iGEM 2020 — Team UCAS-China

[![](https://static.igem.org/mediawiki/2020/8/8b/HQ_page_logo.jpg)](https://2020.igem.org)

# Team:UCAS-China/Notebook

![Team:UCAS-China](https://static.igem.org/mediawiki/2020/4/40/T--UCAS-China--logonsrc.png)

* [Home](https://2020.igem.org/Team%3AUCAS-China)
* Project
  + [Description](https://2020.igem.org/Team%3AUCAS-China/Description)
  + [Design](https://2020.igem.org/Team%3AUCAS-China/Design)
  + [Proof Of Concept](https://2020.igem.org/Team%3AUCAS-China/Proof_Of_Concept)
  + [Demonstrate](https://2020.igem.org/Team%3AUCAS-China/Results)
  + [Future Plan](https://2020.igem.org/Team%3AUCAS-China/Future_Plan)
  + [Engineering](https://2020.igem.org/Team%3AUCAS-China/Engineering)
  + [Contribution](https://2020.igem.org/Team%3AUCAS-China/Contribution)
  + [Parts](https://2020.igem.org/Team%3AUCAS-China/Parts)
* [Hardware](https://2020.igem.org/Team%3AUCAS-China/Hardware)
* [Model](https://2020.igem.org/Team%3AUCAS-China/Model)
  + [Model for the Design](https://2020.igem.org/Team%3AUCAS-China/Model_for_the_Design)
  + [Dynamic of Hp Infection](https://2020.igem.org/Team%3AUCAS-China/Dynamic_Inf)
  + [Dynamic of Hp-SHEEP-Human System](https://2020.igem.org/Team%3AUCAS-China/Dynamic_Sys)
  + [Model for Therapy Method](https://2020.igem.org/Team%3AUCAS-China/Model_TM)
  + [Estimation for Individuality in Stable States](https://2020.igem.org/Team%3AUCAS-China/Estimation_ISS)
* [Human Practices](https://2020.igem.org/Team%3AUCAS-China/Human_Practices)
  + [Integrated Human Practice](https://2020.igem.org/Team%3AUCAS-China/Human_Practices)
  + [Education&Public Engagement](https://2020.igem.org/Team%3AUCAS-China/Education)
  + [Collaborations](https://2020.igem.org/Team%3AUCAS-China/Collaborations)
  + [Implementation](https://2020.igem.org/Team%3AUCAS-China/Implementation)
  + [Partnership](https://2020.igem.org/Team%3AUCAS-China/Partnership)
* Team
  + [Meet The Team!](https://2020.igem.org/Team%3AUCAS-China/Meet_The_Team)
  + [Attributions](https://2020.igem.org/Team%3AUCAS-China/Attributions)
  + [Notebook](https://2020.igem.org/Team%3AUCAS-China/Notebook)
  + [Acknowledgement](https://2020.igem.org/Team%3AUCAS-China/Acknowledgment)

[<div> </div>](https://www.ucas.ac.cn/)[<div> </div>](https://bio.ucas.ac.cn/)[<div> </div>](http://www.ibp.cas.cn/)[<div> </div>](https://www.snapgene.com/)

![footer](https://static.igem.org/mediawiki/2020/b/b2/T--UCAS-China--HomepageBtm.png)

Due to the impact of the COVID-19, team UCAS-China have not had enough time to carry out the experiments this year yet. This part will be edited later.

[![](https://static.igem.org/mediawiki/2020/8/8b/HQ_page_logo.jpg)](https://2020.igem.org)

# Team:UCAS-China/Engineering

![Team:UCAS-China](https://static.igem.org/mediawiki/2020/4/40/T--UCAS-China--logonsrc.png)

