iGEM 2021 — Team OUC-China

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# Team:OUC-China/Notebook

# Notebook

Document the dates you worked on your project. This should be a detailed account of the work done each day for your project.

### What should this page have?

* Chronological notes of what your team is doing.
* Brief descriptions of daily important events.
* Pictures of your progress.
* Mention who participated in what task.

### Inspiration

You can see what others teams have done to organize their notes:

* [2018 Munich](https://2018.igem.org/Team%3AMunich/Notebook)
* [2019 Georgia State](https://2019.igem.org/Team%3AGeorgia_State/Notebook)
* [2019 Newcastle](https://2019.igem.org/Team%3ANewcastle/Notebook)
* [2020 IISER Pune India](https://2020.igem.org/Team%3AIISER-Pune-India/Notebook)
* [2020 Lund](https://2020.igem.org/Team%3ALund/Notebook)
* [2020 NOVA LxPortugal](https://2020.igem.org/Team%3ANOVA_LxPortugal/Notebook)
* [2020 RDFZ China](https://2020.igem.org/Team%3ARDFZ-China/NoteBook)

[![](https://static.igem.org/mediawiki/2021/8/8b/HQ_page_logo.jpg)](https://2021.igem.org)

# Team:OUC-China/Engineering

OUC-China/NAV

* PROJECT
  + [Description](https://2021.igem.org/Team%3AOUC-China/Description)
  + [Design](https://2021.igem.org/Team%3AOUC-China/Design)
  + [Engineering](https://2021.igem.org/Team%3AOUC-China/Engineering)
  + [Contribution](https://2021.igem.org/Team%3AOUC-China/Contribution)
  + [Implementation](https://2021.igem.org/Team%3AOUC-China/Implementation)
  + [Proof of Concept](https://2021.igem.org/Team%3AOUC-China/Proof_Of_Concept)
* WET LAB
  + [Results](https://2021.igem.org/Team%3AOUC-China/Results)
  + [Experiments](https://2021.igem.org/Team%3AOUC-China/Experiments)
  + [Measurement](https://2021.igem.org/Team%3AOUC-China/Measurement)
* DRY LAB
  + [Hardware](https://2021.igem.org/Team%3AOUC-China/Hardware)
  + [Model](https://2021.igem.org/Team%3AOUC-China/Model)
* [PARTS](https://2021.igem.org/Team%3AOUC-China/Parts)
* ![](https://static.igem.org/mediawiki/2021/archive/1/13/20211006161521%21T--OUC-China--team_logo.png)
* HUMAN PRACTICE
  + [Integrated HP](https://2021.igem.org/Team%3AOUC-China/Human_Practices)
  + [Collaborations](https://2021.igem.org/Team%3AOUC-China/Collaborations)
  + [Partnership](https://2021.igem.org/Team%3AOUC-China/Partnership)
  + [Education](https://2021.igem.org/Team%3AOUC-China/Education)
  + [Communication](https://2021.igem.org/Team%3AOUC-China/Communication)
  + [Sustainable](https://2021.igem.org/Team%3AOUC-China/Sustainable)
* TEAM
  + [People](https://2021.igem.org/Team%3AOUC-China/Team)
  + [Attribution](https://2021.igem.org/Team%3AOUC-China/Attributions)
* [MEDAL REQUIREMENTS](https://2021.igem.org/Team%3AOUC-China/Medal_Requirements)

OUC-China/back to top

![](https://static.igem.org/mediawiki/2021/8/80/T--OUC-China--Back_to_top_banner.png)

![]( https://static.igem.org/mediawiki/2021/9/96/T--OUC-China--Engenering_page_banner.png)

![](https://static.igem.org/mediawiki/2021/5/58/T--OUC-China--Engineer.jpg)

This year, OUC-China is devoted in designing a whole cell biosensor for detecting antibiotic in water sample. Our team has noticed the ever increasing contamination of new pollutants such as antibiotic. The needs of detecting, monitoring and controlling antibiotic boomed accordingly. So our original wisdom was to solve environmental contamination problems and creating a cleaner and safer environment for human welfare.

The very first group of people we decided to consult was the teachers in our university, who study the environmental toxicology. Because we believe our project should be problem-solving oriented. Thus, learning about the current situation of environmental pollution is definitely needed first. We then visited the professor Ru Shaoguo.

