iGEM 2021 — Team XMU-China

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# Team:XMU-China/Notebook

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Team:XMU-China/Notebook - 2021.igem.org

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  + [Improve](https://2021.igem.org/Team%3AXMU-China/Improve)
* Human Practices 
  + [Integrated Human Pratice](https://2021.igem.org/Team%3AXMU-China/Human_Practices)
  + [Science Communication](https://2021.igem.org/Team%3AXMU-China/Education)
* Awards 
  + [Best Integrated Human Practices](https://2021.igem.org/Team%3AXMU-China/Human_Practices)
  + [Best Education](https://2021.igem.org/Team%3AXMU-China/Education)
  + [Best Model](https://2021.igem.org/Team%3AXMU-China/Model)
  + [Best Hardware](https://2021.igem.org/Team%3AXMU-China/Hardware)
  + [Best Basic Parts](https://2021.igem.org/Team%3AXMU-China/Parts#Basic)
  + [Best Composite Parts](https://2021.igem.org/Team%3AXMU-China/Parts#Composite)
  + [Best Part Collection](https://2021.igem.org/Team%3AXMU-China/Parts#Collection)

![](https://static.igem.org/mediawiki/2021/5/5a/T--XMU-China--notebook.jpg)

Notebook

* February
* Mars-May
* June
* 7.12-7.18
* 7.19-7.25
* 7.26-8.1
* 8.2-8.8
* 8.9-8.15
* 8.16-8.22
* 8.23-8.29
* 8.30-9.5
* 9.6-9.12
* 9.13-9.19
* 9.20-9.26
* 9.27-10.3
* 10.4-10.10
* 10.11-10.17

![](https://static.igem.org/mediawiki/2021/7/7e/T--XMU-China--blueleftArrow.png)
![](https://static.igem.org/mediawiki/2021/d/d7/T--XMU-China--bluerightArrow.png)

# February

- Our team was formally founded.
- All student members learned the fundamental knowledge of molecular cloning and iGEM.
- All student members looked through the previous excellent projects and learned the basic notion of synthetic biology and iGEM.

# Mars-May

- We decided our project theme.
- We were divided into three groups and polish our project separately (and we reported each's progress every weekend).
- We optimized and constructed the sequences of the desired genes.
- Our team was officially accepted by iGEM.

# June

- All student members came back to school and learned laboratory fundamental qualities and received molecular cloning operation training.

# 7.12-7.18

- [BBa\_K3739031](http://parts.igem.org/Part%3ABBa_K3739031) is constructed.

# 7.19-7.25

- [BBa\_K3739037](http://parts.igem.org/Part%3ABBa_K3739037), [BBa\_K3739040](http://parts.igem.org/Part%3ABBa_K3739040) are constructed successfully.

# 7.26-8.1

- [BBa\_K3739032](http://parts.igem.org/Part%3ABBa_K3739032) is constructed.
- [BBa\_K3739031](http://parts.igem.org/Part%3ABBa_K3739031) is transferred to *Vibrio natriegens* and characterized.
- [BBa\_K3739044](http://parts.igem.org/Part%3ABBa_K3739044), [BBa\_K3739043](http://parts.igem.org/Part%3ABBa_K3739043) are constructed.

# 8.2-8.8

- [BBa\_K3739044](http://parts.igem.org/Part%3ABBa_K3739044) is transferred to *Vibrio natriegens* and characterized.
- [BBa\_K3739033](http://parts.igem.org/Part%3ABBa_K3739033) is successfully constructed via Gibson.
- [BBa\_K3739041](http://parts.igem.org/Part%3ABBa_K3739041), [BBa\_K3739038](http://parts.igem.org/Part%3ABBa_K3739038), [BBa\_K3739061](http://parts.igem.org/Part%3ABBa_K3739061) are constructed.

# 8.9-8.15

- [BBa\_K3739032](http://parts.igem.org/Part%3ABBa_K3739032) is transferred to *Vibrio natriegens* and characterized.
- [BBa\_K3739047](http://parts.igem.org/Part%3ABBa_K3739047), [BBa\_K3739045](http://parts.igem.org/Part%3ABBa_K3739045), [BBa\_K3739050](http://parts.igem.org/Part%3ABBa_K3739050) are constructed successfully.
- [BBa\_K3739046](http://parts.igem.org/Part%3ABBa_K3739046) is successfully constructed by point mutation.

# 8.16-8.22

- [BBa\_K3739048](http://parts.igem.org/Part%3ABBa_K3739048), [BBa\_K3739049](http://parts.igem.org/Part%3ABBa_K3739049), [BBa\_K3739051](http://parts.igem.org/Part%3ABBa_K3739051), [BBa\_K3739052](http://parts.igem.org/Part%3ABBa_K3739052) are constructed successfully.

# 8.23-8.29

- [BBa\_K3739049](http://parts.igem.org/Part%3ABBa_K3739049), [BBa\_K3739061](http://parts.igem.org/Part%3ABBa_K3739061) are transferred to *Vibrio natriegens*.
- [BBa\_K3739034](http://parts.igem.org/Part%3ABBa_K3739034), [BBa\_K3739035](http://parts.igem.org/Part%3ABBa_K3739035), [BBa\_K3739063](http://parts.igem.org/Part%3ABBa_K3739063) are constructed.

# 8.30-9.5

- [BBa\_K3739036](http://parts.igem.org/Part%3ABBa_K3739036) is constructed successfully.
- [BBa\_K3739043](http://parts.igem.org/Part%3ABBa_K3739043), [BBa\_K3739042](http://parts.igem.org/Part%3ABBa_K3739042) are transferred to *Vibrio natriegens* and characterized.
- [BBa\_K3739056](http://parts.igem.org/Part%3ABBa_K3739056), [BBa\_K3739057](http://parts.igem.org/Part%3ABBa_K3739057) are constructed via Gibson.

# 9.6-9.12

- [BBa\_K3739039](http://parts.igem.org/Part%3ABBa_K3739039), [BBa\_K3739054](http://parts.igem.org/Part%3ABBa_K3739054) are constructed successfully.
- [BBa\_K3739046](http://parts.igem.org/Part%3ABBa_K3739046), [BBa\_K3739047](http://parts.igem.org/Part%3ABBa_K3739047) are successfully ligated together to construct [BBa\_K3739053](http://parts.igem.org/Part%3ABBa_K3739053)

# 9.13-9.19

- [BBa\_K3739058](http://parts.igem.org/Part%3ABBa_K3739058), [BBa\_K3739059](http://parts.igem.org/Part%3ABBa_K3739059) are constructed.
- [BBa\_K3739053](http://parts.igem.org/Part%3ABBa_K3739053) is transferred to *Vibrio natriegens* and characterized.