* [Home](https://2020.igem.org/Team%3AUCAS-China)
* Project
  + [Description](https://2020.igem.org/Team%3AUCAS-China/Description)
  + [Design](https://2020.igem.org/Team%3AUCAS-China/Design)
  + [Proof Of Concept](https://2020.igem.org/Team%3AUCAS-China/Proof_Of_Concept)
  + [Demonstrate](https://2020.igem.org/Team%3AUCAS-China/Results)
  + [Future Plan](https://2020.igem.org/Team%3AUCAS-China/Future_Plan)
  + [Engineering](https://2020.igem.org/Team%3AUCAS-China/Engineering)
  + [Contribution](https://2020.igem.org/Team%3AUCAS-China/Contribution)
  + [Parts](https://2020.igem.org/Team%3AUCAS-China/Parts)
* [Hardware](https://2020.igem.org/Team%3AUCAS-China/Hardware)
* [Model](https://2020.igem.org/Team%3AUCAS-China/Model)
  + [Model for the Design](https://2020.igem.org/Team%3AUCAS-China/Model_for_the_Design)
  + [Dynamic of Hp Infection](https://2020.igem.org/Team%3AUCAS-China/Dynamic_Inf)
  + [Dynamic of Hp-SHEEP-Human System](https://2020.igem.org/Team%3AUCAS-China/Dynamic_Sys)
  + [Model for Therapy Method](https://2020.igem.org/Team%3AUCAS-China/Model_TM)
  + [Estimation for Individuality in Stable States](https://2020.igem.org/Team%3AUCAS-China/Estimation_ISS)
* [Human Practices](https://2020.igem.org/Team%3AUCAS-China/Human_Practices)
  + [Integrated Human Practice](https://2020.igem.org/Team%3AUCAS-China/Human_Practices)
  + [Education&Public Engagement](https://2020.igem.org/Team%3AUCAS-China/Education)
  + [Collaborations](https://2020.igem.org/Team%3AUCAS-China/Collaborations)
  + [Implementation](https://2020.igem.org/Team%3AUCAS-China/Implementation)
  + [Partnership](https://2020.igem.org/Team%3AUCAS-China/Partnership)
* Team
  + [Meet The Team!](https://2020.igem.org/Team%3AUCAS-China/Meet_The_Team)
  + [Attributions](https://2020.igem.org/Team%3AUCAS-China/Attributions)
  + [Notebook](https://2020.igem.org/Team%3AUCAS-China/Notebook)
  + [Acknowledgement](https://2020.igem.org/Team%3AUCAS-China/Acknowledgment)

[<div> </div>](https://www.ucas.ac.cn/)[<div> </div>](https://bio.ucas.ac.cn/)[<div> </div>](http://www.ibp.cas.cn/)[<div> </div>](https://www.snapgene.com/)

![footer](https://static.igem.org/mediawiki/2020/b/b2/T--UCAS-China--HomepageBtm.png)

Engineering Success

After brainstorming, we proposed the idea of microbial therapy for *H. pylori*. We believe that the three most important parts of medical microbiology are drug-related metabolic pathway, targeted gene design and biosafety module. In addition, the hardware and mathematical model should be considered in the complete treatment plan. Therefore, we also designed a mathematical model that can accurately control the drug administration and determine the situation of *H. pylori* infection in the stomach to assist in the formulation of personalized treatment plan.

We chose the combination of pfnrs and LL-37 as the drug metabolism pathway. Pfnrs was experimentally validated in our project last year and successfully expressed downstream CD47 nanoantibody under hypoxia.

LL-37 is an antibacterial peptide produced by the human immune system. According to literature research, it can be confirmed that it has inhibitory effect on *H. pylori*, and can be expressed in prokaryotic expression system. On the basis of last year's plasmid, we changed the gene and added the start codon and stop codon. Due to the epidemic situation, we can not finish the experiment before wiki freeze, so we plan to carry out experiments in the future to prove the feasibility of its expression and the antibacterial effect of antimicrobial peptides.

Through previous experiments and literature research, we can prove that this part of the project is successful.

For the design of ammonia sensor in engineering bacteria, we selected the transcription factor NtrC as the NOT gate protein switch, which combined with another downstream NOT gate protein to form an induction switch that can sense the change of ammonia concentration in the environment and turn on the downstream gene transcription. The related design of NtrC is verified by experiments in UCAS-China 2017 project. According to the new literature research and the proof of logic circuit, we can verify the engineering success of this gene circuit.