Prof. Ru almost provided us useful information in all possible aspects(It is so beneficial talking with such good scholars). Despite him, we later got to know more scholars in the field ecotoxicology through New Pollutants Workshop initiated by our team. The conversations with them made us confirmed the necessity and urgency of developing our project.

Professor Ru critically pointed out that the key difficulty of biosensor is to transform the chemical based input signal into electronic output signal. Using fluorescent protein such as GFP as the reporter has been widely applied in synthetic biology. But after professor Ru explained to us that in order to match the requirements of quantitative analysis, reporting the results by electronic signal is a must. And it is also a good way competing test paper analysis, whose results are usually evaluated by naked eyes, thus more of qualitative than quantitative. Being inspired by professors Ru’s recommendation, we eventually designed our hardware which is a portable box containing the whole cell biosensors. We also developed a mini apps to cooperate with the hardware by analyzing the fluorescence intensity of the photo taken through a whole on the hardware, and reporting the corresponding antibiotic concentration.

Prof. Ru recommended us to have interviews with people in sewage treatment plant and sewage treatment plant because they may be the potential users of our products.

We conducted field research in aquaculture farm and sewage treatment plant. Interestingly, these to stakeholders asserted different needs, which we would never consider if not interviewing both of them. The engineer and the director in a local sewage treatment plant named Haibohe told us that what they want was something has broad spectrum detection ability. Because antibiotic concentration was not one of their discharging index, thus there exists no need for knowing the specific concentration of it. But antibiotic, including a lot of other environmental pollutants, even the parameter of water may influence the living condition of anaerobic bacteria which is commonly functional in sewage treating procedure. Thus, they want a testing method which can indicate the general toxicity of sewage water.

However, since Chinese government has promulgated a series of regulations in order to reduce the usage of antibiotic in stock farming and aquaculture farming industry. The local Ministry of environmental protection would conduct spot check from time to time. “Where there exists standards, there exists needs”. When we asked a aquaculture farm owner whether he would be interested in a product which can detect a specific antibiotic’s concentration. He was surprised at that our product was designed to have such specificity. He encouraged us to extend our project into a platform witch can detect a series of antibiotic, exploring a bigger market. And he then emphasized that he would only be interested if the product is affordable. Because for private enterprises, their values always include “profit maximization”. They would usually make a balance between the investments and risks. If a biosensor which is cheep for use and need no hiring technicist can effectively monitor the concentration of antibiotic in tail water, it is probably more beneficial than taking the risk of being fined for discharging tail water containing excessive levels of antibiotics. The conversation with people in sewage treatment plant and aquaculture farm cleared our mind. Our design can not meet everyone’s expectation, because their needs might be controversial. Our design should service a specific group of people. And in order to meet their needs, we have to know them first. We decided that the aquaculture farmers are one of our target clients. And their needs include user-friendly, cheep and platformisation.

Next, in order to get to know how the antibiotics are detected in real world. We consulted the officers in Food and Drug Administration and Environment Bureau. Since in most of the cases, antibiotic is not a target contaminants for regular monitoring, the officer we consulted didn’t know much of antibiotic testing. We then turned to Food and Drug Administration for antibiotic is a traditional substance threatening food safety. The officers in Food and Drug Administration told us, government would entrust a third party testing company for antibiotic concentration.

After knowing this information, we actively contacted an international third party testing company named SGS. We were luckily welcomed and visited their chemical laboratory to learn the process of antibiotic testing. We learned that the commonly used approaches of antibiotic quantitative analyzing are generally based on chemistry. The accuracy is ensuring, but there are still spaces for biology based biosensor to complement or even surpass it. After learning the complicated pre-treatments which must be done to sample, we hope our design can detect the sample in original water sample. For these reason, we collected a series of water sample including sewage in different links of treating procedure, tail water in aquaculture farm and open sea water. We would like to test whether our biosensor can survive and be functional in different scenarios. Besides, in order to overcome the complicated and time consuming testing procedures(which usually takes a bout a week to got the results), we decided to give full play to the advantage of rapid test. Instead of choosing traditional fluorescent protein, we chose 3WJdB which is a RNA based fluorescent aptamer. Without the need of translation, we expected our product to respond to the target even sooner.