# 9.20-9.26

- [BBa\_K3739060](http://parts.igem.org/Part%3ABBa_K3739060), [BBa\_K3739062](http://parts.igem.org/Part%3ABBa_K3739062) are constructed.
- [BBa\_K3739058](http://parts.igem.org/Part%3ABBa_K3739058), [BBa\_K3739059](http://parts.igem.org/Part%3ABBa_K3739059) are transferred to *Vibrio natriegens* and characterized.

# 9.27-10.3

- [BBa\_K3739064](http://parts.igem.org/Part%3ABBa_K3739064), [BBa\_K3739065](http://parts.igem.org/Part%3ABBa_K3739065) are constructed successfully.
- [BBa\_K3739036](http://parts.igem.org/Part%3ABBa_K3739036) is transferred to *Vibrio natriegens* and characterized.

# 10.4-10.10

- [BBa\_K3739055](http://parts.igem.org/Part%3ABBa_K3739055), [BBa\_K3739066](http://parts.igem.org/Part%3ABBa_K3739066) are constructed successfully.
- [BBa\_K3739042](http://parts.igem.org/Part%3ABBa_K3739042), [BBa\_K3739061](http://parts.igem.org/Part%3ABBa_K3739061) are transferred to *Vibrio natriegens* and characterized.

# 10.11-10.17

- [BBa\_K3332020](http://parts.igem.org/Part%3ABBa_K3332020), [BBa\_K3332021](http://parts.igem.org/Part%3ABBa_K3332021), [BBa\_K3332022](http://parts.igem.org/Part%3ABBa_K3332022), [BBa\_K3332023](http://parts.igem.org/Part%3ABBa_K3332023) are successfully ligated together to construct [BBa\_K3739067](http://parts.igem.org/Part%3ABBa_K3739067) to be an improvement.

Xiamen University, Fujian, China

No.422, Siming South Road, Fujian, P.R.China 361005

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

# Team:XMU-China/Engineering

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Team:XMU-China/Engineering - 2021.igem.org

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![](https://static.igem.org/mediawiki/2021/d/d6/T--XMU-China--EngineeringSuccess.jpg)

Engineering
Success

![](https://static.igem.org/mediawiki/2020/1/11/T--XMU-China--right40.png)
[![](https://static.igem.org/mediawiki/2020/c/ca/T--XMU-China--right41.png)
[Overview](#H1)](#H1)
[![](https://static.igem.org/mediawiki/2020/b/bf/T--XMU-China--right42.png)
[Algae treatment system](#H2)](#H2)
[![](https://static.igem.org/mediawiki/2020/d/de/T--XMU-China--right43.png)
[Mussel treatment system](#H3)](#H3)
[![](https://static.igem.org/mediawiki/2020/3/35/T--XMU-China--right44.png)
[Light-induced suicide system](#H4)](#H4)

Overview

This year XMU-China endeavored to tackle and had solved problems during our engineering process, generating expected results. Our SALVAGE project has three main sections: algae treatment system, mussel treatment system and light-induced suicide (kill switch) system (see our [design of SALVAGE project](https://2021.igem.org/Team%3AXMU-China/Design)).

After the several cycles of “brainstorm, design, human practices, design improvement” (see our [Human Practices](https://2021.igem.org/Team%3AXMU-China/Human_Practices)page for details), we decided on several enzyme candidates. In total, 99 new BioBricks were designed for our project (see our [Parts](https://2021.igem.org/Team%3AXMU-China/Parts)page).

Countless efforts are made to ensure our design can function to attain our goals. **Among them, [BBa\_K3739000](http://parts.igem.org/Part%3ABBa_K3739000), [BBa\_K3739014](http://parts.igem.org/Part%3ABBa_K3739014) and [BBa\_K3739030](http://parts.igem.org/Part%3ABBa_K3739030) are typically worth mentioning. These three BioBricks perform well and function as expected.** All the related data are recorded below. Hopefully, they will make some contribution to the iGEM community.

Section 1: Algae treatment system

his-HutH ([BBa\_K3739000](http://parts.igem.org/Part%3ABBa_K3739000))

HutH, which can catalyze *L*-histidine into trans-urocanate, is found in the liver of vertebrates and in bacteria such as *Escherichia coli*, *Salmonella* and *Pseudomonas*. Because of the toxic effect of urocanate to *P. globosa*, we chose the histidine ammonia-lyase (HutH) from *Pseudomonas putida* to solve the algae problem. *J23100-B0030* ([BBa\_K081005](http://parts.igem.org/Part%3ABBa_K081005)) was used to construct the expression circuit ([BBa\_K3739031](http://parts.igem.org/Part%3ABBa_K3739031)), and the HutH protein was purified then used to test the enzyme activity.

1.1 Agarose Gel Electrophoresis

![](https://static.igem.org/mediawiki/2021/d/d7/T--XMU-China--BBa_K3739031.png)

**Fig. 1. DNA gel electrophoresis image of expression circuit for HutH (BBa\_K3739031).**

Result: When we were building this circuit, regular PCR was used to certify that the plasmid was correct. Target bands (1877 bp) can be observed at the position around 2000 bp (Fig. 1).

1.2 SDS-PAGE

![](https://static.igem.org/mediawiki/2021/2/25/T--XMU-China--SDS-PAGE.png)

**Fig. 2. SDS-PAGE analysis of protein HutH in lysate of *Vibrio natriegens* and the eluant.**

Result: *hutH* gene was expressed successfully. Compared with the negative control groups (NC), the target bands of HutH (55.5 kDa) in the eluant (FR) can be observed at around 50 kDa (Fig. 2).

1.3 Enzyme activity

Method: Enzyme activity of purified HutH was measured spectrophotometrically by monitoring the increase of absorbance at 277 nm, due to the formation of *trans*-urocanate. The concentration of *trans*-urocanate was calculated from the standard working curve determined in advance (OD277 was plotted against varying concentrations of *trans*-urocanate).

Result: We got the OD277-time curve of the product trans-urocanate at 28 °C. Then we calculated relevant enzyme activity and drew 1/Enzyme activity and 1/[*L*-histidine] curves (Lineweaver-Burk Plot), from which we can obtain relevant *K*m and *k*cat of purified HutH (Fig. 3).

![](https://static.igem.org/mediawiki/2021/1/18/T--XMU-China--L_histidine.png)

**Fig. 3. The relationship of 1/enzyme activity and 1/concentration of *L*-histidine.**

Section 2: Mussel treatment system

TnaA-his ([BBa\_K3739014](http://parts.igem.org/Part%3ABBa_K3739014))

Mussels can form byssus, a network of biofilms that attach to surfaces. However, indole can prevent mussels from attaching by inhibiting the biofilm formation. TnaA was chosen for its function of converting tryptophan to indole. Similarly, *J23100-B0030* ([BBa\_K081005](http://parts.igem.org/Part%3ABBa_K081005)) was used to construct the expression circuit ([BBa\_K3739044](http://parts.igem.org/Part%3ABBa_K3739044)), and the TnaA protein was purified then used to test the enzyme activity.