For the biosafety switch, we creatively connect the toxin-antitoxin system controlled by the cold-inducible on-switch with the "super DOC".Decline of ambient temperature can turn on the translation of DOC toxin protein and kill cells. The existence of degradation tag greatly reduces the possibility of the leaky expression, and improves the treatment efficiency. The toxin-antitoxin system controlled by temperature-sensitive switch was verified by experiments in last year's project, and it has high reliability. As a new design of this year's project, the new original of DOC toxin protein gene with SsrA degradation tag, after the theoretical proof and literature research, can preliminarily verify the success of the project. In the future, we will directly prove the correctness and reliability of such designs through experiments.

[![](https://static.igem.org/mediawiki/2020/8/8b/HQ_page_logo.jpg)](https://2020.igem.org)

# Team:UCAS-China/Experiments

![Team:UCAS-China](https://static.igem.org/mediawiki/2020/4/40/T--UCAS-China--logonsrc.png)

* [Home](https://2020.igem.org/Team%3AUCAS-China)
* Project
  + [Description](https://2020.igem.org/Team%3AUCAS-China/Description)
  + [Design](https://2020.igem.org/Team%3AUCAS-China/Design)
  + [Proof Of Concept](https://2020.igem.org/Team%3AUCAS-China/Proof_Of_Concept)
  + [Demonstrate](https://2020.igem.org/Team%3AUCAS-China/Results)
  + [Future Plan](https://2020.igem.org/Team%3AUCAS-China/Future_Plan)
  + [Engineering](https://2020.igem.org/Team%3AUCAS-China/Engineering)
  + [Contribution](https://2020.igem.org/Team%3AUCAS-China/Contribution)
  + [Parts](https://2020.igem.org/Team%3AUCAS-China/Parts)
* [Hardware](https://2020.igem.org/Team%3AUCAS-China/Hardware)
* [Model](https://2020.igem.org/Team%3AUCAS-China/Model)
  + [Model for the Design](https://2020.igem.org/Team%3AUCAS-China/Model_for_the_Design)
  + [Dynamic of Hp Infection](https://2020.igem.org/Team%3AUCAS-China/Dynamic_Inf)
  + [Dynamic of Hp-SHEEP-Human System](https://2020.igem.org/Team%3AUCAS-China/Dynamic_Sys)
  + [Model for Therapy Method](https://2020.igem.org/Team%3AUCAS-China/Model_TM)
  + [Estimation for Individuality in Stable States](https://2020.igem.org/Team%3AUCAS-China/Estimation_ISS)
* [Human Practices](https://2020.igem.org/Team%3AUCAS-China/Human_Practices)
  + [Integrated Human Practice](https://2020.igem.org/Team%3AUCAS-China/Human_Practices)
  + [Education&Public Engagement](https://2020.igem.org/Team%3AUCAS-China/Education)
  + [Collaborations](https://2020.igem.org/Team%3AUCAS-China/Collaborations)
  + [Implementation](https://2020.igem.org/Team%3AUCAS-China/Implementation)
  + [Partnership](https://2020.igem.org/Team%3AUCAS-China/Partnership)
* Team
  + [Meet The Team!](https://2020.igem.org/Team%3AUCAS-China/Meet_The_Team)
  + [Attributions](https://2020.igem.org/Team%3AUCAS-China/Attributions)
  + [Notebook](https://2020.igem.org/Team%3AUCAS-China/Notebook)
  + [Acknowledgement](https://2020.igem.org/Team%3AUCAS-China/Acknowledgment)

[<div> </div>](https://www.ucas.ac.cn/)[<div> </div>](https://bio.ucas.ac.cn/)[<div> </div>](http://www.ibp.cas.cn/)[<div> </div>](https://www.snapgene.com/)

![footer](https://static.igem.org/mediawiki/2020/b/b2/T--UCAS-China--HomepageBtm.png)

# 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.

If you made Parts this year, please remember to put all information, characterization, and measurement data on the Part's Main Page on the [Registry](http://parts.igem.org/Main_Page).

