iGEM 2018 — Team OUC-China

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# Team:OUC-China/Notebook

Team OUC-China: Main

jQuery UI 折叠面板（Accordion） - 非自动高度

TOP

* [Main](https://2018.igem.org/Team%3AOUC-China)
* Project
  + [Overview](https://2018.igem.org/Team%3AOUC-China/Description)
  + [Design](https://2018.igem.org/Team%3AOUC-China/Design)
  + [Results](https://2018.igem.org/Team%3AOUC-China/Results)
  + [Demonstrate](https://2018.igem.org/Team%3AOUC-China/Demonstrate)
* Lab WORK
  + [Experiments](https://2018.igem.org/Team%3AOUC-China/Experiments)
  + [Safety](https://2018.igem.org/Team%3AOUC-China/Safety)
  + [InterLab](https://2018.igem.org/Team%3AOUC-China/InterLab)
* [Model](https://2018.igem.org/Team%3AOUC-China/Model)
* Parts
  + [List](https://2018.igem.org/Team%3AOUC-China/Parts)
  + [Improve](https://2018.igem.org/Team%3AOUC-China/Improve)
* HP
  + [Human Practices](https://2018.igem.org/Team%3AOUC-China/Human_Practices)
  + [Public Engagement](https://2018.igem.org/Team%3AOUC-China/Public_Engagement)
* Team
  + [Team Member](https://2018.igem.org/Team%3AOUC-China/Team)
  + [Collaborations](https://2018.igem.org/Team%3AOUC-China/Collaborations)
  + [Attributions](https://2018.igem.org/Team%3AOUC-China/Attributions)
* [Notebook](https://2018.igem.org/Team%3AOUC-China/Notebook)

![banner](https://static.igem.org/mediawiki/2018/0/04/T--OUC-China--notebook.jpg)

## Notebook

### 2.4-2.10

- 1. Completed the team registration.
- 2. Learnt about synthetic biology by reading papers and wikis.
- 3. Tried to set up our project.
- 4. The iGEM team from SDU-China came to our meeting room for communication.

### 2.11-2.17

- 1. Learnt about synthetic biology by reading papers and wikis.
- 2. Tried to set up our project.
- 3. Looked for the PI of our project.

### 2.18-2.24

- 1. Sorted out the previous projects, and made the first selection according to the feasibility of each project.
- 2. The principles of all projects had been further explored.
- 3. We asked our instructor for advice.
- 4. Began to learn microbial experiment operation, and everyone in the team carried out the test of experiment operation.

### 2.25-3.3

- 1. Started communication and collaboration with iGEM teams in other schools.
- 2. Began to familiarize ourselves with laboratory equipment and other arrangements.
- 3. Had applied for the 5th Conference of China iGEMer Community（CCiC）.
- 4. Set up some more projects.

### 3.4-3.10

- 1. The previous project was classified again, and our team members were divided into different groups to dig deeper into the principles of the project.
- 2. Started learning molecular experimentation.
- 3. Started preparing the bid report of the 5th Conference of China iGEMer Community（CCiC）.

### 3.11-3.17

- 1. Had temporarily identified two alternative projects and confirmed their circuit, components, related reagents as well as equipment, analyzed their feasibility in detail, continuously filled and enriched the project content, and tried to design experiments.
- 2. Contacted the authors of the project-related papers, inquired about the details, and received many suggestions from the original paper’s team.
- 3. Asked many teachers and our seniors at the same time, and got their advice.
- 4. We were learning about molecular experimentation.
- 5. Introduced team status, project progress and other issues to our PI.
- 6. Set daily management rules for our team.

### 3.18-3.24

- 1. Referred to the opinions of the teachers and members of the previous team, finally chose one of the two projects according to their feasibility.
- 2. Purchased the required strains: RP6666 and RP437.
- 3. Contacted the equipment needed for our project.
- 4. Sorted out the materials of the 5th Conference of China iGEMer Community (CCiC) and communicated with our school, finally won the support of the school.

### 3.25-3.31

- 1. Preserved RP6666 and RP437.
- 2. Modified the existing project, proposed a new project.
- 3. Made a detailed exploration of the principle, circuit, parts and equipment of the project and sorted out a file package.
- 4. Got in touch with the original author of the paper, Mr. Du Pei, and got his help.