2.1 Agarose Gel Electrophoresis

![](https://static.igem.org/mediawiki/2021/b/b9/T--XMU-China--TnaA.png)

**Fig. 4. DNA gel electrophoresis image of expression circuit of TnaA (BBa\_K3739044).**

Result: After transferring the correct plasmid into *Vibrio natriegens*, colony PCR was used to select the positive clones. Target bands (1739 bp) can be observed at the position around 1500 bp (Fig. 4).

2.2 SDS-PAGE

![](https://static.igem.org/mediawiki/2021/6/6b/T--XMU-China--SDS-PAGE2.png)

**Fig. 5. SDS-PAGE analysis of protein TnaA in lysate of *Vibrio natriegens* and the eluant.**

Result: *tnaA* gene was expressed successfully. Compared with the negative control groups (NC), the target bands of TnaA (53.5 kDa) in the eluant (FR) can be observed at around 50 kDa (Fig. 5).

2.3 Enzyme activity

Method: The amount of indole produced by TnaA from tryptophan in a certain time was subsequently measured by indole reagent, and the concentration of indole was calculated from the standard working curve determined in advance (OD570 was plotted against varying concentrations of indole).

![](https://static.igem.org/mediawiki/2021/b/b4/T--XMU-China--indole.png)

**Fig. 6. The relationship of 1/enzyme activity and 1/concentration of indole.**

Result: We got the OD570-time curve of the product indole at 28 °C. Then we calculated relevant enzyme activity and drew 1/Enzyme activity and 1/[indole] curves (Lineweaver-Burk Plot), from which we can obtain relevant *K*m and *k*cat of purified TnaA (Fig. 6).

Section 3: Light-induced suicide system
(kill switch)

BlrA-GFP ([BBa\_K3739030](http://parts.igem.org/Part%3ABBa_K3739030))

To facilitate biosafety and biocontainment, BlrA, originated from *Bacillus subtilis*, was chosen for its function of producing ROS (reactive oxygen species) upon blue-light irradiation, which can cause cell death of the bacteria. For this part, a GFP sequence was fused to the C-terminal of BlrA in order to direct view the expression level of the toxin gene, and *blrA-gfp* was cloned into to pET-28a(+) vector. To verify the cytotoxicity of BlrA, colony forming units (CFUs) were counted for indicating cell viability as time progressed after inducing.

3.1 Agarose Gel Electrophoresis

![](https://static.igem.org/mediawiki/2021/c/cf/T--XMU-China--RegularPCR.png)

**Fig. 7. The result of regular PCR (plasmid was added as the template for PCR).**

Result: When we were building this circuit, regular PCR was used to certify the correct plasmids. Target bands (1542 bp) can be observed at the position around 1500 bp (Fig. 7).

3.2 Measurement for killing effects of BlrA

Method: During the cultivation, inducer IPTG was used to control the expression of *blrA-gfp*. After the addition of IPTG, blue-light was also supplied to activate BlrA at the same time. Samples were taken out by every 2 hours after inoculation, and IPTG was added at the fourth hour of cultivation. GFP fluorescence intensity and CFUs of every sample were measured to represent the expression level and killing effects of BlrA, respectively.

![](https://static.igem.org/mediawiki/2021/2/2c/T--XMU-China--cytotoxicity.png)

**Fig. 8. The cytotoxicity of BlrA was verified.** (**A**) Variation tendency of normalized fluorescence density (RFUGFP/OD600) of *Vibrio natriegens* harboring *blrA-gfp*\_pET-28a(+) with or without adding IPTG. (**B**) Variation tendency of CFUs after *Vibrio natriegens* harboring *blrA-gfp*\_pET-28a(+) were induced with and without IPTG. Gray areas represent dark state while the blue areas represent illumination state.

Result: *blrA* gene was expressed successfully under the inducement of IPTG (Fig. 8A). Meanwhile, CFUs of induced group decreased sharply after IPTG addition compared to the non-induced group, of which CFUs arose continuously (Fig. 8B). The difference of CFUs between these two groups has verified that BlrA can be used as a toxic protein for killing engineered bacteria.

Xiamen University, Fujian, China

No.422, Siming South Road, Fujian, P.R.China 361005

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

# Team:XMU-China/Experiments

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Team:XMU-China/Experiments - 2021.igem.org

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  + [Improve](https://2021.igem.org/Team%3AXMU-China/Improve)
* Human Practices 
  + [Integrated Human Pratice](https://2021.igem.org/Team%3AXMU-China/Human_Practices)
  + [Science Communication](https://2021.igem.org/Team%3AXMU-China/Education)
* Awards 
  + [Best Integrated Human Practices](https://2021.igem.org/Team%3AXMU-China/Human_Practices)
  + [Best Education](https://2021.igem.org/Team%3AXMU-China/Education)
  + [Best Model](https://2021.igem.org/Team%3AXMU-China/Model)
  + [Best Hardware](https://2021.igem.org/Team%3AXMU-China/Hardware)
  + [Best Basic Parts](https://2021.igem.org/Team%3AXMU-China/Parts#Basic)
  + [Best Composite Parts](https://2021.igem.org/Team%3AXMU-China/Parts#Composite)
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Experiments

Molecular Cloning Handbook

#### [Molecular Cloning Handbook 6.0 BETA of XMU-China](#collapse0)

[Molecular\_Cloning\_Handbook.pdf](https://static.igem.org/mediawiki/parts/6/68/T--XMU-China--Molecular_Cloning_Handbook-7.0.pdf)

The Algae part

Protocol

#### [I. Materials](#collapse11)

1. MD® Spectra max190 Molecular Device

2. 96 well microtiter plates

3. *L*-histidine, Tris-HCl buffer, MnCl2, reduced glutathione (GSH) and water

4. Purified HutH enzyme

#### [II. Before Experiment](#collapse114_1_1)

Prepare aqueous solution below.

