iGEM 2022 — Team ouc-china

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# Team:OUC-China

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#

![Group Photo](https://static.igem.wiki/teams/4193/wiki/mediawiki/header-photo/2022-ouc-china-notebook-header-photo.png)

### Notebook

This notebook contains the timeline of our iGEM journey.

![](https://static.igem.wiki/teams/4193/wiki/other/2022-ouc-china-notebook-teamdiary.png)

### Protocol

* Part 1 Transporter RNA Construction and Testing
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  + PCR
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    Library：

    ![](https://static.igem.wiki/teams/4193/wiki/other/2022-ouc-china-notebook-pcr-library.png)

    Pool\_fwd has T7 promoter sequence: TAATACGACTCACTATAGGG

    Still use Pool\_fwd and Pool\_rev primer in reverse transcription
    Run 20 circles. Monitor size and quality of the PCR product on a 1.5% agarose gel. Take the optimum number of cycles for 123bp.

    ![](https://static.igem.wiki/teams/4193/wiki/other/2022-ouc-china-notebook-pcr-system.png)
  + Construction of RNA library (using T7 transcription kit)
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    ![](https://static.igem.wiki/teams/4193/wiki/other/2022-ouc-china-notebook-t7-system.png)

    1.Aliquot the 30 μL to sterile and RNase-free tubes, then centrifuge to prevent drops from hanging on the wall. Incubate overnight (16h) at 37 °C.
    2.Add 1/10 vol 3M NaAc(pH=6.5).
    3.Add 4ul Glycoblue.
    4.Add 2.5 vol ethanol pre-cooled at 20 °C, then Incubate the reaction for 1 h.
    5.Centrifuge at 4°C for 15min, remove the supernatant, dry for 5 min in the super clean bench, then resuspend by 20ul DEPC.
    6.Measure the RNA purity.
  + SELEX
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    1.Mix 10uL RNA, 10uL 2nM CO and 40uL 5×SELEX buffer, add DEPC to 200uL.
    2.Heat the solution for 5 min at 65 °C, then put at 21°C for 30min to form RNA-CO complex.
    3.Take 20uL pellets and wash the pellets thrice with 200uL B&W buffer (5 mM Tris–HCl, pH 7.5, 1 M NaCl, 0.5 M EDTA), then wash once with 200μL 1×SELEX buffer.
    4.Mix the washed pellets and RNA-CO complex and incubate for 1h at 37°C.
    5.Remove supernatant (i.e. unbound RNA and CO) after placing the tube on a magnetic rack for 1min.
    6.Resuspend by 200uL 1×SELEX buffer, then shake it gently, then incubate for 5 min. Remove the supernatant.
    7.Repeat step 6 for 2 times.
    8.Resuspend pellets by 100uL 300mM mannitol and 100uL 1×SELEX buffer, then incubate for 1h. Collect the supernatant.
    9.Add 20uL 3M pH5.5 NaAc and 2.5 times volume 100% absolute alcohol, then add 4uL ClycoBlue and store at -20°C for 1h.
    10.Centrifuge at 4°C for 15 min and remove supernatant. Dry for 5 min and then add 20uL DEPC to dilute.
    11.Use reverse transcription kit to conduct RT-PCR.
  + RNA Antisense Purification
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    Conduct RNA Antisense Purification (RAP) at round 3, 6 and 9. Almost all the steps are same as above except that we should add a step after step 7:

    Add 100 μL 1mM sorbitol and 100 μL 1×SELEX buffer to resuspend the pellets, then incubate for 5 min. Remove supernatant.
  + Assembly of Transporting RNA
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    1.Desperately dissolve RNA9, RNA10-1 dry powder using DEPC water.
    2. 4 μM RNA9 and 2 μM RNA10 was incubated at room temperature with 50 mM HEPES (pH 7.0) , 50 mM NaCl, 5 mM MGCL2, and 2 mM Cacl2 for 15 min. (the whole volume of this system is about 1 ml). Obtain the corresponding transfer RNA1 and transfer RNA2.
  + Aptamer Kd measurement
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    1.select 13 concentrations of RNA（0，0.1，0.2，0.3，0.4，0.5，0.75，1，1.5，2.5，5，7.5，10μM）
    2.Add 100μL 300mMGABA and 10μL 1×SELEX buffer, incubate for 1h under RT.
    3. Use microplate reader to measure the fluorescence intensity of cells with excitation wavelength at 485 nm and emission wavelength at 525 nm.
  + In vitro transport efficiency and rate measurement
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    Group 1: GABA (30mM) , transporting RNA (0.6 μM) and exosome (15 μM) were added to the buffer 50mM HEPES (pH 7.0) respectively, which contains 50mM Naci and 7mM CaCl2. The buffer was prepared with DEPC water.

    Group 2: GABA (20 mM) , transporting RNA (0.6 μM) and exosome (15 μM) were added to buffer 50 mM HEPES (pH 7.0) respectively, which contains 50 mM NaCI and 7 mM CaCl2. The buffer was prepared with DEPC water.

    Group 3: GABA (10 mM) , Transporting RNA (0.6 μM) and exosome (15 μM) were added in buffer 50mM HEPES (pH 7.0) respectively, which contains 50mM Naci and 7mM CaCl2. The buffer was prepared with DEPC water.

    Zero calibration group: Transporting RNA1(1.5 μM) and exosome (15 μM) suspensions were added to buffer 50 mM HEPES (pH 7.0) respectively, which contains 50 mM NACI, 7 mM CaCl2. The buffer was prepared with DEPC water.
    PS: The final concentration is shown in parentheses. The system size of the four groups was the same, and each group had 7 parallel samples for different sampling time.

    1.The system was transferred into 4 ° C incubator.
    2.Before sampling, the mixture was slightly oscillated and 5 ml was sampled every 20 min within 120 min.
    3.After sampling, the exosomes were removed by centrifugation. (The exosomes have to be completely removed.)
    PS: This step can be regarded as a termination step, sample processing can be stopped at this step
    4.The concentration of GABA in 1 ml supernatant was detected by BERTHERLOT method.
    i.4 ml pH 9.0 borate buffer and 1.4 ml 6% phenol were added respectively into the test tube and mixed.
    ii. Put it in a 40℃water bath, add 2.2mL 5% sodium hypochlorite solution by drop, and stay 20mins for coloration at a constant temperature.
    iii. Add DEPC water and dilute to 10 ml.
    5.The absorbance at 645 nm wavelength was measured in order of GABA concentration from low to high (Use zero calibration Group to calibrate).
    PS: the absorbance of Group 1 and Group 2 should be measured after zero calibration of the corresponding Group 3 samples.

* Part 2 Quorum Sensing
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  + Preparation of electrocompetent cells
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    1. Microscopy: to ensure that the sample is not infected with bacteria. Place sorbitol in a chromatography cabinet; and shovel a certain mass of ice.
    2. 40mL solution was taken to a large centrifuge tube and centrifuged at 5000r for 5min at 4℃
    3. At room temperature, the supernatant was removed, resuspended with 40mL of pretreatment solution and 400mL DTT for 30min
    4. It was centrifuged at 5000r for 5min at 4℃
    5. The supernatant was discarded and 10-15mL of sorbitol was added for cleaning. After three replicates, the samples were centrifuged at 5000r for 5min at 4℃
    6. According on the bacterial volume, 1 – 2mL of sorbitol was added for resuspension
    7. Add the mixture of 200 μL bacterial solution (80 μL) and 30 μL plasmid into an EP tube , then transfer it into an electric cup, stand on ice for 10-15min.
    8. A 1mL EP tube, 500 μL YPD and 500 μL sorbitol was taken
    9. Electric turn
    10. In the clean bench, 1 mL solution of mixing YPD with sorbitol was added and the solution from the transfer cup was transferred to the sterilized empty EP tube
    11. Body resuscitation: knockdown for 1.5-2h, extinction resistance for 1h, and expression
    12. Carry out plate coating
  + Preparation of 2-IP solution
    *arrow\_downward*