The engineer in SGS told us that the current concentration of antibiotic in environment is on the level of µg/kg. Thus, if our product can’t reach this detection limit, our results would be all negative. We felt thankful for this advice, and decided to try our best to reduce the detection limit. We then decided to add another DNA component, whose corresponding RNA product can complement to 3WJdB(the reporter we just mentioned). Supplemented by CRISPRi system, we expect our design can reduce the background leakage, increase dynamic rangeand amplify the signal.

In conclusion, according to a series of interviewing with scholars and stakeholders, as well as field research, we decided that we would like to design a whole cell biosensor which can detect antibiotic in original water sample, which low leakage, high dynamic range and amplified signal. We also like to enabling this design to effectively detect different antibiotics, so that it can be extended into a platform in the future. Combining the features mentioned above, we named our project--A.L.L.P.A.S.S, which stands for amplifying low leakage antibiotic biosensor. (See more detail in our design page).

After we constructed the circuit for detecting antibiotics, we carried out experiments and found that our experimental results can prove that the fluorescence intensity increases with the increase of antibiotic concentration in a certain range. However, its detection limit does not reach the desired result. We are thinking about the possible problems in the process of loop construction. Our laboratory administrator found that many of the bacterial solutions of the group with the reporter gene sfGFP were already green before our bacteria were cultured overnight for fluorescence measurement. After our team met and discussed, it was considered that it was the leakage of T7 promoter. We also consulted our PI teacher Liu. He has the same idea as us.

Then our team began to look for ways to reduce the leakage of T7 promoter by adding a certain concentration of glucose during culture. We conducted experiments and found that T7 leakage was indeed reduced, but a new problem was ushered in. The growth of engineering bacteria was poor. We tried to solve the problem of T7 promoter leakage from another angle. We later added lacI to our constructed loop. Surprisingly, lacI can inhibit T7 leakage and will not have a great impact on the growth of engineering bacteria.
So that we can detect antibiotic with more flexible dynamic range and high sensitivity.

[![](https://static.igem.org/mediawiki/2021/8/8b/HQ_page_logo.jpg)](https://2021.igem.org)

# Team:OUC-China/Experiments

OUC-China/NAV

* PROJECT
  + [Description](https://2021.igem.org/Team%3AOUC-China/Description)
  + [Design](https://2021.igem.org/Team%3AOUC-China/Design)
  + [Engineering](https://2021.igem.org/Team%3AOUC-China/Engineering)
  + [Contribution](https://2021.igem.org/Team%3AOUC-China/Contribution)
  + [Implementation](https://2021.igem.org/Team%3AOUC-China/Implementation)
  + [Proof of Concept](https://2021.igem.org/Team%3AOUC-China/Proof_Of_Concept)
* WET LAB
  + [Results](https://2021.igem.org/Team%3AOUC-China/Results)
  + [Experiments](https://2021.igem.org/Team%3AOUC-China/Experiments)
  + [Measurement](https://2021.igem.org/Team%3AOUC-China/Measurement)
* DRY LAB
  + [Hardware](https://2021.igem.org/Team%3AOUC-China/Hardware)
  + [Model](https://2021.igem.org/Team%3AOUC-China/Model)
* [PARTS](https://2021.igem.org/Team%3AOUC-China/Parts)
* ![](https://static.igem.org/mediawiki/2021/archive/1/13/20211006161521%21T--OUC-China--team_logo.png)
* HUMAN PRACTICE
  + [Integrated HP](https://2021.igem.org/Team%3AOUC-China/Human_Practices)
  + [Collaborations](https://2021.igem.org/Team%3AOUC-China/Collaborations)
  + [Partnership](https://2021.igem.org/Team%3AOUC-China/Partnership)
  + [Education](https://2021.igem.org/Team%3AOUC-China/Education)
  + [Communication](https://2021.igem.org/Team%3AOUC-China/Communication)
  + [Sustainable](https://2021.igem.org/Team%3AOUC-China/Sustainable)
* TEAM
  + [People](https://2021.igem.org/Team%3AOUC-China/Team)
  + [Attribution](https://2021.igem.org/Team%3AOUC-China/Attributions)
* [MEDAL REQUIREMENTS](https://2021.igem.org/Team%3AOUC-China/Medal_Requirements)