5 mmol•L-1 L-histidine, 100 mmol•L-1 Tris-HCl buffer pH 9.0, 0.1 mmol•L-1 MnCl2, 1.7mmol•L-1 reduced glutathione (GSH)

Prepare these reaction systems according to the table below. (Unit: μL)

|  | *L*-histidine | Tris-HCl buffer | MnCl2 | GSH | H2O | Purified HutH enzyme | BSA |
| --- | --- | --- | --- | --- | --- | --- | --- |
| A | 80 | 16 | 0.16 | 8 | 55.84 | 40 | 0 |
| B | 32 | 16 | 0.16 | 8 | 103.84 | 40 | 0 |
| C | 16 | 16 | 0.16 | 8 | 119.84 | 40 | 0 |
| D | 8 | 16 | 0.16 | 8 | 127.84 | 40 | 0 |
| E | 3.2 | 16 | 0.16 | 8 | 132.84 | 40 | 0 |
| F | 16 | 16 | 0.16 | 8 | 119.84 | 0 | 40 |

#### [III. protocols](#collapse115_1_1)

1. Start testing immediately after preparing the reaction system. Record the value of fluorescence in real time.

2. Analyze the slope.

Culturing *Phaeocystis globosa*

#### [I. Materials & Apparatus](#collapse113_1)

Erlenmeyer flask; Hemocytometer; Optical microscope; Incubator; light source; 0.22µm membrane; filter; Milli Q water; Sea salt; Penicillin-Streptomycin Solution

#### [II. Before Experiment](#collapse12)

Get sea water ready: add commercial sea salt to Milli Q water to get 3 % seawater, which is filtered through a 0.22µm pore filter membrane to prevent contamination by algae, sterilized at 110℃ for 30 minutes, and cooled at room temperature for later usage. The other components of L1-Si medium need to be filtered through 0.22µm membrane filters, and the whole preparation process needs to be aseptically operated in a Flow Clean Bench.

#### [III. Protocol](#collapse13_13)

Prepare L1-Si medium (Silicate-free L1 medium):

The Mussel part

Measurement of enzyme activity of TnaA

#### [I. Materials：](#collapse131)

●Purified TnaA enzyme

●TECAN® Infinite M200 Pro Microplate Reader

●96-well microtiter plates

#### [II. Protocol](#collapse132)

A. Measurement of standard curve

1. 5 mg indole powder was dissolved in toluene and prepared into 0.1 mg/mL solution.

2. Dilute in turn and prepare solutions of concentration of 1, 2, 3, 4, 5 µg/mL.

3. Add 0.4 mL distilled water, 3 mL mixed chromogenic solution (5% PDAB, 5% concentrated HCl) into 1 mL of each indole solution prepared in step 2, stand for 30 min.

4. Measure the samples at 570 nm using microplate reader.

5. Draw the OD570 - Concentration curve.

B. Determination of enzyme activity

1. Add 20 µL 200 µg/mL PLP solution, 10 µL 5 mM GSH solution, and 270 µL enzyme solution in a 15mL centrifuge tube in order.

2. Add 1.0 mL toluene to cover the mixed solution in step 1.

3. Stand for 5 min at 37℃.

4. Add 100 µL tryptophan solution of different concentration (0.25, 0.5, 1, 2.5, 5, 10, 20 mg/mL)

5. Incubate the solution at 37 °C with agitation at 200 r.p.m for 10 min.

6. Add 0.4 mL distilled water, 3 mL mixed chromogenic solution (5% PDAB, 5% concentrated HCl), stand for 30min.

7. Measure the samples at 570 nm using microplate reader.

Rhamnolipid production

#### [I. Material & Apparatus](#collapse33)

●Dry bath

●Shaker

●Rotary evaporator

●Benchtop centrifuge

●Vortex mixer

●Microplate reader

●96-well microtiter plates

●0.22-µm filter

#### [II. Before Experiment](#collapse34)

Inoculate 200 µL bacteria glycerol stock into 10 mL LB liquid medium with 0.1 mg/mL kanamycin, 37 ℃, 200 rpm overnight.

Determine standard curve of rhamnose: Prepare 100 µL rhamnose solution of different concentration (0.01, 0.05, 0.10, 0.25, 0.05, 0.1, 0.2, 0.5 mM), 100 µL of 1.6% orcinol solution and 800 µL of 60% sulphuric acid in distilled water was added in each sample. The mixture was incubated at 80 ℃, 1000 rpm for 30 min, then cooled to room temperature. Measure the absorbance of the samples at 420 nm using microplate reader.

#### [III. Protocol](#collapse1313)

1. Inoculate 1 mL of the culture into 100 mL LB liquid medium with 0.1 mg/mL kanamycin, culture at 37 ℃, 200 rpm for 72 hours, take 200 µL of culture at 0 h, 3 h, 5 h, 7 h, 9 h, 12 h, 18 h, 24 h, 36 h, 48 h, centrifuge at 16000×*g* for 5 min, then filter the supernatant with 0.22-µm filter, finally store the samples at 4 ℃ for further treatment.

2. 100 µL culture was extracted three times with 500 µL of ethyl acetate. The sample was mixed by vortex mixer for 2 min, then centrifuge at 10000 rpm for 1 min. The upper phase was collected in rotary evaporator sample bottles.

3. The organic solvent was removed by evaporation, the remaining was dissolved in 100 µL distilled water, subsequently 100 µL of 1.6% orcinol solution and 800 µL of 60% sulphuric acid in distilled water was added.

4. The mixture was incubated at 80 ℃, 1000 rpm for 30 min, then cooled to room temperature.

5. Measure the absorbance of the samples at 420 nm using microplate reader.

The suicide part

Validation of the killing effect of blrA protein

#### [I. Materials and apparatus](#collapse21)

500-mL conical flasks; LBv2 medium; LB medium; Agar; LB agar plates; 96-well microtiter plates;

#### [II. Groups](#collapse22)

IPTG-induced blrA-GFP light \* 3

IPTG non-induced blrA-GFP light \* 3

#### [III. Protocol](#collapse220)

1.Add 100 mL LBv2 medium to each of the six 500-mL conical flasks to sterilize.

2. Add 300 mL LB medium and 3 g agar to both of the 500-mL conical flasks to sterilize.

3. Preculture 15 mL bacteria culture to OD600 > 0.6 in dark.

4. Take samples of the bacteria culture in dark to measure the OD600 value until it is between 0.8 and 1.0.

5. Add kanamycin (100 µg/mL) at a dilution of 1:1000 in conical flasks and mix. Replace the tinfoil wrapped around the bottleneck of the flask with sterile six-layer gauze.

6. Inoculate the bacteria culture into the conical flasks at a volume ratio of 2-3%

7. Take 200 µL of samples twice at the same time after manual mixing into two wells of 96-well microtiter plates to measure OD600 and GFP, and take average of the measurement results.

8. Incubate the culture at 37 ℃ with agitation at 200 r.p.m., then take samples every two hours to measure OD600 and GFP fluorescence intensity, and replace them to the shaker as soon as possible after sampling.

9. Samples are serially diluted in LBv2 over a 6-log range and spot 100µL bacteria solution onto LBv2 solid plate.

11. Repeat step 10 after two hours, add IPTG at a dilution of 1:1000 to the induced group, at the same time turn on the blue light.

10.Take samples every two hours after induction, measure OD600, GFP fluorescence intensity and repeat step 10 to measure the colony forming unit (CFU). Repeat those steps 3-4 times in total.

Validation of the blue light induced promoter for its inducing capacity

#### [I. Materials & Apparatus](#collapse201)

LBv2 medium; LB medium; Agar; LB agar plates; 96-well microtiter plates

#### [II. Groups](#collapse202)

Light induced J23106-EL222-pBLind-GFP\*3;

Light non-induced blrA-GFP\*3;

Light induced pBLind-EL222-pBLind-GFP\*3;

Light non-induced pBLind-EL222-pBLind-GFP\*3.