    50mg 2-IP was dissolved in 50ml of distilled water, and the bacteria were removed by 0.22 μm filter membrane in the clean bench, and stored at 4 ° C.
  + Determination of growth curve and background expression intensity of SSRE promoter of Aureobasidium melanogenum P16
    *arrow\_downward*

    1.activation of strain: 100μL of preserved glycerol stock Aureobasidium melanogenum P16 strain fluid was cultured at 28°C 175rpm for 24 hours in rocking device.
    2.Culture of the strain: The 100μL of Aureobasidium melanogenum P16 strain fluid was inoculated into 8 tubes of 5ml YPD liquid medium and labeled, then cultured in rocking device at 28°C,175rpm for 8h.
    3.Measurement: The culture tubes of 0,2,4,6,8,10,12,14 hours were taken out in turn. Next, using microplate reader to measure the concentration of the culture at 600 nm and the fluorescence intensity of the green fluorescent protein at 485 nm and 520 nm , respectively.
    4.Draw the curve: The growth curve of Aureobasidium melanogenum P16 can be drawn by plotting a set of OD values and the corresponding incubation time. The background expression intensity of SSRE promoter was plotted as the ratio of the measured fluorescence intensity to the OD value and the corresponding time.
  + Determination of dynamic regulatory range of SSRE promoter
    *arrow\_downward*

    1.Activation of strain: 100μL of preserved glycerol stock Aureobasidium melanogenum P16 strain fluid was cultured at 28°C,175rpm for 24 hours in rocking device.
    2.Culture of the strain: The 100μL of Aureobasidium melanogenum P16 strain fluid was inoculated into 40 tubes of 5ml YPD liquid medium and labeled, then cultured in rocking device at 28°C,175rpm for 8h(The OD value was 0.4).
    3.10 tubes of cultured bacteria solution were taken out and different volumes of 2-IP solution were added to make the final concentration of 2-IP reach 0,0.5, 1,1.5, 2μmol/L.
    4.Measurement: Add different concentrations of 2-IP to 200uL of bacteria solution, each concentration was incubated for 0,0.5, 1.25, and 1.5h. Then, measuring the express intensity of the green fluorescent protein at 485 nm and 520 nm , respectively.

* Part 3 Kill Switch
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  + RNA SELEX
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    Seen in Part 1
  + Functional Verification of Riboswitch
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    1.Add IPTG to induce protein expression (2.39mL 210mMIPTG per liter media).
    2.Add 50μl 0,50,100,150,200,250,300 mM GABA in LB liquid mediums respectively.
    3.Cells were grown in shaking LB liquid media overnight at 37℃.
    4.The culture was diluted 1/100 fold, cultured for 3 hours at 37℃.
    5.The fluorescence intensity of cells was measured by microplate reader with excitation wavelength at 485 nm and emission wavelength at 525 nm.
    6. Negative control experiment and blank control experiment were carried out according to the above steps (Negative control: Do not add the IPTG and GABA; Blank control: Add IPTG but do not add GABA).

* Part 4 Other experiments
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  + Agarose Gel Electrophoresis
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    1. Fold the weighing paper along the diagonal, weigh 0.2g agarose, then pour it into the conical flask.
    2. Pour 20mL TAE buffer into the flask and put it in the microwave oven for 3 minutes, until the agarose is completely dissolved.
    3. Inject 2μL GelRed into the liquid.
    4. Inset the “comb” into the liquid. Let it stand until the gel is solidified.
    5. Transfer the gel into the electrophoresis chamber, then pull out the “comb” gently. Pour into enough TAE buffer until the gel is submerged.
    6. Carefully use the pipette to transfer a 3μL marker into the first hole, then add 2μL PCR products in the rest holes.
    7. Set the voltage to 100V and the current to 100mA, turn on the power and wait for 40 minutes.
    8. Take out the gel and place it into the UV transilluminator and turn on the UV light.
    9. Compare with the marker, and determine the length of DNA.
    10.Recover the gel.

##### DISP

A project by the OUC-China & Research iGEM 2022 team.