### What should this page contain?

* Protocols
* Experiments

### 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)

[![](https://static.igem.org/mediawiki/2020/8/8b/HQ_page_logo.jpg)](https://2020.igem.org)

# Team:UCAS-China/Results

![Team:UCAS-China](https://static.igem.org/mediawiki/2020/4/40/T--UCAS-China--logonsrc.png)

* [Home](https://2020.igem.org/Team%3AUCAS-China)
* Project
  + [Description](https://2020.igem.org/Team%3AUCAS-China/Description)
  + [Design](https://2020.igem.org/Team%3AUCAS-China/Design)
  + [Proof Of Concept](https://2020.igem.org/Team%3AUCAS-China/Proof_Of_Concept)
  + [Demonstrate](https://2020.igem.org/Team%3AUCAS-China/Results)
  + [Future Plan](https://2020.igem.org/Team%3AUCAS-China/Future_Plan)
  + [Engineering](https://2020.igem.org/Team%3AUCAS-China/Engineering)
  + [Contribution](https://2020.igem.org/Team%3AUCAS-China/Contribution)
  + [Parts](https://2020.igem.org/Team%3AUCAS-China/Parts)
* [Hardware](https://2020.igem.org/Team%3AUCAS-China/Hardware)
* [Model](https://2020.igem.org/Team%3AUCAS-China/Model)
  + [Model for the Design](https://2020.igem.org/Team%3AUCAS-China/Model_for_the_Design)
  + [Dynamic of Hp Infection](https://2020.igem.org/Team%3AUCAS-China/Dynamic_Inf)
  + [Dynamic of Hp-SHEEP-Human System](https://2020.igem.org/Team%3AUCAS-China/Dynamic_Sys)
  + [Model for Therapy Method](https://2020.igem.org/Team%3AUCAS-China/Model_TM)
  + [Estimation for Individuality in Stable States](https://2020.igem.org/Team%3AUCAS-China/Estimation_ISS)
* [Human Practices](https://2020.igem.org/Team%3AUCAS-China/Human_Practices)
  + [Integrated Human Practice](https://2020.igem.org/Team%3AUCAS-China/Human_Practices)
  + [Education&Public Engagement](https://2020.igem.org/Team%3AUCAS-China/Education)
  + [Collaborations](https://2020.igem.org/Team%3AUCAS-China/Collaborations)
  + [Implementation](https://2020.igem.org/Team%3AUCAS-China/Implementation)
  + [Partnership](https://2020.igem.org/Team%3AUCAS-China/Partnership)
* Team
  + [Meet The Team!](https://2020.igem.org/Team%3AUCAS-China/Meet_The_Team)
  + [Attributions](https://2020.igem.org/Team%3AUCAS-China/Attributions)
  + [Notebook](https://2020.igem.org/Team%3AUCAS-China/Notebook)
  + [Acknowledgement](https://2020.igem.org/Team%3AUCAS-China/Acknowledgment)

[<div> </div>](https://www.ucas.ac.cn/)[<div> </div>](https://bio.ucas.ac.cn/)[<div> </div>](http://www.ibp.cas.cn/)[<div> </div>](https://www.snapgene.com/)

![footer](https://static.igem.org/mediawiki/2020/b/b2/T--UCAS-China--HomepageBtm.png)

Demonstrate

Microbial Therapeutics (demonstrate of drug production module)

Because the oxygen concentration in gastric environment is generally low, we connected the gene of LL-37 cathelicidin behind the anaerobic induced promoter, PfnrS, to ensure its expression is only in gastric environment.

glnAp2+riboJ+RBS (demonstrate of ammonia sensor)

According to the work done by team UCAS in 2017 and the reference [1], we have faith in the availability of NtrC-regulated promoter glnAp2, their work gives valid evidence that glnAp2 is a reliable promotor regulated by NtrC.

The figure below shows that when in high-nitrogen circumstance (ammonium as the nitrogen substance), the promoter will work after an obvious delay time therefore suppress the transcription, while in low-nitrogen circumstance, the promoter will start the transcription rather sensitive.

Since the behavior of the promoter glnAp2 changes linearly with the variation of concentration of ammonium, we believe it will work as a predictable way we expected in the design part.