#### [III. Protocol](#collapse203)

1. Preculture 15 mL bacteria culture to reach an OD600 value between 0.8 and 1.0 in dark.

2. Add kanamycin (100 µg/mL) at a dilution of 1:1000 in conical flasks and mix. Replace the tinfoil wrapped around the bottleneck of the flask with sterile six-layer gauze.

3. Inoculate the bacteria culture into the conical flasks at a volume ratio of 2-3%

4. Take 200 µL of samples twice at the same time after manual mixing into two wells of 96-well microtiter plates to measure OD600 and GFP, and take average of the measurement results.

5. Incubate the culture at 37 ℃ with agitation at 200 r.p.m, then take samples every two hours to measure OD600 and GFP fluorescence intensity, and replace them to the shaker as soon as possible after sampling.

6. When OD600 value exceeds 0.6, turn on the blue light for the light group after sampling.

7. Keep the blue light on, continuously measure the OD600 and GFP fluorescence intensity for 10-12 hours with a 2-hour interval.

Verify the killing effect of blue light induced suicide switch combination circuit

#### [I. Materials & Apparatus](#collapse204)

LBv2 medium; LB medium; Agar; LBv2 agar plate, 96-well microtiter plates

#### [II. Groups](#collapse205)

J23106-EL222-pBLind-blrA-GFP

pBLind-EL222-pBLind-blrA-GFP

#### [III. Protocol](#collapse206)

1. Preculture 15 mL bacteria culture to reach an OD600 value between 0.8 and 1.0 in dark.

2. Add 10 mL LBv2 liquid culture into the 50 mL centrifuge tube, add Kanamycin (100 µg/mL) at a dilution of 1:1000 and mix. Cover the tube but do not tighten it, wrap the tube with tin foil.

3, Incubate the bacteria culture into the conical flasks at a volume ratio of 2-3%.

4. Inoculate the culture at 37 ℃ with agitation at 220 r.p.m, for 5 h until OD600=1.0-1.4.

5. The samples are diluted 107 times. Individually spot 100 µL of each sample onto four LBv2 agar plates, wrap two of them in tin foil, expose the other two to blue light and culture at 37℃ for 10-12 h.

6. Observe and compare the differences between the illuminated group and the non-illuminated group.

Measurement of RFUsfGFP/OD600 as time progressed

I. Material & Apparatus

#### [I. Materials & Apparatus](#collapse41)

Molecular Devices SpectraMax® iD3 Multi-Mode Microplate Reader

Corning® Costar 96-Well Black flat-Bottom Plates

LB liquid medium with 0.1 mg/mL chloramphenicol

#### [II. Before Experiment](#collapse42)

Prepare two groups of 50-mL conical flask, wrapped with tinfoil and newspaper to make it light-proof. 8-layer of sterilized gauze covers the bottleneck for shadowing. 10 mL of LB liquid medium with 0.1 mg/mL chloramphenicol was added to each flask (**for strictly shadowed condition**).

Prepare two groups of 50-mL conical flask, breathable sterile sealing film covers the bottleneck. 10 mL of LB liquid medium with 0.1 mg/mL chloramphenicol was added to each flask (**for light-induced condition**).

#### [III. Protocols](#collapse402)

1. Inoculate 200 µL bacteria glycerol stock into 10 mL LB liquid medium with 0.1 mg/mL chloramphenicol, 37 ℃, 200 rpm overnight.

2. Inoculate 200 µL bacteria culture of experimental and control group into each group of flasks, shake the flasks and immediately take 200 µL out for detection.

3. When cultured at 37 ℃, 200 rpm for 20 hours, take 200 µL of culture at 1 h, 2 h, 3 h, 4 h, 5 h, 8 h, 11 h and 19 h for detecting OD600 and fluorescence intensity.

#### [IV. Notes](#collapse403)

1. Parameter settings for measuring fluorescence intensity in microplate reader:

2. Relative fluorescent unit (RFU) = Fluorescence Intensity of Sample – Fluorescence Intensity of Blank (LB medium)

The translocation part

Quantify cell's adhesion ability

#### [I. Material & Apparatus](#collapse404)

—96-well microtiter plates

—LAS buffer

—PBS buffer

—Shaking incubator of 37 °C

#### [II. Before Experiment](#collapse405)

1. Prepare a clean 96-well microtiter plates. (Note: choose non-polymer plate, such as quartz plate)

2. Prepare LBv2 culture medium (3.7 g LB broth, 1.19 g NaCl, 0.031 g KCl and 0.47 g MgCl2·6H2O per 100ml).

3. Prepare PBS buffer (pH=7.4)

4. Prepare 0.05 mM LAS buffer and 0.25 mM LAS buffer (dissolved in PBS buffer, pH=7.4)

#### [III. Protocol](#collapse406)

1. Cells are inoculated in fresh LBv2 medium at a dilution of 1:100 (overnight culture/fresh medium) and incubated overnight at 30 ℃ with agitation at 200 r.p.m.

2. Add ten 1 mm PP balls into each well in the first column of the plate. Add 200μL culture into the wells. For negative control, add 200 μL LBv2.

3. Incubate at 30 ℃ with agitation at 200 r.p.m. for 30 min.

4. Move the beads to the next column, add 500 μL PBS buffer, incubate with agitation at 200 r.p.m. for 1 min, discard the solution, repeat 5 times.

5. Move the beads to the next column, add 500 μL 0.05mM LAS buffer, incubate with agitation at 200 r.p.m. for 1 min.

6. Repeat step 5 once.

7. Move the beads to the next column, add 500 μL 0.05mM LAS buffer, incubate with agitation at 200 r.p.m. for 1 min.

8. Move the beads to the next column, add 200 μL LBv2 culture medium, incubate at 30 ℃ with agitation at 200 r.p.m. for 30 min.

8. Take 10 μL of culture from each well in step 7, move it to the next column, add 190 μL LBv2, measure the samples at 600 nm using microplate reader.

Verify the adhesion of LCI-KR2

#### [I. Material & Apparatus](#collapse407)

—96-well microtiter plates

—PBS buffer

#### [II. Before Experiment](#collapse408)

1. Prepare LBv2 culture medium (3.7 g LB broth, 1.19 g NaCl, 0.031 g KCl and 0.47 g MgCl2·6H2O per 100ml).

2. Prepare PBS buffer (pH=7.4)

#### [III. Protocol](#collapse521)

1. Cells are inoculated in fresh LBv2 medium at a dilution of 1:100 (overnight culture/fresh medium) and incubated overnight at 30 ℃ with agitation at 200 r.p.m.