##### Contact

*mail\_outline*
[OUCiGEM@163.com](OUCiGEM%40163.com)

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DISP 2022

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# Team:OUC-China

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#

![Design Header](https://static.igem.wiki/teams/4193/wiki/mediawiki/header-photo/2022-ouc-china-engineering-header-photo.png)

## Background

Nowadays, strain quality has become a major factor limiting the yield of fermentation industry. The engineering bacteria degenerate into wild-type due to mutation or some unknown endogenous regulation. They have more growth advantages than the production engineering bacteria and gradually occupy resources to become the dominant species. And the extraction process of the products by breaking the engineering bacteria is very complicated. We plan to design a platform that automatically regulates the production and screening of strains to extract product simply to ensure high-quality strains and improve the fermentation yield.

### Quorum sensing

#### Quorum sensing

**design**

We use cytokinin-mediated quorum sensing circuit from Arabidopsis thaliana makes our engineered microorganisms only reaches a certain population density will start fermentation process, used for decoupling competition between cell growth and fermentation.

![](https://static.igem.wiki/teams/4193/wiki/2022-ouc-china-quorum-sensing-system.jpg)

*Figure1. Signal molecule IP activates the AtCRE1-Ypd1-Skn7 phosphorylation pathway*

When the signal molecule IP reaches a certain concentration, it activates the SSRE promoter and increases the expression of production gene controlled by SSRE promoter.Therefore, when the number of engineered bacteria in the fermentation tank is small, the expression of the production genes will remain very low, which makes the engineered bacteria have higher competitiveness to the wild bacteria.

**Test**

In our experiment,to better characterize SSRE promoter and quorum sensing circuit,we put GFP gene at the downstream of SSRE promoter then detect them in Aureobasidium  melanogenum.

**Learn**

We also construct a gene circuit to help maintain or increase the copies of production gene compared with common production bacteria,which brings out a contradiction between increase of expression and regulatory of expression.We built a system biology model to simulate the interaction between components in the quorum sensing system.It can prove the feasibility of our quorum sensing system, because although the number of plasmid copies is gradually increasing, the expression of SSRE promoter does not increase significantly, which means that the SSRE promoter can indeed play a role in regulating the start of fermentation at the early stage of fermentation and tactfully handle the contradiction with the number of copies at the same time.

![](https://static.igem.wiki/teams/4193/wiki/model/cellular/enge/fi2.png)

Figure2.The expression of SSRE promoter related to the number of copies

To have a great insight for quorum sensing,we simulated the behavior of three proteins before and after phosphorylated in quorum sensing system.

![](https://static.igem.wiki/teams/4193/wiki/model/cellular/enge/fig3.png)

Figure3.the behavior of three proteins and three phosphorylated proteins in quorum sensing system

**Design**

To add more parts with various of dynamic ranges, we simulated the expression of different subtypes SSRE promoters based on our learning result,which will provide users of our platform with more choices.Because SSRE promoters of different subtypes have different dynamic ranges, different SSRE promoters can be selected according to the simulation results to the needs of different fermenter sizes.Then the expression intensity of different SSRE promoter was simulated in our system

![](https://static.igem.wiki/teams/4193/wiki/model/cellular/enge/fig4.png)

Figure4. The expression of different subtypes SSRE promoter

#### RNA transporter and hardware

We decided to design a transport aptamer which can target membrane structures so that they can play a role in cells as transporters. But because of the epidemic situation, our collaborative team did not have time to help us with in vitro transport experiments using exosomes. Our hardware can adjust the outflow rate of the fermentation liquid through the regulation of the microvalve controller, thus ensuring that the transport RNA will only have minimal probability of transporting the product back into the cell again.And the transport aptamer works along with the hardware, so we decided to analyze the performance of both together.

**Test**

We obtaines some aptamers and measured the aptamers binding abilities to the product molecule . In other words, we got aptamers with different Kd values.

**Learn**

We learned the sequence of these aptamers and the ability to specifically bind the product molecules.Because Disp is a platform, all of our parts need to be designed for different fermentation situations, thus requiring aptamers with different Kd values.