### 4.1-4.7

- 1. Sent the package of documents to PI, teachers and seniors, and listened to their opinions to constantly improve and perfect the project.
- 2. Started trying to contact teachers who could provide guidance or help for our projects（For example, teachers from the Chinese academy of sciences）.
- 3. We were divided into two groups, one responsible for experimental design, the other responsible for circuit design, determining the detailed details of the project, and trying to build the model.
- 4. Submitted the CCiC bid report.

### 4.8-4.14

- 1. Communicated with Mr. Du Pei and got the plasmid we need.
- 2. The first validation experiment was conducted with plasmid.
- 3. In the plasmid transformation experiment, our bacteria did not grow well on the plate. We conducted a detailed analysis on the operation of the experiment, and sought the help of the teacher, formulated the remedial plan.
- 4. The bid for the 5th Conference of China iGEMer Community (CCiC) ended in the voting, and we will hold CCiC as the co-organizer.
- 5. The division of duties of the team was determined.
- 6. Contacted biotechnology companies to sequence plasmids and bought new laboratory reagents.

### 4.15-4.21

- 1. With our persistence and efforts, we successfully enriched the plasmid.
- 2. Commissioned the company to synthesize the circuit.

### 4.22-4.28

- 1. Measured the first system fluorescence with a microplate reader, but the data was not very good.
- 2. Gave the first popular science lecture on synthetic biology.
- 3. Measured the growth curves of three strains of the first system.

### 4.29-5.5

- 1. Asked whether other control groups could be set up, except for the cultured strains without IPTG.
- 2. The control group was redesigned.
- 3. Gave our first intramural lecture on synthetic biology.
- 4. Designed the experimental scheme of Interlab.

### 5.6-5.12

- 1. Started building a new circuit to complement our project.
- 2. Analyzed the experimental scheme of measuring fluorescence last time, summarized the problem, replaced the M9 medium, remeasured the first system, and obtained better experimental data.
- 3. We are going to design a game related to our project, and started the game art design.
- 4. The preliminary experiment of Interlab was carried out.
- 5. The primer design of the standardized experiment was carried out.

### 5.13-5.19

- 1. Began to design the medium of the miniToe Motility detection system.
- 2. Identified the primers for standardized experiments and commissioned the company to synthesize them.
- 3. Completed the pre-experiment of the project.
- 4. Designed a formal experimental scheme.
- 5. Started writing part descriptions.
- 6. Started writing the project overview.
- 7. Started the recruitment of the next team.
- 8. Built the basic model of the first system.

### 5.20-5.26

- 1. We submitted the application for the summer social practice project to our school, and we want to use the platform of the school to promote synthetic biology to more people.
- 2. Did a preliminary design of the web page.
- 3. Finished the first draft of our cartoon.

### 5.27-6.2

- 1. Designed the uniforms of our team.
- 2. Performed transformation of four mutant enzyme plasmids.
- 3. Carried out the pre-experiment of the standardized experiment.
- 4. Completed the fluorescence measurement of four kinds of mutant Csy4 enzymes combined with wild type of hairpin.

### 6.3-6.9

- 1. Applied for visa.
- 2. Standardized Csy4-WT.

### 6.10-6.16

- 1. Contacted many biotechnology companies in Qingdao.
- 2. Our summer social practice program was supported by the school and we planned to better promote synthetic biology.

### 6.17-6.23

- 1. Sorted out what we have done so far and put forward future arrangements and plans.
- 2. Named for our project.

### 6.24-6.30

- 1. Checked out the experimental materials and bought some new ones.
- 2. The tasks of the experiment were divided, and each member of the team would undertake some of the experimental tasks.

### 7.1-7.7

- 1. Took the school final exam.

### 7.8-7.14

- 1. We visited the Qingdao huada gene research institute.
- 2. Our game was already in infancy and was awaiting further modification.
- 3. Design the brochure of our project.
- 4. Started planning the camp of life science and technology jointly with the iGEM team of SDU-China.
- 5. Started the Interlab experiment.
- 6. Optimized the experimental scheme and carried out the pre-experiment, the experimental results were very successful.

### 7.15-7.21

- 1. We visited the molecular development laboratory of the Institute of Oceanology, Chinese Academy of Sciences.
- 2. Visited Qingdao Bright Moon Seaweed Group Co. LTD.
- 3. We co-organized a summer camp of Qingdao no.2 middle school with Marine Life Science Association Science Technology in our university, and gave a lecture to promote synthetic biology and iGEM to more people.
- 4. Our life science and technology camp had started the recruitment of campers.
- 5. We prepared all the materials needed for the experiment, such as culture medium, strains, etc.