OUC-China/back to top

![](https://static.igem.org/mediawiki/2021/8/80/T--OUC-China--Back_to_top_banner.png)

OUC-China/Result

![](https://static.igem.org/mediawiki/2021/7/7d/T--OUC-China--Experiment_page_banner.png)

[We used the following protocols to build our circuits, and each circuit has amazing results. You can click here to see the results.](https://2021.igem.org/Team%3AOUC-China/Results)

# 1. Elementary Operation

[![](https://static.igem.org/mediawiki/2021/8/8b/HQ_page_logo.jpg)](https://2021.igem.org)

# Team:OUC-China/Results

OUC-China/NAV

* PROJECT
  + [Description](https://2021.igem.org/Team%3AOUC-China/Description)
  + [Design](https://2021.igem.org/Team%3AOUC-China/Design)
  + [Engineering](https://2021.igem.org/Team%3AOUC-China/Engineering)
  + [Contribution](https://2021.igem.org/Team%3AOUC-China/Contribution)
  + [Implementation](https://2021.igem.org/Team%3AOUC-China/Implementation)
  + [Proof of Concept](https://2021.igem.org/Team%3AOUC-China/Proof_Of_Concept)
* WET LAB
  + [Results](https://2021.igem.org/Team%3AOUC-China/Results)
  + [Experiments](https://2021.igem.org/Team%3AOUC-China/Experiments)
  + [Measurement](https://2021.igem.org/Team%3AOUC-China/Measurement)
* DRY LAB
  + [Hardware](https://2021.igem.org/Team%3AOUC-China/Hardware)
  + [Model](https://2021.igem.org/Team%3AOUC-China/Model)
* [PARTS](https://2021.igem.org/Team%3AOUC-China/Parts)
* ![](https://static.igem.org/mediawiki/2021/archive/1/13/20211006161521%21T--OUC-China--team_logo.png)
* HUMAN PRACTICE
  + [Integrated HP](https://2021.igem.org/Team%3AOUC-China/Human_Practices)
  + [Collaborations](https://2021.igem.org/Team%3AOUC-China/Collaborations)
  + [Partnership](https://2021.igem.org/Team%3AOUC-China/Partnership)
  + [Education](https://2021.igem.org/Team%3AOUC-China/Education)
  + [Communication](https://2021.igem.org/Team%3AOUC-China/Communication)
  + [Sustainable](https://2021.igem.org/Team%3AOUC-China/Sustainable)
* TEAM
  + [People](https://2021.igem.org/Team%3AOUC-China/Team)
  + [Attribution](https://2021.igem.org/Team%3AOUC-China/Attributions)
* [MEDAL REQUIREMENTS](https://2021.igem.org/Team%3AOUC-China/Medal_Requirements)

OUC-China/back to top

![](https://static.igem.org/mediawiki/2021/8/80/T--OUC-China--Back_to_top_banner.png)

OUC-China/Result

![](https://static.igem.org/mediawiki/2021/archive/9/9c/20211021063153%21T--OUC-China--Results_banner.png)

* [Deciding how to incubate the bacteria for the Plate-reader experiment](#area-1)
* [Verifying the function of Basic Circuits](#area-2)
* [Verify the function of Antibiotic resistance gene](#area-3)
* [Verify the construction of ‘improved circuit’](#area-4)
* [Verify the strand replacement reaction between KB2 and 3WJdB](#area-5)
* [Verify CRISPRi](#area-6)

## 1. Deciding how to incubate the bacteria for the Plate-reader experiment

Before we start formal experiments, we want to know how to incubate our engineered bacteria to ensure that they perform at their best during the plate-reader experiment. When the overnight culture was diluted and then re-grown to OD600=0.6-0.8, we adopted three different methods of incubation including shock culture in a flask and EP tubes, sampling every half hour for fluorescence detection, shock culture directly on a 96-well plate with vibrating plate device, and continuous sample measurement.

![](https://static.igem.org/mediawiki/2021/7/7d/T--OUC-China--3_way--to--incubate.png)

Fig. 1

As the result shows, we found 96-well plate will not affect the expression of our bacteria and finally chose 96 well plate as the most important way.