**Design**

We used molecular docking and the relationship between the sequence and the Kd value to add more aptamers with different Kd values. These aptamers were used for the modification and were designed as different transport aptamers to meet the different fermentation conditions. Then we used aptamers with different Kd value to construct transport RNA that can bind to membrane structure and specifically to the product by simply replacing the product binding aptamer sequence in RNA10,then transport the product along the concentration gradient.

**Test**

Using the above transport RNA constructed with different Kd value aptamers, we simulated different cases of transport aptamers through simulation, which is sufficient to prove the feasibility of our hardware.

#### Kill switch

**Design**

To enable the engineered microorganism to initiate suicide at low yields, we designed kill switch whose aptamer faction binding to the product would activate ribozyme activity, causing the designed mRNA to self-cleavage and preventing YopE (kill protein)expression.

**Build**

Firstly, the model was constructed to analyze the content of products (inside and outside cells) in the whole system after a period of fermentation and we could see that the level of the products was stable after a period of fermentation, which also verified the feasibility of the riboswitch.
Because under normal conditions, the level of intracellular products is basically unchanged, and if some mutations or other factors cause the production to decline, the riboswitch will work. This also provided confidence for us to further test the function of the kill switch.

**Test**

We examined the performance of the suicide switch by replacing upstream of the ribozyme riboswitch to the eGFP gene. However, the experimental data of the riboswitch is not ideal due to detection equipment failure, so we used simulation modeling to verify its feasibility. We constructed a systems biology model, which first changed the yield in the system, and we can see that with the higher production rate, the YopE protein expression decreased significantly by figure6A, and its production rate increased significantly. After continuing the exploration, we found that there is another element in the system that can affect the yield change in the system, which is the ability of the riboswitch to bind to the ligand. By changing the binding capacity of the aptamer, it can be seen that changing the binding capacity of the aptamer can effectively inhibit the expression of YopE protein, thus reducing the manslaughter phenomenon.

#### Overall test

Because we can't use the large fermentation tank used in the company's production, it is difficult for us to know whether the platform we designed has the function of successfully enabling the engineering bacteria to automatically adjust the production and growth continuously ferment to increase production.After adjusting the components of each component to achieve the most effective, it is obvious that engineered bacteria with CHIP introduced have higher production and have better fermentation effect under the same number of engineering bacteria by using cellular automata to conduct integrity analysis

![](https://static.igem.wiki/teams/4193/wiki/model/cellular/model/1000-3.png)

Figure5:Comparison of fermentation of engineered bacteria containing CHIP and original engineered bacteria in fermenter

##### DISP

A project by the OUC-China & Research iGEM 2022 team.

##### Contact

*mail\_outline*
[OUCiGEM@163.com](OUCiGEM%40163.com)

![](https://static.igem.wiki/teams/4193/wiki/other/2022-ouc-china-home-sponsor-png.png)

DISP 2022

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Team:OUC-China\_experiments-2022.igem.org

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# Team:OUC-China

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#

![Group Photo](https://static.igem.wiki/teams/4193/wiki/mediawiki/header-photo/2022-ouc-china-experiments-header-photo.png)

This year,we use the following protocols to complete our experiments.

### Part 1 Transporter RNA Construction and Testing

[PCR](https://2022.igem.wiki/ouc-china/notebook%20)

[Construction of RNA library (using T7 transcription kit)](https://2022.igem.wiki/ouc-china/notebook%20)

[SELEX](https://2022.igem.wiki/ouc-china/notebook%20)

[RNA Antisense Purification](https://2022.igem.wiki/ouc-china/notebook%20)

[Assembly of Transporting RNA](https://2022.igem.wiki/ouc-china/notebook%20)

[Aptamer Kd measurement](https://2022.igem.wiki/ouc-china/notebook%20)

[In vitro transport efficiency and rate measurement](https://2022.igem.wiki/ouc-china/notebook%20)