### 7.22-7.28

- 1. We visited Qingdao Youdu Biotechnology Co. LTD.
- 2. Completed the recruitment of science and technology campers.
- 3. Began to prepare flow cytometry and western blot.
- 4. We found some problems with standardized experiments and overcame them.
- 5. Commissioned the company to synthesize the circuit of the third system.
- 6. Prepared a summer training program for the members of the next team.
- 7. Completed the Interlab experiment and passed it successfully.

### 7.29-8.4

- 1. Planned the joint research on the transformation of basic research results with the iGEM team of Tianjin, XJTU-China, NUDT-China and JLU-China, which has been carried out.
- 2. Measured most of the data in the second system.
- 3. Had preliminarily built the mathematical models of the first system and the second system.
- 4. Planned to have a project communication with the iGEM team of Peking, Tsinghua, BIT, BNU-China and USTC-China on August 7. We start to prepare PPT to introduce our project.
- 5. Prepared the materials needed by the life science and technology camp.

### 8.5-8.11

- 1. Went to Beijing for visa.
- 2. Had a project communication with the iGEM team of Peking, Tsinghua, BIT, BNU-China and USTC-China.
- 3. Went to the institute of microbiology of the Chinese academy of sciences to consult Pr. Du, and got many constructive Suggestions, improved our experimental methods.
- 4. Started writing wiki content.
- 5. The life science and technology camp that we run jointly with SDU-China opened.
- 6. Started the pre-experiment of the miniToe Motility detection system.
- 7. Designed the experimental scheme of flow cytometer.
- 8. Promoted the comic book drawn by the previous team, and commissioned teams from different countries to translate them into different languages. At present, we have made contact with teams from five countries.
- 9. Bought tickets for Jamboree.

### 8.12-8.18

- 1. The life science and technology camp ended successfully. We introduced iGEM and synthetic biology to more college students.
- 2. The preliminary experiment of flow cytometry was carried out.
- 3. Prepared PPT for CCiC.
- 4. Completed the CSS template for the web page and completed the team member page.
- 5. Standardized Csy4-Y176F and Csy4-H29A.
- 6. The pre-experiment of miniToe Motility detection system was carried out to test the solidification of different media, the diffusion of molecules in the medium was simulated with methylene blue, and the movement of DH5α, RP437, RP6666 in the medium of casein medium, LB medium and peptone medium was tested.
- 7. The first circuit of the polycistron is synthesized.

### 8.19-8.25

- 1. Carried out the experiments of flow cytometry.
- 2. Prepare the materials needed by CCiC, including the poster, presentation and after iGEM's speech draft and PPT, and record video.
- 3. Completed the pre-experiment of miniToe Motility detection system, and the effects of tryptophan medium and casein peptone medium on the motion of RP6666 and RP437 were compared on the plate. In addition, puncture culture in the EP tube was also done, but the experimental results were not good.
- 4. Standardized miniToe test system No.2.
- 5. Plasmid construction of miniToe Motility detection system and polycistron system is under way.
- 6. Completed the material summary of summer social investigation.

### 8.26-9.1

- 1. Went to Shanghai to participate in CCiC.
- 2. Introduced our comic book to the competition organizing committee and the teams from over 100 different schools on CCiC.
- 3. Measured the flow cytometry of all 30 bacteria in the second system, and measured the fluorescence growth curve of wild type × wild type bacteria.
- 4. Perfected the design of the control group.
- 5. Standardized miniToe test system No.3.
- 6. Bought new experimental supplies.

### 9.2-9.8

- 1. Began the experiment of the polycistron system.
- 2. Finished protocol for the miniToe Motility detection system.
- 3. Contacted four different teams (NWU-China, SKLMT-China, SDU-China, Tianjin) for collaboration.
- 4. Completed the second round of wiki writing.
- 5. Standardized Csy4-Q104A.
- 6. Finished the attribution page.

### 9.9-9.15

- 1. Repeated the experiment with the flow cytometer of the second system.
- 2. Sent off collaboration lab materials to other teams.