## 2. Verifying the function of Basic Circuits

The basic circuit is composed of a constitutively expressed allosteric Transcription Factor（aTF）a corresponding inducible promoter, and a reporter gene (see more details on Design page).

### 2.1 tetR-T7(tetO)-3WJdB, tetR-T7(tetO)-sfGFP

![](https://static.igem.org/mediawiki/2021/f/f6/T--OUC-China--result--result1.png)

Fig. 2.1.1 the fluorescence of tetR-T7(tetO)-3WJdB incubated in different concentrations of tetracycline.

TetR-T7(tetO)-3WJdB showed recognizable response to different concentrations of tetracycline, which means this WCB is able to detect tetracycline as low as 0.01μg/ml. Moreover, the result shows 3WJdB enable the WCB to have a faster response, as the fluorescence intensity matched the concentration gradient well only after 2 hours.

![](https://static.igem.org/mediawiki/2021/c/c5/T--OUC-China--result--Tsf.png)

Fig. 2.1.2 the fluorescence of tetR-T7(tetO)-sfGFP incubated in different concentrations of tetracycline.

The fluorescence intensity decreased with the increase of tetracycline concentration, contrary to our expectation. Moreover, it shows max fluorescence intensity with the absence of tetracycline, which means huge leakage of output signal which may be caused by the large leakage rate of T7 promoter. Since tetracycline inhibits protein production, sfGFP is also difficult to translate and thus decreases with increasing concentrations.

To further characterize these two circuits, we invited our partnership team ZJUT （see more in partnernership page）to test them in a cell-free system.

![](https://static.igem.org/mediawiki/2021/2/2d/T--OUC-China--result--vitro_3WJdB.png)

Fig 2.1.3 tetR-T7(tetO)-3WJdB characterization in cell free system

![](https://static.igem.org/mediawiki/2021/a/a5/T--OUC-China--%E4%BD%93%E5%A4%96_sfGFP.png)

Fig 2.1.4 tetR-T7(tetO)-sfGFP characterization in cell free system

Here are the analysis and speculation of this phenomenon: There exists the phenomenon that the gradient dependent fluorescence trend may be different (usually opposite) within different concentration range. We can see from the graph below that the trend in the second testing point (1 ng/µl) has already opposite to our expectation. One possible speculation is that the cell free system is ultra-sensitive, and 1 ng/µl of antibiotic has already exceeded the operating range. We can also see that the fluorescence intensity corresponding to 0ng/µl antibiotic is also 0. This fact indicated that the basal leakage of our genetic circuit is pretty good in cell free system. In the future, we would also like to try cell free system to achieve our goal for it have better basal leakage rate and other adorable attributes such as controllability and safety. And to further prove the concept, we have to design more testing point with lower antibiotic concentration range (at least below 1 ng/µl according to the data ZJUT provided)

### 2.2 ctcS- T7(ctcO)-3WJdB

![](https://static.igem.org/mediawiki/2021/9/9c/T--OUC-China--result--85ctcs-3wjdb.png)

Fig 2.2.1 the fluorescence of ctcS- T7(ctcO)-3WJdB incubated in different concentrations of chlortetracycline.

CtcS- T7(ctcO)-3WJdB can also quickly detect different concentrations of chlortetracycline. However, the fluorescence reached the maximum at the concentration of 1μg/ml, then gradually decreased, which shows the potential operating range of this circuit is between 0.05-1μg/ml.

### 2.3 mphR-ermC- T7(mphO)-3WJdB, mphR-ermC-T7(mphO)-sfGFP

![](https://static.igem.org/mediawiki/2021/6/64/T--OUC-China--result--mphrbc.png)

Fig 2.3.1 the fluorescence of mphR-ermC- T7(mphO)-3WJdB incubated in different concentrations of erythromycin.

Unfortunately, mphR-ermC- T7(mphO)-3WJdB showed no erythromycin - induced effects at all. The downward trend was caused by the growth of OD600 and constant fluorescent intensity. We changed different promoters in this circuit to find the reason of of no response on later experiments.

![](https://static.igem.org/mediawiki/2021/8/86/T--OUC-China--mphR-sfGFP.png)

Fig 2.3.2 the fluorescence of mphR-ermC- T7(mphO)-sfGFP incubated in different concentrations of erythromycin.

There was still no significant difference between the experimental groups.