2. Take 10 mL culture and cleave the cell, centrifuge at 5000 rpm for 20 min in 4 times to extract protein.

3. Condense the protein, drop 1 mL solution onto the 96-well microtiter plates placed horizontally, and stand for 10 min.

4. Wash 96-well microtiter plates with PBS buffer from the same direction for 1 min continuously.

5. Measure the fluorescence intensity under blue light.

#### [Molecular Cloning Handbook 6.0 BETA of XMU-China](#collapse550)

[Molecular\_Cloning\_Handbook.pdf](http://parts.igem.org/File%3AT--XMU-China--Western_Blotting_protocol.pdf)

Xiamen University, Fujian, China

No.422, Siming South Road, Fujian, P.R.China 361005

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

# Team:XMU-China/Results

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Result

![](https://static.igem.org/mediawiki/2020/1/11/T--XMU-China--right40.png)
[![](https://static.igem.org/mediawiki/2020/c/ca/T--XMU-China--right41.png)
[Remove of Phaeocystis globosa](#H1)](#H1)
[![](https://static.igem.org/mediawiki/2020/b/bf/T--XMU-China--right42.png)
[Remove of Mytilus edulis](#H2)](#H2)
[![](https://static.igem.org/mediawiki/2020/d/de/T--XMU-China--right43.png)
[translocation protein](#H3)](#H3)
[![](https://static.igem.org/mediawiki/2020/3/35/T--XMU-China--right44.png)
[Kill switch system](#H4)](#H4)

Remove of *Phaeocystis globosa*

## What work we have done:

We have verified that his-HutH enables the conversion L-histidine to trans-urocanate.

We have verified that his-CBM-GFP could bind to cellulose

We have verified that his-CBM-HutH could bind to cellulose and conversion L-histidine to trans-urocanate.

## Work has not finished yet:

The function of sp-3SpyTag-lecA wasn't verified

The function of sp-3SpyCatcher wasn't verified

### his-HutH

The *hutH* gene from Pseudomonas putida was heterologously expressed in chassis bacteria to produce histidine ammonia-lyase (HutH), which could catalyze *L*-histidine to *trans*-urocanate. Promoter (BBa\_J23100), RBS (BBa\_B0030), *hutH* gene, and terminator (BBa\_B0010) were assembled into pET-28a(+) plasmid backbone to express the HutH.

The constructed plasmid was transformed into *Vibrio natriegens* through electroporation. Positive colonies were selected by kanamycin preliminarily and then verified by regular PCR (Fig. 1) and sequencing. Then, the colony with the corrected sequence was cultivated to express HutH target protein. After ultrasonication broke and centrifugation, GE AKTA Prime Plus FPLC System was employed to purify the HutH from the supernatant. Purified protein was verified by electrophoresed on a sodium dodecyl sulfate (SDS)-10% (wt/vol) polyacrylamide gel, followed by Coomassie blue staining (Fig. 2).

![](https://static.igem.org/mediawiki/2021/b/b9/T--XMU-China--result--fig1.png)

**Fig. 1.** The result of regular PCR. Plasmid pET-28a(+).

![](https://static.igem.org/mediawiki/2021/d/da/T--XMU-China--result--fig2.png)

**Fig. 2.** SDS-PAGE analysis of protein in lysate of *Vibrio natriegens* and the eluant. Target bands (55.7 kDa) can be observed at the position around 50 kDa.

The absorbance of *L*-histidine was measured at 277 nm (ε = 18000 (mol⋅L-1)-1⋅cm-1) to obtain the data of concentration, which was used to calculate the kinetic constants of Km and kcat (Fig. 3).

Thus, these data demonstrate that HutH works well to catalyze *L*-histidine to *trans*-urocanate.

![](https://static.igem.org/mediawiki/2021/7/7e/T--XMU-China--result--fig3.png)

**Fig. 3.**The relationship of 1/Enzyme activity and 1/concentration of *L*-histidine.

### his-GFP-CBM

### Proof of expression

Cellulose is a part of the structure on the surface of the *Phaeocystis globose*, and we realized that it could be a target for our protein 1. As described in the literature, as a part of the cellulolytic multi-enzyme protein, CBM owns the ability of cellulose-binding, which guides our functional proteins to bind around the single cell of *Phaeocystis globose*. We also call CBM the cellulose (or carbohydrate) binding module 2. So, the genetic circuit of his-GFP-CBM (K3739033) was designed, the expression product could bind to cellulose with fluorescence. Promoter (BBa\_J23100), RBS (BBa\_B0030), his-GFP-CBM gene and terminator (BBa\_B0010) were assembled into pET-28a(+) plasmid backbone to express the his-GFP-CBM.

The constructed plasmid was transformed into *Vibrio natriegens* through electroporation. Positive colonies were selected by kanamycin preliminarily and then verified by colony PCR (Fig. 4) and sequencing. Then, the colony with the corrected sequence was cultivated to express his-GFP-CBM. After ultrasonication broke and centrifugation, GE AKTA Prime Plus FPLC System was employed to purify the CBM from the supernatant. Purified protein was verified by electrophoresed on a sodium dodecyl sulfate (SDS)-10% (wt/vol) polyacrylamide gel, followed by Coomassie blue staining (Fig. 5).

![](https://static.igem.org/mediawiki/2021/9/94/T--XMU-China--result--fig4.png)

**Fig. 4.** The result of colony PCR. Plasmid pET-28a (+).

![](https://static.igem.org/mediawiki/2021/e/e1/T--XMU-China--result--fig5.png)

**Fig. 5.** SDS-PAGE analysis of protein his-CBM-HutH. Target bands can be seen at about 50.9 kDa.

Target bands can be seen at about 50.9 kDa, which verified the successfully expressed of His-GFP-CBM in *Vibrio natriegens*. The purified GFP-CBM and BSA were added to the quantitative filter paper (equality of mass), and the changes of protein concentration were measured after being incubated for a specific time. The data could indicate the activity of his-GFP-CBM. The experimental steps are as follows:

Firstly, quantitative filter paper fragments with an equal weight of 0.16 g were weighed and placed in the glass container, 2 mL GFP-CBM and BSA were added, respectively (Fig. 6). After being incubated on a shaker (20 min, 60 rpm) at room temperature, the changes of protein concentrations of GFP-CBM and BSA were measured using Bradford reagent (Fig. 7)

![](https://static.igem.org/mediawiki/2021/6/6d/T--XMU-China--result--fig6.png)

**Fig. 6.** Schematic diagram of experimental process.

![](https://static.igem.org/mediawiki/2021/6/6a/T--XMU-China--result--fig7.png)

**Fig. 7.** The concentration changes of BSA and his-CBM-GFP. \*\* = p < 0.01

### his-CBM-HutH

In order to eliminate threat from *Phaeocystis globose* to the nuclear power plant cooling system, we design a genetic circuit which could express protein aim at this alga. The cellulose binding module (CBM) and (histidine ammonia-lyase) HutH from *Pseudomonas Putida* were fused to protein CBM-HutH. The fusion protein could bind to the cellulose on the surface of *P. globosa* and catalyzes *L*-histidine, secreted by *P. globosa* to *trans*-urocanate. which then elevates the ROS around the *P. globosa* to damage the algal cells.