### 9.16-9.22

- 1. Started the miniToe Motility detection system experiment.
- 2. Conducted the polycistron system experiment, testing the 24-hour growth curve and fluorescence expression curve of the experimental group and many control groups.
- 3. Started the collaboration experiment with SKLMT-China to help them test the promoter strength.
- 4. Planned our trip to America.

### 9.23-9.29

- 1. Standardized Csy4-F155A.
- 2. Modified the experimental scheme of miniToe Motility detection system.
- 3. Helped build part of the model for XJTU-China and finished the mathematical model of our last system.
- 4. Finished the pages of safety and notebook.
- 5. Received comic books in Turkish, and our current comic book is available in 12 languages.
- 6. Arranged our work for the next month.

### 9.30-10.6

- 1. Performed a repeat experiment with flow cytometry.
- 2. Conducted IPTG induction experiments for miniToe Motility detection system and tested the motility of RP6666 strain containing motA plasmid.
- 3. Repeat experiment of the polycistron system.
- 4. Send parts.
- 5. We were writing the contents of the wiki.
- 6. Printed the project manual.
- 7. Uploaded the overview, design, results, demonstrate, safety, miniToe, part improve, human practice and education page.

### 10.7-10.17

- 1. The effects of medium and IPTG on the experimental group were verified in the miniToe Motility detection system.
- 2. Repeated a flow cytometry experiment with miniToe family and Csy4-WT.
- 3. Repeated a fluorescence microscope experiment.
- 4. Finished the judging form.
- 5. Wrote the part.
- 6. Wrote the contents of the wiki.
- 7. Uploaded the main page, experiment, interlab, model, overview, miniToe family, polycistron and part page.
- 8. Designed the poster.
- 9. Summarized the Q&A of the project.
- 10. Started writing PPT and lecture notes for the project.
- 11. Printed comic books in 13 languages.

/>

Contact Us : oucigem@163.com | ©2018 OUC IGEM.All Rights Reserved.
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# Team:OUC-China/Experiments

Team OUC-China: Main

TOP

* [Main](https://2018.igem.org/Team%3AOUC-China)
* Project
  + [Overview](https://2018.igem.org/Team%3AOUC-China/Description)
  + [Design](https://2018.igem.org/Team%3AOUC-China/Design)
  + [Results](https://2018.igem.org/Team%3AOUC-China/Results)
  + [Demonstrate](https://2018.igem.org/Team%3AOUC-China/Demonstrate)
* Lab WORK
  + [Experiments](https://2018.igem.org/Team%3AOUC-China/Experiments)
  + [Safety](https://2018.igem.org/Team%3AOUC-China/Safety)
  + [InterLab](https://2018.igem.org/Team%3AOUC-China/InterLab)
* Model
  + [Overview](https://2018.igem.org/Team%3AOUC-China/Model)
  + [miniToe](https://2018.igem.org/Team%3AOUC-China/miniToe)
  + [miniToe Family](https://2018.igem.org/Team%3AOUC-China/miniToe%20Family)
  + [polycistron](https://2018.igem.org/Team%3AOUC-China/polycistron)
* Parts
  + [List](https://2018.igem.org/Team%3AOUC-China/Parts)
  + [Improve](https://2018.igem.org/Team%3AOUC-China/Improve)
* HP
  + [Human Practices](https://2018.igem.org/Team%3AOUC-China/Human_Practices)
  + [Public Engagement](https://2018.igem.org/Team%3AOUC-China/Public_Engagement)
* Team
  + [Team Member](https://2018.igem.org/Team%3AOUC-China/Team)
  + [Collaborations](https://2018.igem.org/Team%3AOUC-China/Collaborations)
  + [Attributions](https://2018.igem.org/Team%3AOUC-China/Attributions)
* [Notebook](https://2018.igem.org/Team%3AOUC-China/Notebook)

![banner](https://static.igem.org/mediawiki/2018/a/ab/T--OUC-China--experiment.jpg)

## Experiments

###

This year, our project can be divided into four systems. Between the different systems, there are different protocols. And we have sorted out these experimentals in detail.

