bacteria keep division in an environment without antibiotics, which would make the bacteria unable to perform designated functions.
To prevent this from happening, we designed a plasmid protection module. We want to block the expression of a housekeeping gene of *E. coli*, and insert its coding sequence in one of our vectors. As a result, the bacteria can only survive when harboring this vector.

##### 1. The genes *alr* and *dadX*

D-alanine is one of the essential components needed by bacteria to synthesize cell wall peptidoglycan layer. Hence, the bacteria without D-alanine will not undergo cell division. *E. coli* has two genes related to the synthesis of alanine racemase. One is the *alr* gene with constitutive expression, while the other one is the *dadX* gene under control of the dad-operon with induced expression. Therefore, the knockout of the *alr* and *dadX* gene will lead to a strict dependence of D-alanine, so the bacteria with these mutations are only able to grow in the medium supplied with D-alanine or carrying a plasmid expressing the alanine racemase.

##### 2. CRISPR/Cas9

CRISPR (Clustered regularly interspaced short palindromic repeat) is an adaptive immune system and can provide protection against mobile genetic element by guided specific DNA cleavage. Cas9 protein belongs to the type II CRISPR system and exhibits strong DNA cleavage activity. In order to accomplish site specific cleavage, Cas9 protein must be guided by crRNA (CRISPR-derived RNA
) and tracrRNA(trans-activating crRNA).

![](https://static.igem.org/mediawiki/2019/1/19/T--NEFU_China--pla_1.png)

Fig. 25. The schematic of the knockout technique we used. The plasmid pCas9 used by us can constitutively express Cas9 with induced expression of bet, gam protein and exonuclease by arabinose and a sgRNA-pTargetF targeting the plasmid pTargetF that can be induced by IPTG. And the plasmid pTargetF can constitutively transcribe sgRNA-X targeting the gene X in the genomic DNA.

#### Methods:

1. We designed two primers at about 500 bp upstream and downstream of the target gene for PCR amplification, and then used two fragments as the homologous arms for homologous recombination. When CRISPR/Cas9 cleaved the target site in the bacterial genome, the homologous arms would recombine with the corresponding genomic locus leading to the knockout of the target gene.

2. Preparation of electrocompetent cells pCas9/BW
(1) The pCas9 plasmid was transformed into the *E. coli BW* strain.
(2) We inoculated the bacteria into 100 ml LB medium. After being cultured at 30 ℃, 200 rpm for about 12 h, the bacteria were inoculated at 1/50 ratio into 100 ml fresh LB medium. After culture for another 0.5 h, L-arabinose was added to a final concentration of 0.2 %.
(3) After culture for 2-2.5 h, the OD600 would reach about 0.55-0.6 (too high or too low bacterial density might seriously affect the efficiency of electric-to-inductive state). The conical flask of the bacteria was taken out and placed on ice for 30 min.
(4) The bacteria were centrifuged in a precooled centrifuge tube at 4,200 rpm, 4 ℃ for 10 min.
(5) The bacteria were carefully resuspended with ice-cold sterile 10 % glycerol and were centrifuged again at 4,200 rpm, 4 ℃ for 10 min.
(6) After the supernatant was carefully discarded, the bacteria were kept on ice.
(7) We repeated steps (5) and (6) for 3 times. Ten milliliters of sterilized 10 % glycerin was used for the first two washes of the bacteria each time. The final bacterial pellet was resuspended in 0.5 ml glycerin.
(8) The bacterial suspension was aliquoted as 100 μl in each pre-cooled tube. Electroporation for transformation could be carried out immediately or the aliquoted bacteria could be quickly frozen and stored in a -80 ℃ refrigerator.

3. The construction of Δ*alr*/BW strain
(1) We loged on to https://crispy.secondarymetabolites.org/#/input, searched for a suitable gRNA affinity site, and selected a site near the 5' -end of the ATG for the target gene.
(2) We constructed a pTargetF-alr plasmid that can express sgRNA targeting the *alr* gene.
(3) We obtained the homologous fragment upstream and downstream of the *alr* gene by PCR, which could be used as homologous arms for DNA recombination.
(4) The pTargetF-alr plasmid and homologous arms of the *alr* gene were transformed into pCas9/BW strain by electroporation.
(5) We picked colonies for PCR screening.
(6) We sequenced and alignment through a biological company.

4. The plasmid pTargetF-alr(str+) of Δ*alr*/BW strain was eliminated and competent cells of pCas9/Δ*alr*BW strain were prepared.

5. Construction of the Δ*alr*Δ*dadX*/BW strain
The method was the same as the first gene knockout.

6. The construction of the PRB1a-alr/ Δ*alr*Δ*dadX* BW strain
(1) The coding sequence of the *alr* gene was subcloned into the plasmid PRB1a.
(2) The plasmids of pCas9 (Kan+) and pTargetF-alr (str+) of Δ*alr*Δ*dadX*/BW strain were eliminated and competent cells of Δ*alr*Δ*dadX* /BW strain were prepared.
(3) We transformed the plasmid PRB1a-alr into the Δ*alr*Δ*dadX* /BW strain competent cells.