Promoter (BBa\_K525998), RBS (BBa\_B0030), his-CBM-HutH gene (BBa\_K3739071), and terminator (BBa\_B0010) were assembled into pET-28a(+) plasmid backbone to express the his-CBM-HutH. The constructed plasmid was transformed into *Vibrio natriegens* through electroporation. Positive colonies were selected by kanamycin preliminarily and then verified by colony PCR (Fig. 8) and sequencing. Then, the colony with the corrected sequence was cultivated to express HutH target protein. After ultrasonication broke and centrifugation, GE AKTA Prime Plus FPLC System was employed to purify the CBM from the supernatant. Purified protein was verified by electrophoresed on a sodium dodecyl sulfate (SDS)-10% (wt/vol) polyacrylamide gel, followed by Coomassie blue staining (Fig. 9). Because CBM own the ability of plastic binding activity, tube and beaker used after ultrasonication are made from glass or Teflon.

![](https://static.igem.org/mediawiki/2021/a/a9/T--XMU-China--result--fig8.png)

**Fig. 8.** The result of colony PCR. Plasmid pET-28a (+).

![](https://static.igem.org/mediawiki/2021/a/a7/T--XMU-China--result--fig9.png)

**Fig. 9.** SDS-PAGE analysis of protein his-CBM-HutH. Target bands can be seen at about 69 kDa.

The absorbance of *trans*-urocanate was measured at 277 nm to obtain the data of concentration. Same concentration and volume of HutH and CBM-HutH solutions were added to the *L*-histidine solution, and the value OD277 was monitored and recorded. As shown in Fig. 10, the OD277 in two group was rising continuously, suggesting that CBM-HutH has the ability to catalyzes *L*-histidine to *trans*-urocanate.

![](https://static.igem.org/mediawiki/2021/8/87/T--XMU-China--result--fig10.png)

**Fig. 10.** Time course of the value of OD277.

Remove of *Mytilus edulis*

## What work we have done:

We have verified that tnaA-his enables the conversion of tryptophan to indole.

We have verified that polyphenol oxidase (PPO) can be expressed in E. coli and Vibrio natriegens.

We have verified that PPO could catalyze dopamine to dopaquinone.

We have verified the plastic binding ability of Vibrio natriegens with plastic-binding proteins on their surface.

## Work has not finished yet:

The function of rhlAB wasn't verified.

The function of LCIKR2-GFP wasn't verified.

### TnaA-his

As we know, mussels can form byssus, a biofilm that attaches to the solid surface. At the same time, indole can prevent mussels from attaching by inhibiting biofilm formation3. Thus, the tnaA-his brick (BBa\_K3739044) was designed and constructed, which converts tryptophan to indole and thus inhibits the formation of mussel byssus.

![](https://static.igem.org/mediawiki/2021/e/e5/T--XMU-China--tnaA.jpg)

**Fig. 11.** Gene circuit of tnaA-his.

The constructed plasmid was transformed into *E. coli* DH5α. Positive colonies were selected by chloramphenicol preliminarily and then verified by colony PCR (Fig. 12). The colony, confirmed by sequencing finally, was cultivated to amplification the plasmid. Then, the plasmid was transformed into the *Vibrio natriegens* by electroporation. The colony, verify by colony PCR (Fig. 13) and confirmed by sequencing finally, was cultivated to express the target protein.

Moreover, the total protein was gained from the supernatant after ultrasonication and centrifugation. GE AKTA Prime Plus FPLC System was used to harvest purified protein from the broken supernatant. An apparent protein peak in the AKTA FPLC System demonstrated the correct purified protein. Purified protein was verified by sodium dodecyl sulfate (SDS)-12% (wt/vol) polyacrylamide gel electrophoresis and Coomassie blue staining (Fig. 14).

![](https://static.igem.org/mediawiki/2021/9/98/T--XMU-China--result--fig12.png)

**Fig. 12.** Colony PCR of pET-28a(+)-J23100-RBS-tnaA-his in *E. coli* DH5α.

![](https://static.igem.org/mediawiki/2021/7/7d/T--XMU-China--result--fig13.png)

**Fig. 13.** Colony PCR of pET-28a(+)-J23100-RBS-tnaA-his in *Vibrio natriegens.*

As shown in SDS-PAGE of TnaA-his, the target protein (53.5 kDa) could be observed at the position around 50 kDa on the purified protein lanes and broken supernatant lanes, but not in the control groups. These results demonstrated that the tnaA with His-Tag was successfully expressed in *Vibrio natriegens* and purified using GE AKTA Prime Plus FPLC System.

![](https://static.igem.org/mediawiki/2021/d/df/T--XMU-China--result--fig14.png)

**Fig. 14.** SDS-PAGE analysis of TnaA-his by Coomassie blue staining.

The purified tnaA enzyme activity was measure by the amount of indole produced from tryptophan within a specific time. The concentration of indole was calculated through the standard curve, which draws as the method as fellow:

After dissolving 5 mg of indole in a small amount of toluene, transfer the solution 50 mL volumetric flask, dilute with toluene to tick marks, and mix. Transfer 10, 20, 30, 40, and 50 µL solution above into five clean tubes respectively and dilute with toluene to 1.0 mL, then added 0.4 mL distilled water, 3mL PDAB (4-Dimethylaminobenzaldehyde) (5%, v/v) and n-butanol (5%, v/v) sulfate mixed chromogenic solution, and mix. Stand for 30 min and measure the absorbance at 570 nm. Taking the absorbance as the X-axis, the concentration as the Y-axis, draw the standard curve and obtain the equation.

20 µL PLP solution (0.20 mg/mL), 10 µL GSH solution (0.005 mol/L), and 270 µL tnaA enzyme solution were added in several 15 mL tubes respectively and then covered with 1.0 mL toluene. After keeping the temperature at 37 °C for 5 min, 100 µL tryptophan solution with different concentrations was added into the 15 tubes respectively, which were put in a shaker for 10 min at 37 °C. Then 3 mL of PDAB (5%, v/v) and n-butanol sulfate mixed chromogenic solution (5%, v/v) was added to stop the reaction. After 30 min, absorbance was measured at 570 nm, which was used to calculate the Km and kcat. (Fig. 14)

![](https://static.igem.org/mediawiki/2021/2/29/T--XMU-China--result--fig15.png)

**Fig. 15.** The relationship of 1/enzyme activity and 1/concentration of Indole.

### PPO-his

## Proof of expression:

The mussel can adhere to a solid surface mainly through 3,4-dihydroxyphenylalanine (DOPA), the side chain in mucoprotein. We designed a genetic circuit to express polyphenol oxidase (PPO), which could degrade the DOPA. According to the literature, DOPA could be catalyzed by PPO to generate dopaquinone and significantly weaken mucoprotein viscosity. Thus, we express the PPO in *Vibrio natriegens* and characterization its enzyme activity, which is derived from *Bacillus Megaterium* (PDB ID: 3NM8). This PPO does not require a caddie protein for activity and possesses superior catalytic activity in the mixture of water and organic reagents. The brick of BBa\_K3739015 has assembled into pET-28a(+) plasmid backbone with T7 promoter to express the PPO-his. After being verified by SDS-PAGE (Fig. 16), PPO was successfully expressed in *E. coli* BL21(DE3) and *Vibrio natriegens*.