### Part 2 Quorum Sensing

[Preparation of electrocompetent cells](https://2022.igem.wiki/ouc-china/notebook%20)

[Preparation of 2-IP solution](https://2022.igem.wiki/ouc-china/notebook)

[Determination of growth curve and background expression intensity of SSRE promoter of Aureobasidium melanogenum P16](https://2022.igem.wiki/ouc-china/notebook%20)

[Determination of dynamic regulatory range of SSRE promoter](https://2022.igem.wiki/ouc-china/notebook)

### Part 3 Kill Switch

[RNA SELEX](https://2022.igem.wiki/ouc-china/notebook)

[Functional Verification of Riboswitch](https://2022.igem.wiki/ouc-china/notebook)

### Part 4 Other experiments

[Agarose Gel Electrophoresis](https://2022.igem.wiki/ouc-china/notebook)

##### DISP

A project by the OUC-China & Research iGEM 2022 team.

##### Contact

*mail\_outline*
[OUCiGEM@163.com](OUCiGEM%40163.com)

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DISP 2022

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# Team:OUC-China

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#

![Results Header](https://static.igem.wiki/teams/4193/wiki/mediawiki/header-photo/2022-ouc-china-results-header-photo.png)

## Overview

How can we build a platform that ensures high production? By ensuring the system has the ability to recognize specific molecules and eliminate engineered bacteria that don’t have high production, our system can produce, identify, transport efficiently. We will introduce the results of these functions in the following aspects: number of copies, quorum system, kill switch and transport RNA.

### Number of copies

We knocked out the phosphofructokinase gene essential for the growth of the engineered bacteria and planned to introduce it at the downstream of the production gene.We successfully construct production engineered bacteria.

#### Knock out PFK gene

To verify that we knocked out the phosphofructokinase gene in *Aureobasidium  melanogenum P16*, we extracted the genome of P16 and P16 with PFK gene knocked out then amplified PFK gene.As shown in figure1, lane 2 has the exact length of PFK gene but lane 1 dose not,which indicated we successfully knocked out PFK gene.

![](https://static.igem.wiki/teams/4193/wiki/other/results/2022-ouc-china-results-fig1.png)

*Figure1: Gel electrophoresis results of P16 genome with PFK gene knocked out(left) and P16 genome(right)*

We also incubated Aureobasidium  melanogenum on YNB solid medium for two days, using glucose (Figure2.A) and lactic acid as carbon source(Figure2.B). 1, 2 and 3 are three phosphofructokinase knocked out strains . From left to right, the inoculated liquid decreased gradually.The results told us strains with PFK gene knocked out could hardly grow on the medium using glucose as carbon source but could grow on the medium as carbon source,which matches well with our expectation.

![](https://static.igem.wiki/teams/4193/wiki/other/results/2022-ouc-china-results-fig2-a.png)

Figure2.A: YNB solid medium results A: glucose as carbon source;

![](https://static.igem.wiki/teams/4193/wiki/other/results/2022-ouc-china-results-fig2-b.png)

Figure2.B: YNB solid medium results,B: glucose and lactic acid as carbon source.

#### The activity of strain knocked out PFK

According to the results of measuring the growth curves of strains without PFK gene knockout and three strains with PFK gene knockout in YPD liquid and YPGL liquid medium, it can be seen that the three strains with PFK gene knockout can hardly grow in the medium with glucose as the main carbon source, while in the medium with lactic acid and glycerol as the carbon source, there is only a difference before and after a logarithmic period compared with the strains without PFK gene knocked out, At 60h, the same stable period was reached, which showed that the three strains with PFK gene knocked out had roughly the same production activity as the strains without PFK gene knocked out.

![](https://static.igem.wiki/teams/4193/wiki/other/results/2022-ouc-china-results-fig3.png)

Figure3.The growth curve of P16（△PFK）YPD

![](https://static.igem.wiki/teams/4193/wiki/other/results/2022-ouc-china-results-fig4.png)

Figure4.The growth curve of P16（△PFK）YPGL

### Quorum sensing

n our platform when the signal molecule IP reaches a certain concentration, it activates the SSRE promoter and increases the transcription of production gene. We transformed our fragments into the genome of our engineering bacteria and measured the intensity of SSRE promoter.