![](https://static.igem.org/mediawiki/2020/e/ee/T--UCAS-China--desmon9.png)

The figure is from Part: BBa\_K2287001

Hence the downstream of glnAp2 of the genetic circuit in the ammonia concentration switch is orthogonal to the original genes of *L. acidophilus*, the last question is whether the lysis part will work.

![](https://static.igem.org/mediawiki/2020/c/c1/T--UCAS-China--demon10.png)

The table (from [2]) above shows antimicrobial activity of endolysin P28 against several Gram-positive bacteria. It is a strong evidence that the endolysin will work in L. acidophilus as a Gram-positive bacterium.

![](https://static.igem.org/mediawiki/2020/5/5c/T--UCAS-China--demon11.png)

Shown is SDS-PAGE (15%) of the cellular proteins from 80 μL cultures of bacteria. The gel was stained by Coomassie brilliant blue R-250 to visualize the bands. *E. coli* BL21 (DE3; pET-26b-ORF8) before induction (lane 1) and 6 h after induction (1 mM IPTG; lane 2). The overexpressed oligohistidine-tagged endolysin P28 was purified (lane 3). M, molecular masses of reference proteins (Thermo Scientific; in kilodaltons).[2]

In addition, the figure above shows the overproduction of endolysin P28 in *E. coli* BL21 (DE3) cells, which validate that the gene expresses P28 has good compatibility in different kinds of bacteria.

In one word, we testified not only the circuit will work as expectation, but also the production protein will carry out its function.

Doc-SsrA (demonstrate of biosafety part)

Toxin-antitoxin system is an ingenious and effective strategy for biosafety, an appropriate toxin protein can effectively control the escape of engineering bacteria. The robustness of the system depends on the effectiveness of the toxin-antitoxin and the sensitivity of the upstream switch.

As for the trigging factors of the switch, temperature stands out for its unique advantages in microbial therapy: non-invasive nature, good penetrability and reversibility. In 2019, UCAS's iGEM team designed a cold-inducible on-switch on the base of the evolved transcription factor and the evolved protease, which can be activated below 37℃, to control the expression of toxin protein (Fig. 1). We characterized the tendency of fluorescence change of a series of cold-inducible ON-switches using different TEVts mutants under different temperatures and chose two kinds of switches consisting of TEVts#6 and TEVts#18 (Fig. 2). Then we measure their temperature response curve more precisely by flow cytometer in TOP10 strain. From Figure 3 we can see that two groups both show narrow transition ranges and have about 100-fold induction within the range of ten degrees.

![](https://static.igem.org/mediawiki/2020/0/0c/T--UCAS-China--BS1.png)

Figure 1. The cold-inducible ON-switch is encoded on two plasmids. The circuit is shown with the genetic parts and relationships among them.

![](https://static.igem.org/mediawiki/2020/a/ad/T--UCAS-China--BS2.png)

Figure 2. The tendency of fluorescence change of a series of TEVts mutants under different temperatures (measured by ELIASA)

![](https://static.igem.org/mediawiki/2020/a/ac/T--UCAS-China--BS3.png)

Figure 3. The induction curve of the cold-inducible ON-switches (TOP10)

Through the above experiments, we have proved that the cold induced switch has high sensitivity. We also measured the escape rate of the bacteria with DOC gene in solid and liquid medium (Fig4, 5).

In solid medium, we measured the escape rate, and the 20h average solid escape frequency is 2.318×10^(-2). In liquid medium, we measured the growth curve of the strains with DOC gene and strains without it at different temperatures, the different growth of the two strains at 37℃ showed that the leakage of the system exists.

![](https://static.igem.org/mediawiki/2020/a/a4/T--UCAS-China--BS4.png)
![](https://static.igem.org/mediawiki/2020/d/db/T--UCAS-China--BS5.png)
![](https://static.igem.org/mediawiki/2020/f/fa/T--UCAS-China--BS6.png)

Figure 4. The escape rate of bacteria on solid LB plate after 20h. The escape rate is calculated using the formula per Escape Frequency=(Colonies on nonpermissive plate×dilution)/(Colonies on permissive plate×dilution)[6] , the escape rate of 3 groups are (1×10^7)/(2×10^8 )=5×10^(-2) , (11×10^7)/(6×10^10 )=1.83×10^(-2) and (2×10^7)/(16×10^9 )=1.25×10^(-3) respectively(from above to below). And the Escape Frequency=Average Escape Frequency±standard deviation[6]=2.318 ×10^(−2) ± 0.122.