#### 1.Elementary Operation

（1）[Conservation](https://static.igem.org/mediawiki/2018/d/d2/T--OUC-China--conserva.pdf)

  （2）[Gel Electrophoresis](https://static.igem.org/mediawiki/2018/9/9d/T--OUC-China--gel.pdf)

  （3）[Gel Extraction](https://static.igem.org/mediawiki/2018/e/e2/T--OUC-China--gel2.pdf)

  （4）[Inoculation](https://static.igem.org/mediawiki/2018/1/12/T--OUC-China--ino.pdf)

  （5）[Ligation system](https://static.igem.org/mediawiki/2018/f/fe/T--OUC-China--liga.pdf)

  （6）[medium](https://static.igem.org/mediawiki/2018/1/11/T--OUC-China--medium.pdf)

  （7）[Plasmid extraction](https://static.igem.org/mediawiki/2018/3/35/T--OUC-China--plas1.pdf)

  （8）[Polymerase Chain Reaction](https://static.igem.org/mediawiki/2018/d/d2/T--OUC-China--poly.pdf)

  （9）[Restriction Enzyme Digestion](https://static.igem.org/mediawiki/2018/6/6e/T--OUC-China--res.pdf)

  （10）[Sterilization](https://static.igem.org/mediawiki/2018/9/98/T--OUC-China--ster.pdf)

  （11）[Transformation of Escherichia coli](https://static.igem.org/mediawiki/2018/8/86/T--OUC-China--transr.pdf)

#### 2.Systems Detection

（1）[Assay of Gene Expression by Flow Cytometry](https://static.igem.org/mediawiki/2018/6/61/T--OUC-China--assay.pdf)

  （2）[Fluorescence measurement](https://static.igem.org/mediawiki/2018/b/b1/T--OUC-China--fluo.pdf)

  （3）[miniToe Polycistron Fluorescence measurement](https://static.igem.org/mediawiki/2018/d/dd/T--OUC-China--minitoe.pdf)

  （4）[The application of miniToe——Regulation of motA](https://static.igem.org/mediawiki/2018/f/f6/T--OUC-China--thepp.pdf)

Contact Us : oucigem@163.com | ©2018 OUC IGEM.All Rights Reserved.
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# Team:OUC-China/Results

Team OUC-China: Main

TOP

* [Main](https://2018.igem.org/Team%3AOUC-China)
* Project
  + [Overview](https://2018.igem.org/Team%3AOUC-China/Description)
  + [Design](https://2018.igem.org/Team%3AOUC-China/Design)
  + [Results](https://2018.igem.org/Team%3AOUC-China/Results)
  + [Demonstrate](https://2018.igem.org/Team%3AOUC-China/Demonstrate)
* Lab WORK
  + [Experiments](https://2018.igem.org/Team%3AOUC-China/Experiments)
  + [Safety](https://2018.igem.org/Team%3AOUC-China/Safety)
  + [InterLab](https://2018.igem.org/Team%3AOUC-China/InterLab)
* Model
  + [Overview](https://2018.igem.org/Team%3AOUC-China/Model)
  + [miniToe](https://2018.igem.org/Team%3AOUC-China/miniToe)
  + [miniToe Family](https://2018.igem.org/Team%3AOUC-China/miniToe%20Family)
  + [polycistron](https://2018.igem.org/Team%3AOUC-China/polycistron)
* Parts
  + [List](https://2018.igem.org/Team%3AOUC-China/Parts)
  + [Improve](https://2018.igem.org/Team%3AOUC-China/Improve)
* HP
  + [Human Practices](https://2018.igem.org/Team%3AOUC-China/Human_Practices)
  + [Public Engagement](https://2018.igem.org/Team%3AOUC-China/Public_Engagement)
* Team
  + [Team Member](https://2018.igem.org/Team%3AOUC-China/Team)
  + [Collaborations](https://2018.igem.org/Team%3AOUC-China/Collaborations)
  + [Attributions](https://2018.igem.org/Team%3AOUC-China/Attributions)
* [Notebook](https://2018.igem.org/Team%3AOUC-China/Notebook)

![banner](https://static.igem.org/mediawiki/2018/7/73/T--OUC-China--proresults.jpg)

* [1. THE RESULTS OF FIRST SYSTEM: MINITOE](#tips1)
* [2. THE RESULTS OF SECOND SYSTEM: MINITOE FAMILY](#tips2)
* [3. THE RESULT OF THIRD SYSTEM: MINITOE POLYCISTRON](#tips3)
* [4. THE RESULT OF FOURTH SYSTEM: MINITOE BASED MOTILITY DETECTION SYSTEM](#tips4)

## Results

![](https://static.igem.org/mediawiki/2018/2/29/T--OUC-China--project-_overview-upupup.jpg)

To help you enjoy our result in a more convenient and smooth way, here are some key concepts in our discussion.