#### Genome transformation

We inserted the gene circuit of the quorum sensing system to the genome of *Aureobasidium  melanogenum*  pHPTX13-loxp, which was given to us from our PI. To verify if the DNA segments were inserted into the genome correctly，we amplified the target gene after extracting the genome of each strain. We selected 583bp specific fragment of AtCRE1 gene (Lane 1 ) and the whole length of PTP2+GFP (Lane 4) to test transformation. The electrophoresis gel show that the gene fragments were properly inserted into the genome.

![](https://static.igem.wiki/teams/4193/wiki/other/results/2022-ouc-china-results-fig5.png)

Figure 5. Gel electrophoresis results of P16 genome amplifying AtCRE1 gene(Lane 1), transformed strain genome amplifying AtCRE1(Lane 2), P16 genome amplifying PTP2+GFP(Lane 3) and transformed strain genome amplifying PTP2+GFP(Lane 4)

#### SSRE promoter

At first, we measured the background expression intensity of SSRE promoter while measuring the growth curve of the transformed strain.

![](https://static.igem.wiki/teams/4193/wiki/other/results/2022-ouc-china-results-fig6.png)

Figure6. growth curve of the transformed Aureobasidium  melanogenum and the background expression intensity of SSRE promoter

For IP dose–response experiments, all strains were precultured for 8h with terminal OD600 of 0.4, then treated with IP for 0，0.5，1，1.25，1.5h at 28℃, 175 rpm, and the fluorescence intensity of the strains was measured. Different concentrations of IP can increase the expression of SSRE promoter. After rough statistical analysis, the expression intensity of SSRE promoter was significantly correlated with IP concentration, showing that SSRE promoter can fulfill our assumption to regulate the production and growth. In order to obtain more SSRE promoters with different dynamic regulation ranges, we obtained various of subtypes of SSRE promoters through modeling and analysis

![](https://static.igem.wiki/teams/4193/wiki/other/results/2022-ouc-china-results-fig7.png)

Figure7. Effect of different concentrations of IP on SSRE promoter induction

#### Other findings

The results of the background expression intensity of SSRE promoter showed that the expression intensity of SSRE decreased at the 6th hour. The results of repeated experiments are the same. Therefore, we suspect that there may be a protein with similar function to AtlPT4 in the genome of *Aureobasidium  melanogenum.*

### Kill switch

To make sure our platform always maintains high production, our team used a ribozyme riboswitch to identify the product molecule. And through further engineering, we used the aptamer to construct a kill switch that only lets the engineered bacteria live when the product molecule is produced in high intensity.

#### SELEX

Our primary part is to screen RNA aptamers that could bind specifically to our product. We decided to perform SELEX 12 times, but we failed in the early rounds and there was no band on the electrophoresis gel. We figured out that the RT-PCR step didn’t work as we expected. So, we changed our experimental protocol, we divided reverse transcription and amplification into two steps during PCR, and we got aptamers that combined specifically and strongly with mannitol.

![](https://static.igem.wiki/teams/4193/wiki/other/results/2022-ouc-china-results-fig8.png)

Figure8.Verification of the SELEX products in a 1% agarose gel. Lanes 1,2,3,4,5,6,7 correspond to the SELEX results of rounds 2,4,6,8,10,11,12. The designed aptamer is about 123bp, the results of electrophoresis were as expected.

#### Dissociation Constant(Kd) of RNA Aptamer

Firstly,we simulated the relationship between the expression of Yope and the dissociation constant of RNA aptamer,which showed that if we want to express YopE while the production is low,we must get the aptamer with low Kd value.
Our team simulated RNA aptamer with high specificity for γ- aminobutyric acid(GABA) by modeling method. We then synthesized the target RNA aptamer sequence with fluorescence 3’ FAM. Therefore, the dissociation constant(Kd) of the aptamer was determined by fluorescence spectroscopy. We incubated the aptamer with a range of concentrations of GABA for two hours at room temperature, respectively. Next, we measured the fluorescence intensity at 525nm of each group and used the following formula to characterize the Kd value.