7. The construction of the PRB1a-alr/ Δ*alr*Δ*dadX* BW strain
(1) The coding sequence of the *alr* gene is subcloned into the plasmid PRB1a.
(2) The plasmids of pCas9 (Kan+) and pTargetF-alr (str+) of Δ*alr*Δ*dadX*/BW strain were eliminated and competent cells of Δ*alr*Δ*dadX* /BW strain were prepared.
(3) Transform the plasmid PRB1a-alr into the Δ*alr*Δ*dadX* /BW strain competent cells.

#### Results:

##### 1. Knockout

We used CRISPER/Cas9 technology to knock out the *alr* and *dadX* genes in the genome of wild type **E. coli BW 25113** successively. The results of DNA sequencing were as followed.

![](https://static.igem.org/mediawiki/2019/a/af/T--NEFU_China--pla_2.png)

Fig. 26. The diagram of the gene *alr* and *dadX* sequencing and alignment.

##### 2. Verification

(1) As shown in Fig. 27, the bacteria with two alanine racemase related genes knocked out (i.e. Δ*alr*Δ*dadX*/BW) could only survive when the LB medium was supplied with D-alanine or the bacteria carrying the plasmid expressing the gene *alr*. Thus, we have generated the bacterial strain Δ*alr*Δ*dadX*/BW with growth dependence of D-alanine.

![](https://static.igem.org/mediawiki/2019/8/8e/T--NEFU_China--pla_3.png)

Fig. 27. The growth curves of wild type BW and Δ*alr*Δ*dadX*/BW. In the condition of “+D-ala”, D-alanine was added to a final concentration of 1 mg/ml. The error bars represent the standard deviations.

(2) In the absence of any antibiotic, the strain PRB1a-GFP-alr/Δ*alr*Δ*dadX* BW was cultured for 10 days. The bacteria were photographed with the same exposure to check the green fluorescent signal.
As shown in Fig. 28, the proportion of bacteria capable of expressing GFP was almost 70% in the first generation of PRB1a-GFP-alr/ Δ*alr*Δ*dadX* BW strain and after ten generations the proportion became 60%. The result showed that our plasmid with *alr* can be stable.

![](https://static.igem.org/mediawiki/2019/6/61/T--NEFU_China--pla_4.png)

Fig. 28. Fluorescence microscopy of PRB1a-GFP-alr/ Δ*alr*Δ*dadX* BW strain. a and b are the images of the PRB1a-GFP-alr/Δ*alr*Δ*dadX* BW strain cultured for one day; c and d are the same bacterium stain continuously cultured for ten days. These results indicated, the bacteria Δ*alr*Δ*dadX*/BW carrying the plasmid expressing the gene *alr* would survive in the condition without any antibiotic selection pressure.

#### References

[1] Ryan, R.M., et al., Bacterial delivery of a novel cytolysin to hypoxic areas of solid tumors. Gene Ther, 2009. 16(3): p. 329-39.
[2] Wu, Y., et al., Lactate, a Neglected Factor for Diabetes and Cancer Interaction. Mediators Inflamm, 2016. 2016: p. 6456018.
[3] Bell, A. and S. Busby, Location and orientation of an activating region in the Escherichia coli transcription factor, FNR. Mol Microbiol, 1994. 11(2): p. 383-90.
[4] Goers, L., et al., Whole-cell Escherichia coli lactate biosensor for monitoring mammalian cell cultures during biopharmaceutical production. Biotechnol Bioeng, 2017. 114(6): p. 1290-1300.
[5] Aguilera, L., et al., Dual role of LldR in regulation of the lldPRD operon, involved in L-lactate metabolism in Escherichia coli. J Bacteriol, 2008. 190(8): p. 2997-3005.
[6] Moser, F., et al., Dynamic control of endogenous metabolism with combinatorial logic circuits. Mol Syst Biol, 2018. 14(11): p. e8605.
[7]Liu, X., et al., Engineering Genetically-Encoded Mineralization and Magnetism via Directed Evolution. Sci Rep, 2016. 6: p. 38019.
[8].Huber, B.E., et al., In vivo antitumor activity of 5-fluorocytosine on human colorectal carcinoma cells genetically modified to express cytosine deaminase. Cancer Res, 1993. 53(19): p. 4619-26.
[9]Liang, C., et al., Development of a novel uric-acid-responsive regulatory system in Escherichia coli. Appl Microbiol Biotechnol, 2015. 99(5): p. 2267-75.
[10]Papakostas, K. and S. Frillingos, Substrate selectivity of YgfU, a uric acid transporter from Escherichia coli. J Biol Chem, 2012. 287(19): p. 15684-95.
[11] Old, L.J., Tumor necrosis factor (TNF). Science, 1985. 230(4726): p. 630-2.
[12] Watanabe, N., et al., Toxic effect of tumor necrosis factor on tumor vasculature in mice. Cancer Res, 1988. 48(8): p. 2179-83.
[13] Folli, S., et al., Tumor-necrosis factor can enhance radio-antibody uptake in human colon carcinoma xenografts by increasing vascular permeability. Int J Cancer, 1993. 53(5): p. 829-36.

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