1) miniToe system
2) miniToe family system (The miniToe family refers to the whole second system below)
3) miniToe polycistron system (The miniToe polycistron refers to the whole third system below)
4) miniToe based motility detection system

The first system (miniToe system) is the basics of other three systems. As description we introduced above, on the level of **DNA**, we design a **miniToe part** and construct it to circuits. On the level of **RNA**, we focus on **miniToe structure**. In the miniToe family system, we focus on Csy4 **hairpin** which is a part of **miniToe part**. In fact, the Csy4 hairpin is the miniToe target region. **A cis-repressive RNA (crRNA)** served as a translation suppressor by pairing with RBS is critical part of miniToe structure.

![](https://static.igem.org/mediawiki/2018/7/72/T--OUC-China--Pic.jpg)

Also, the miniToe system is a kind of **translation activator**. In miniToe family, the **capabilities** of different Csy4 mutants reflect their **activation efficiency** to turn on translation. So the fluorescence intensity reflect **activation efficiency** which is a comprehensive ability including the capabilities of recognition and cleavage.

![](https://static.igem.org/mediawiki/2018/f/ff/T--OUC-China--project-_overview.png )

### 1. The results of first system: miniToe

#### 1.1 Plasmid construction

First, we use an inducible promoter P*tac*  to regulate the expression of Csy4 (pCsy4). Without the inducer isopropyl-β-d-thiogalactoside (IPTG), no Csy4 is produced. Otherwise, Csy4 can produce. As for another plasmid pRepoter, the superfolder green fluorescent protein (sfGFP) is the reporter gene to reflect output of our system under miniToe regulation, the expression of this gene is driven by a constitutive promoter named J23119 from Anderson family. The Csy4 hairpin is inserted between RBS and cis-repressive RNA region.

![](https://static.igem.org/mediawiki/2018/6/68/T--OUC-China--res1.png)

Fig.1-1 The two plasmids of miniToe system. The pCsy4 is constructed for the expression of Csy4. The pReporter contains miniToe part.

#### 1.2 The measurement of growth rates

After circuit construction to get two plasmids pCsy4 and pReporter, we transformed them into *E. coli* DH5 Alpha and got the recombinant strain with miniToe system successfully. For the sake of functional test, 5 different groups are set, the control group *E. coli* DH5 Alpha, the pCsy4 only group, the pReporter only group, the pCsy4&pReporter with IPTG group and the pCsy4&pReporter without IPTG group. As preliminary experiment, the growth rate measurements are essential. The curve below demonstrates all the groups have almost the same tendency of OD600 with the negative control strain during the entire cultivation period. It means that miniToe system has no negative influence on the growth of recombinant strain. The metabolic stress by two plasmids is not harmful to the recombinant strains.

![](https://static.igem.org/mediawiki/2018/d/d3/T--OUC-China--JCod1.png)

Fig.1-2 Growth curve of strains we used in experiments. Error bars represent standard deviation of four biological replicates. (Measured by microplate reader)

#### 1.3 Proof of function

The microplate reader is used to test the intensity of superfolder green fluorescent protein (sfGFP) which is changed over time. The aim is to prove that miniToe system can control the downstream gene expression during the whole cultivation period.

The following chart shows the dynamic curve measured by microplate reader every two hours. The yellow line refers to the test group which is recombinant strain (with the whole miniToe system) with IPTG (0.125mM). The blue line shows the change of fluorescence intensity in recombinant strain (with the whole miniToe system) without IPTG (0mM). The green line refers to another control group which only has pReporter without the pCsy4 in strain. The results help us to prove two problems in miniToe system.

![](https://static.igem.org/mediawiki/2018/e/ed/T--OUC-China--res3.png)

### 2. The results of second system: miniToe family

#### 2.1 Plasmid construction

After building the ODE model, we use it to simulate the dynamics of sfGFP. Comparing with the experimental data, it fits perfectly, which indicates that the model is reliable about first system. By analyzing the sensitivity of the GFP level in the system to cleavage rate by model, it is not difficult to predict that the cleavage rate has an influence in the expression of sfGFP. It means we may change the expression level of sfGFP if we employ different mutants of Csy4 proteins.

There are two ways in second system which can help to achieve our goal. One is to design some Csy4 mutants and the other is to design the miniToe mutants. There are all the plasmids we used in Fig.2-1 and Fig.2-2.