---

## 3. Verify the function of Antibiotic resistance gene

We linked antibiotic resistance genes into the Basic circuit by molecular cloning. TetM was assembled downstream of ctcS or tetR and controlled by the same constitutive promoter (Pc2). Successful colonies were verified by colony PCR then confirmed with sequencing.

![](https://static.igem.org/mediawiki/2021/7/7a/T--OUC-China--result--tetM1.jpeg)

![](https://static.igem.org/mediawiki/2021/e/e4/T--OUC-China--result--tetM2.jpeg)

Fig. 3 Gel run of samples from colony PCR product of tetM. The lanes of correct length are marked with ‘√’.

### 3.1 tetR-tetM- T7(tetO)-3WJdB

![](https://static.igem.org/mediawiki/2021/d/d6/T--OUC-China--tetR-tetM-T7%28tetO%29-3WJdB.png)

Fig. 3.1 the fluorescence of tetR-tetM- T7(tetO)-3WJdB incubated in different concentrations of tetracycline.

TetM gene confers antibiotic resistance by producing ribosomal protection proteins, as an result, the WCB will have a wider operating range and keep robust when detecting relatively high concentration of antibiotics. The engineered bacteria were incubated in tetracycline at a maximum of 50 μ g/mL, and still maintained high fluorescence after 4 hours, indirectly reflecting the protective effect of tetM.

### 3.2 ctcS-tetM- T7(ctcO)-sfGFP

![](https://static.igem.org/mediawiki/2021/7/7f/T--OUC-China--ctcs-tetM-sfGFP.png)

Fig. 3.2 the fluorescence of ctcS-tetM- T7(tetO)-sfGFP incubated in different concentrations of chlortetracycline.

With the insertion of tetM gene, the reversed induction effect of ctcS-tetM-T7 (tetO)-sfGFP was corrected. In the absence of tetM, the inhibition of tetracycline or chlortetracycline on protein production is more significant than its induction. However, in the presence of tetM, the inhibition of sfGFP translation was reduced, and the normal induction effect become dominant.

---

## 4. Verify the construction of ‘improved circuit’

To construct the ‘improved circuit’ the KB2 and sgRNA are linked to both sides of the ‘basic circuit’. Successful colonies were verified by colony PCR then confirmed with sequencing.

![](https://static.igem.org/mediawiki/2021/a/aa/T--OUC-China--result--kb2.png)

Fig. 4 Gel run of samples from colony PCR product of KB2-tetR-3WJdB-sgRNA. The lanes of correct length are marked with ‘√’.

---

## 5. Verify the strand replacement reaction between KB2 and 3WJdB

To repress the leakage of 3WJdB, KB2 (a small antisense RNA which can bind to 3WJdB) is inserted downstream of a constitutive promoter (Pc1). By tuning the strength of Pc1, the output signal can be regulated

![](https://static.igem.org/mediawiki/2021/thumb/6/68/T--OUC-China--Model--structure--TetR-ODE-Verifying_strand_replacement_reaction.png/800px-T--OUC-China--Model--structure--TetR-ODE-Verifying_strand_replacement_reaction.png)

Fig. 5 Verifying the strand replacement reaction. Promoter J23104 is much stronger than J23110. TetR-T7(tetO)-3WJdB has no KB2, so it is set as a negative control.

The result of this experiment proved that it’s valid to repress the fluorescence in vivo, which strongly support the feasibility of the ‘improved circuit’.

---

## 6. Verify CRISPRi

![](https://static.igem.org/mediawiki/2021/thumb/b/bc/T--OUC-China--Everyfying_CRISPRi.png/800px-T--OUC-China--Everyfying_CRISPRi.png)

Fig. 6 the result of verifying the CRISPRi in our WCB.

We constructed a testing circuit to verify the CRISPRi in our WCB. An sfGFP gene was set downstream of the sgRNA binding site (bs2). The expression of dCas9 on another plasmid can be induced by 3OC6HSL (a kind of quorum sensing signal molecule) with a Plux promoter. The result shows that the fluorescence decreased with the increasing concentration of 3OC6HSL. It was proved that CRISPRi works properly.

From now on, the output signal suppression and de-suppression functions of the ‘improved Circuit’ in our whole project have been verified respectively.