![](https://static.igem.wiki/teams/4193/wiki/mediawiki/header-photo/2022-ouc-china-measurement-kd.png)

Here, F0 and F represent the fluorescence intensity of the fluorescent groups in the presence and absence of GABA.

![](https://static.igem.wiki/teams/4193/wiki/other/results/2022-ouc-china-results-fig9.png)

Figure9.Measurement of dissociation Constant of Aptamer GABA-1:Kd=6.143e+04(-2.915e+08,2.917e+08);GABA-2:Kd=1.741e+05(-9.327e+10,9.327e+10)

GABA-1 is the aptamer we designed in silicon.But it have worse GABA-binding ability compared to GABA-2.Although the result of Kd value of GABA-1 aptamer is not very good,we decreased it by learning the experiment data to design another aptamer called GABA-2.It has a stronger binding ability,showing we could adapt this sequence to ribozyme riboswitch.

#### Toxic protein

At the beginning of the project design, we chose YopE as the suicide protein of our engineering bacteria. Injection of YopE into eukaryotic cells induces depolymerization of actin stress fibres. After communicating with other teams, we found that using YopE as the suicide protein in our design not only posed a security threat to the platform's R&D personnel, but also posed a greater security problem to the staff of the microbial factory. After communicating with the DUT-China team to test the sacB toxic protein in their project, we decided to use sacB as the final suicide protein of our platform because of its good killing effect and better biosafety.
As shown in figure 10(on the left), we selected 183bp specific fragment of SacB gene and genome PCR to test transformation. Lane 1 is genome PCR of Aureobasidium  melanogenum P16 transformed with empty genome transformation plasmid. Lane 2 is genome PCR of Aureobasidium  melanogenum P16 transformed with genome transformation plasmid including SacB gene ,which shows we introduced SacB gene successfully.

![](https://static.igem.wiki/teams/4193/wiki/other/results/2022-ouc-china-results-fig10.png)

Figure10. The transformation of SacB gene

As shown in figure 10(on the right), area 1 is Aureobasidium melanogenum with empty genome transformation plasmid; area2 is Aureobasidium  melanogenum with SacB gene transformed into the genome;area3 is Aureobasidium  melanogenum with SacB gene transformed into the genome and cultured with 5mM sucrose, showing SacB has a great ability to kill Aureobasidium melanogenum.
Because Aureobasidium melanogenum has a strong sugar production capacity, SacB protein has a certain toxicity to it when fructose is not added to the culture medium. In the future, we will optimize the fermentation conditions of Aureobasidium melanogenum and add appropriate sucrose to the culture medium, so that the SacB protein expressed by Aureobasidium melanogenum has an ideal killing effect on it after the low yield triggers the kill switch.

![](https://static.igem.wiki/teams/4193/wiki/other/results/2022-ouc-china-results-fig11.png)

Figure11.The killing effect of SacB on Aureobasidium melanogenum

### Transport RNA

In order to transport the products out of the engineering bacteria in time and break through the limitation of limited types of protein transporters, OUC-China decided to apply the aptamer to the transmembrane transport of products. This RNA molecule with transporter function consists of three strands of two different RNA. Among them, one RNA is responsible for targeting the cell membrane structure, and one RNA is responsible for specifically binding products. In order to simply test the transport performance of the transduced RNA, OUC-China decided to use the extracellular sealing membrane structure to incubate the transduced RNA and product molecules, and to test the transport performance of the transduced RNA by detecting the molecular residue of the product in the supernatant of the membrane structure.
Due to the epidemic situation, our partner NJU-China did not have enough time to use their exosomes to detect the performance of RNA transport. We believe that we can enrich our wikis and parts after verifying the performance of RNA transport after the competition, which will inspire the following iGEM teams to flexibly apply RNA in different aspects.

##### DISP

A project by the OUC-China & Research iGEM 2022 team.

##### Contact

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DISP 2022

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