![](https://static.igem.org/mediawiki/2018/4/4f/T--OUC-China--res21.png)

Fig.2-1 The plasmids of Csy4 mutants. The pCsy4 is a plasmid which contains Csy4 and we also use it in first system. The pCsy4-Q104A is a plasmid which contains Csy4-Q104A. The pCsy4-Y176F is a plasmid which contains Csy4-Y176F. The pCsy4-F155A is a plasmid which contains Csy4- F155A. The pCsy4-H29A is a plasmid which contains Csy4- H29A.

![](https://static.igem.org/mediawiki/2018/8/8b/T--OUC-China--res22.png)

Fig.2-2 The plasmids of miniToe mutants. The pReporter is a plasmid which contains miniToe-WT and we also use it in first system. The pReporter-1 is a plasmid which contains miniToe-1. The pReporter-2 is a plasmid which contains miniToe-2. The pReporter-3 is a plasmid which contains miniToe-3. The pReporter-4 is a plasmid which contains miniToe-4. The pReporter-5 is a plasmid which contains miniToe-5.

![](https://static.igem.org/mediawiki/2018/d/d3/T--OUC-China--pCsy4.jpg)

![](https://static.igem.org/mediawiki/2018/5/51/T--OUC-China--pRep.jpg)

After plasmid construction, we proved the functions of Csy4 mutants by wet experiments first.

#### 2.2.1 Proof of functions about Csy4 mutants

In this part, three kinds of experiments help us to confirm the functions of Csy4 mutants including recognition and cleavage. Our expectation is that by using new Csy4 mutants, the expression level of sfGFP vary with Csy4s' capabilities. It means that miniToe family members present various expression of target genes.

#### Prediction

Before the experiments, model proved our ideas. The predication shows the possibilities of different expression levels by different Csy4 mutants. It is not difficult to predict that the cleavage rate has an influence in the expression of sfGFP. The models help us go further this year!

![](https://static.igem.org/mediawiki/2018/c/ca/T--OUC-China--res23.png)

Fig.2-3 The predication: the fluorescence intensities by different Csy4 mutants along with time

We designed three kinds experiments to test the capabilities of five Csy4 mutants by putting them into miniToe system. So the recombination strains for test both have same pReporter but different Csy4 mutants plasmids in the following. The recombination strains to test the functions of Csy4 are strain-Csy4 (pCsy4&pReporter), strain-Csy4-Q104A (pCsy4-Q104A&pReporter), strain-Csy4-Y176F (pCsy4-Y176F&pReporter), strain-Csy4-F155A (pCsy4-F155A&pReporter), strain-Csy4-H29A (pCsy4-H29A&pReporter). At the same time, we have a control strain named strain-miniToe-only which only has pReporter.

![](https://static.igem.org/mediawiki/2018/7/7f/T--OUC-China--plasmid.jpg)

#### 2.2.2 The result by Microscope

First, we tested the capabilities of five Csy4 mutants by Fluorescent Stereo Microscope Leica M165 FC. The sfGFP accumulated during the cultivation period so the fluorescence can be observed by microscope after 8 hours. Because the five Csy4 mutants have different capabilities of cleavage, the distinguishing intensities of fluorescent can be seen by naked eyes. The five test strains have same miniToe part but different Csy4 mutant genes. In Fig.2-4, there are fluorescence images by fluorescent microscope which indicate strain-Csy4, strain-Csy4-Q104A, strain-Csy4-Y176F, strain-Csy4-F155A and strain-Csy4-H29A in sequence. The visible distinctions have shown in these images. The fluorescence intensities decrease one by one from top to bottom which means the Csy4s' capabilities of cleavage decrease one by one. The Csy4-WT has the strongest capability of cleavage when the Csy4-H29A is a kind of dead-Csy4 (dCsy4) which is hardly to find the fluorescence by microscope. The qualitative experiment is a basis of further experiments.

![](https://static.igem.org/mediawiki/2018/2/28/T--OUC-China--JCYWT.png)

Fig.2-4-1 The expression of sfGFP by strain-Csy4.

![](https://static.igem.org/mediawiki/2018/1/19/T--OUC-China--JCYQ2.png)

Fig.2-4-2 The expression of sfGFP by strain-Csy4-Q104A.

