iGEM 2017 — Team ZJUT-China

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# ZJUT-China

IGEM

![](https://static.igem.org/mediawiki/2017/a/a5/Zjut2017fgi.jpg)

**Important events:**

* 6.26: Got the synthetic parts and started experiments.
* 7.23: Lysin’s test had been finished.
* 7.29: Light-controlled system had been found wrong.
* 9.3: Newly synthetic light-controlled system arrived.
* 9.17: New light-controlled system had been tested.
* 9.29: Two parts had been combined.
* 10.11:The whole system was tested successfully.
* 10.22:All submission parts were ready to be sent.

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# Team:ZJUT-China/Experiments

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# ZJUT-China

IGEM

![](https://static.igem.org/mediawiki/2017/a/a5/Zjut2017fgi.jpg)

Our experiments include all four parts: the test of light-controlled system, the test of functional gene lysin, the combination of two parts, and the test of the whole system.

At first we used the plasmid from Shanghai Jiao Tong University which used mRFP as reporter gene. For convenient test, we replaced mRFP with eGFP ,which can be lnminated in UV. But when we tested, we found the fluorescence intensity of experimental group with light sensing system was obscure compared with the control group without it. We then added a strong constitutive promoter BBa\_J23100 before YF1 but with little help.

### Light-controlled System Test

Referring to previous thesis, we found the FixK2 promoter reported in the thesis was different from we used for light sensing system:BBa\_K592006. We finally decided to synthesize a plasmid with primitive FixK2 promoter, we also used BBa\_J23100 promoter and added a terminator before FixK2 promoter. The circuit is as it shows in the picture.

![](https://static.igem.org/mediawiki/2017/c/cf/Light-controlled_System_Test.png)

The result is shown in the figure below:![](https://static.igem.org/mediawiki/2017/a/a4/ZJUT-light-test.png)

Figure 1: The result of light-controlled system test

### Lysin test

Lysin was directly synthesized and was tested in pGLO, regulated by araBAD promoter. We tried to gain the lysis rate by testing OD600. But then we found it difficult to get an accurate lysis rate though this way. So we changed to dilution-spread method and gained the lysis rate successfully.

The results of OD600 testing and dilution-spread method are shown in the figures below.

①![](https://static.igem.org/mediawiki/2017/9/9b/Lysin-od.png )

②![](https://static.igem.org/mediawiki/2017/b/be/Lysin-rate.png )

Figure 2: ①The results of OD600 testing ;②Spread plate method results

### Combination

After testing two parts of the whole system, we started to combine to parts. All two parts and linearized vector were prepared by PCR and connected by One Step Cloning after cleaning up.

### Final Test

The completed recombined plasmids were transferred into E.coli BL21 (DE3), and tested under blue light induction.

The result is shown in the figure below.

![](https://static.igem.org/mediawiki/2017/9/9c/ZJUT-light-lysin-test.png)

Figure 3: Final test of recombined systerm results

### Important Protocols

##### LB medium (solid, 1L = 50 dishes) :

* 2 g / 100ml (in the conical flasks)
* 10 g tryptone
* 5 g yeast extract
* 5 g NaCl
* Water

##### One Step Cloning and Transform:

 [ClonExpress II One Step Cloning Kit-Vazyme](https://static.igem.org/mediawiki/2017/6/69/ZJUT-One_Step_Cloning_and_Transform-.pdf%20)

##### Plasmids extraction:

 [96 Plasmid Kit-Axyprep](https://static.igem.org/mediawiki/2017/2/21/ZJUT-plasmid.pdf)

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# Team:ZJUT-China/Results

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# ZJUT-China

IGEM

![](https://static.igem.org/mediawiki/2017/a/a5/Zjut2017fgi.jpg)

### Experiments

1. The functional gene *lysin* (BBa\_K2277000) was tested using OD600 testing&dilution-spread method and works well.
2. The functional gene *bcsB* (BBa\_K2277110) has been tested and being abandoned.
3. The reporter gene *eGFP* (BBa\_K2277033) works well as reporter gene.
4. The new FixK2 promoter (BBa\_K2277233) has improved the efficiency of the existing part BBa\_K1065303.
5. The light-controlled part (BBa\_K2277555) has been tested using eGFP as the reporter gene and works well.
6. The whole light-controlled lysis system (BBa\_K2277666) is constructed successfully and was tested using OD600 testing.

The whole system’s constitution is shown in the picture.

![](https://static.igem.org/mediawiki/2017/a/a4/ZJUT-System.png )

Cells will self-lyse in the absence of blue light.

### Device

| Light Source | Light Intensity |
| --- | --- |
| When we were making the circuit boards using as the light source, function had a priority.According to the requirements of our cells culture, which is, a fixed wavelength of the blue light irradiation which could start our lysin gene.Therefore, in the process of experimental variables involved, we controlled the wavelength and strength of light source. Then we built bottom fixed wavelength light source in the completely dark black case. In order to control conveniently, we designed a multi paralleled DC circuit which was simple, stable and switchable. Battery powered LED lamps were chosen as the light source so that the device would be easy to carry out. | 34lux |

| Compositions | Parameters | Notes |
| --- | --- | --- |
| Bottom: pvc cover | ф7.6cmcm\*0.5cm |  |
| Bottom: thick cardboard | ф7.6cm\*0.5cm |  |
| Cover | ф7.6cm\*0.5cm |  |
| Tube | Cylindrical shell ф7.5cm\*0.2cm pvc height 17cm | "Wrapped with tin foil, completely opaque. With strong and proper diameter, it can be placed in the shaker to work." |
| Internal fixing plate | "ф7.5cm\*0.5cm,Within the decentralized 4 \* ф2.5 hollow hole" | Four test tubes can be central placed tightly, tube bottom keep direct contact with the cardboard. |
| Light source plate. | ф7.5cm\*0.5cm | Light (circuit board) is fixed at the bottom, and the switch is fixed outside.When being used, the test tube should be placed from the top into the device, then cap the tube in order to make sure it’s completely opaque. |

### Modeling

Based on our experiments data and minimized stimulation device, we created a fermenter model of our light-induced self-lysis system. Our model informed the possible positions for company to input the light source. And it also provide cost budget in fermentation process for reference. This model, presenting below, consists of the ideal figure of fermenter and calculating process of the cost budget.

**More details are in the Modeling Part.**

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