![](https://static.igem.org/mediawiki/2019/e/ee/T--UCAS-China--biosafety6.png)

Figure 5. The growth curve of bacteria on liquid LB. Incubated in 4 mL volume in 24-deep-well plate.

It can be seen that the leakage of DOC toxin protein is the cause of high escape rate. To improve the performance of cold-inducible kill switch, we chose to reduce the leaky expression of Doc toxin by fusing a hybrid tevS/ssrA tag to the C-terminal domain of Doc toxin (Figure 6c). The ssrA tag is used for proteasome-dependent degradation of Doc toxin by endogenous proteases ClpAP and ClpXP42, while tevS for the proteolytic removal of ssrA tag. When the temperature is lower than 34°C, TEVts, as a component of cold-inducible switch, will be highly expressed, and cleave tevS to release ssrA tag from Doc protein. By contrast, as the expression of TEVts is repressed by cI434ts at 37°C, the ssrA tag cannot be removed and Doc toxin is degraded.

We characterized this advanced version of kill switch (Doc-SsrA) in E. coli TOP10 and EcN at different temperatures. The escape frequency of TOP10 Doc-SsrA and EcN Doc-SsrA at 25°C were (2.68±1.56)×10^(-6) and (1.47±0.78)×10^(-6) respectively, which were comparable to the lowest escape frequency achieved by previously reported thermal sensitive kill switch, much lower than Doc without SsrA. Similarly, the escape frequency of TOP10 Doc-SsrA and EcN Doc-SsrA at 30°C were (1.13±0.68)×10^(-5) and (9.78±7.34)×10^(-6), two to three orders of magnitude lower than 10^(-3) described in previous research by Stirling et al(Fig. 6). Growth curves in liquid culture also showed that TOP10 Doc-SsrA and EcN Doc-SsrA suffered significant growth arrest at both 25°C and 30°C (Fig. 7).

![](https://static.igem.org/mediawiki/2020/3/3c/T--UCAS-China--BS7.png)

Figure 6. The construction and characterization of cold-inducible kill switch. (a) Genetic circuit design of cold-inducible kill switch ver1.0. The protease tevts is cloned under pR to a p15A plasmid. The transcription factor is cloned under a strong promoter J23119 with toxin doc cloned under pR downstream to a pSC101 plasmid. (b) The escape rate of bacteria on solid LB plate after 20h. The escape rate is calculated as below: per Escape Frequency = (Colonies on nonpermissive plate ∙ dilution) / (Colonies on permissive plate ∙ dilution) 19. The escape rate of three groups are (1×107)/(2×108)=5×10-2 (i) ,(11×107)/(6×1010)=1.83×10-2 (ii) and (2×107)/(16×109)=1.25×10-3 (iii) respectively. And the Escape Frequency = Average Escape Frequency ± standard deviation = 2.318 ×10-2 ± 0.122.

![](https://static.igem.org/mediawiki/2020/f/f9/T--UCAS-China--BS8.png)

Figure 7.The construction and characterization of double-status switch. (a) Genetic circuit design of the double-status switch. The heat-inducible switch that has mRFP as the output is placed on the same p15A1 plasmid as the protease TEVts.

References

[1] Reitzer, Lawrence J., and Boris Magasanik. Transcription of glnA in E. coli is stimulated by activator bound to sites far from the promoter. Cell 45.6 (1986): 785-792.

[2] Dong, H., Zhu, C., Chen, J., Ye, X., & Huang, Y. P. (2015). Antibacterial Activity of Stenotrophomonas maltophilia Endolysin P28 against both Gram-positive and Gram-negative Bacteria. Frontiers in microbiology, 6, 1299. https://doi.org/10.3389/fmicb.2015.01299

[3] Huo, Yi‐Xin, et al. "Protein induced DNA bending clarifies the architectural organization of the σ54‐dependent glnAp2 promoter." Molecular microbiology 59.1 (2006): 168-180.