![](https://static.igem.org/mediawiki/2018/b/bb/T--OUC-China--JCYY.png)

Fig.2-4-3 The expression of sfGFP by strain-Csy4-Y176F.

![](https://static.igem.org/mediawiki/2018/8/88/T--OUC-China--JCYF.png)

Fig.2-4-4 The expression of sfGFP by strain-Csy4-F155A.

![](https://static.igem.org/mediawiki/2018/f/fd/T--OUC-China--JCYH.png)

Fig.2-4-5 The expression of sfGFP by strain-Csy4-H29A.

Fig.2-4 The fluorescence images by fluorescent microscope. From top to bottom, the images shows the expression of sfGFP by strain-Csy4, strain-Csy4-Q104A, strain-Csy4-Y176F, strain-Csy4-F155A and strain-Csy4-H29A in sequence. The plotting scale is on the right corner of images. The images on the left shows *E. coli* without fluorescence excitation. The images on the right represent situation when fluorescence excitation.

#### 2.2.3 The result by flow cytometer

 The qualitative experiment is not enough to analyze the Csy4 mutants. So we tested miniToe family system by flow cytometer. The expression of sfGFP by strain-Csy4, strain-Csy4-Q104A, strain-Csy4-Y176F, strain-Csy4-F155A and strain-Csy4-H29A is showed in Fig.2-5, and their intensities of fluorescence are from strong to weak.

![](https://static.igem.org/mediawiki/2018/e/e8/T--OUC-China--fig2-5z.png)

Fig.2-5 The fluorescence intensities of sfGFP about Csy4 mutants by flow cytometer. Histograms show distribution of fluorescence in samples with strain-Csy4 (Black), strain-Csy4-Q104A (Orange), strain-Csy4-Y176F (Red), strain-Csy4-F155A (Blue), strain-Csy4-H29A (Green). Crosscolumn number shows fold increase of sfGFP fluorescence.

![](https://static.igem.org/mediawiki/2018/d/da/T--OUC-China--JCPE.png)

Fig.2-6 The Gate Mean of flow cytometer. Histograms show the relative expression of sfGFP. The five test groups present different fluorescence intensities from high to low which prove that they have different capabilities of cleavage.

#### 2.2.4 The result by microplate reader

Besides all the works before, we also need to know more information about the Csy4 mutants in entire cultivation period. Even though we known that our Csy4 mutants have differentiated expression level in ten-hour-culture, the expression of whole cultivation period is also a reference for us to know if our system can work as expectations.

### 3. The result of third system: miniToe polycistron

#### 3.1 The purpose of experiment

The miniToe polycistron is a new method designed by OUC-China this year. By inserting miniToe hairpins between intergenetic regions, it will tune the translation level of corresponding proteins.
1) First, sfGFP and mCherry is used as a test system in bi-cistron circuit.
2) Then we selected some miniToe parts and inserted them between, before and behind sfGFP and mCherry. For example, for bi-cistron, then three miniToe parts will be inserted. for three genes in polycistron, then four miniToe parts will be inserted, and so on.

This year, we have two kinds of miniToe polycistron, miniToe polycistron-A and miniToe polycistron-B. In the future, we will test more miniToe polycistron based on miniToe family.

![]( https://static.igem.org/mediawiki/2018/a/a6/T--OUC-China--res31.jpg)

Fig.3-1 The two test groups. Group A is the control group without miniToe system. Group B is the test group with miniToe system.

### 4. The result of fourth system: miniToe based Motility detection system

#### 4.1 The purpose of designing the experiment

As is shown in the first system miniToe, we created a new method to regulate the downstream gene expression. Furthermore, we proved that our system can be enlarged and then we created miniToe family system based on the mutation of miniToe part and Csy4 mutants. As and translation regulation tool, MiniToe can also be used in application scenario of molecular mechanism research. Sometimes scientists may puzzle with the functions of certain gene or protein without in-depth study. One general method to study them is knock-out or knock-in methods. In this way, organisms show some phenotypic change related to particular gene. However, if we want to know better about the functions of the gene, we may need more tool to change gene expression at different levels.

By using our system, the motility of *E. coli* can be regulated. We transformed the miniToe system into *E. coli* whose motility is regulated by the motor protein, MotA. MotA provides a channel for the proton gradient required for generation of torque. Δ*motA* strains (the *motA*-deletion strain) can build flagella but are non-motile because they are unable to generate the torque required for flagellar rotation.

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