![](https://static.igem.org/mediawiki/2021/9/98/T--XMU-China--result--fig16.png)

**Fig. 16.** The result of regular PCR. Plasmid pET-28a(+).

## Quantitative analysis of dopaquinone

The constructed plasmid was transformed into *Vibrio natriegens* through electroporation. Positive colonies were selected by kanamycin preliminarily and then verified by colony PCR and sequencing. Then, the colony with the corrected sequence was cultivated to express PPO. After ultrasonication broke and centrifugation, GE AKTA Prime Plus FPLC System was employed to purify the PPO from the supernatant. Purified protein was verified by electrophoresed on a sodium dodecyl sulfate (SDS)-10% (wt/vol) polyacrylamide gel, followed by Coomassie blue staining (Fig. 17).

![](https://static.igem.org/mediawiki/2021/5/51/T--XMU-China--result--fig17.png)

**Fig. 17.** SDS-PAGE analysis of PPO. Target bands (34.9kD kDa) can be observed at the position around 50 kDa.

The time course of the absorbance of dopaquinone was measured at 475 nm to obtain the data to calculate the enzyme activity.

Test system: sodium acetate buffer (170 µL), DOPA solution (20 µL, 50 mM) and PPO solution (10 µL) were added to the 96-well plate to measure the absorbance during the catalytic process through ECAN ® Infinite M200 Pro instrument. Parallel experiments were performed three times.

## Kinetics of enzyme catalysis

The effects from different concentration of DOPA (0.01 mM, 0.02 mM, 0.05 mM, 0.1 mM and 0.15 mM) on the PPO catalytic activity were investigated with 0.1 M sodium acetate buffer (pH=6.0). And the catalytic kinetic parameters of PPO were also obtained through calculation.

![](https://static.igem.org/mediawiki/2021/7/78/T--XMU-China--result--fig18.png)

**Fig. 18.** The relationship of 1/Enzyme activity and 1/concentration of DOPA.

### lpp-OmpA-LCI KR2-GFP

We attempt to display the plastic-binding protein on the surface of *Vibrio natriegens* through OmpA, which could endow the ability of plastic-binding to *Vibrio natriegens*. The gene of OmpA-LCI KR-2-GFP (Fig. 19A, BBa\_K3739025) was assembled into the plasmid backbone and transformed into *Vibrio natriegens* through electroporation. After being verified by agarose gel electrophoresis, the plasmids have been transformed into *Vibrio natriegens* successfully (Fig. 19B).

![](https://static.igem.org/mediawiki/2021/e/ec/T--XMU-China--result--fig19.png)

**Fig. 19.** Gene circuit and agarose gel electrophoresis. (A) Gene circuit of *OmpA-LCI KR-2-GFP* (BBa\_K3739025). (B) Target bands of OmpA-LCI KR-2-GFP (black arrow, 2100 bp).

After successful construction and transformation, the experiment was carried out to verify whether the OmpA-LCI KR-2-GFP was expressed and located on the membrane of *Vibrio natriegens*. Thus, the total membrane proteins were extracted to verify the expression of the OmpA-LCI KR-2-GFP. However, the complicated component of the membrane proteins also seriously hinders the analysis of SDS-PAGE (Fig. 20). The western blot analysis was performed, in which the GFP in fusion protein serves as the antigen, and the experimental results are shown in Figure 21. The targeted band was observed in the precalculated position in the western blot results, suggesting that the plastic-binding protein (OmpA-LCI KR-2-GFP) was successfully anchored on the surface of *Vibrio natriegens*.

![](https://static.igem.org/mediawiki/2021/1/17/T--XMU-China--result--fig20.png)

**Fig. 20.** SDS-PAGE analysis of LCI KR-2-GFP by coomassie blue staining. Target bands of OmpA-LCI KR-2-GFP (red box, 61.7 kDa).

![](https://static.igem.org/mediawiki/2021/f/fc/T--XMU-China--result--fig21.png)

**Fig. 21.** Western Blot analysis of OmpA-LCI KR-2-GFP. Target bands of OmpA-LCI KR-2-GFP (white arrow, 61.7 kDa).

![](https://static.igem.org/mediawiki/2021/6/69/T--XMU-China--result--fig22.png)

**Fig. 22.** Binding ability of engineered *Vibrio natriegens* to plastic. (**A**) The effect from adding plastic microsphere on the OD600 in experiment group (OmpA-LCI KR-2-GFP display on the surface of Vibrio natriegens) and control group (OmpA-GFP display on the surface of *Vibrio natriegens*). (**B**) Time course of ΔOD600.ΔOD600: The value of OD600 in each time minus the value of OD600 at the time of 0. \*\*\*\* = p < 0.0001.

translocation protein

Since some proteins in our project are intended to function in extracellular environment, how to get out of the cells is vital for realization of their functions. Signal peptide is a short amino acid sequence on secreted protein, which is a natural helper of protein to get out of cytoplasm. However, signal peptides can lead protein go to not only the extracellular environment but also the periplasm and membrane. There are several signal peptides selected for *V. natriegens*, but these are all directed into periplasm. 4 Although there is a signal peptide of alginate lyase from *V. natriegens* able to direct heterogenous protein to extracellular environment in *E. coli* chasis, It is not clear that there is a signal peptide conducting protein to extracellular environment in *V. natriegens*. 5

### Selected procedure

Here, we use a tip to select the signal peptide. Download the translated CDS information of *V. natriegens* from NCBI Assembly. Through analysis of SignalP 5.0, we found out several candidate signal peptides which have significant likelihood to work with corresponding secretion system. According to the function of proteins containing proposed signal peptide, we can exclude the signal peptides targeting to the periplasm and cell membrane. Finally, we find out the best candidate signal peptide, LMT signal peptide, from our chassis.

### Qualification results

To clarify whether the signal peptides we selected are able to direct the fused proteins to extracellular environment, we constructed Aly01 and LMT single peptides with BBa\_K2560063 which is from 2018 Marburg iGEM program and transformed the pET-28a(+) plasmid with composite parts BBa\_K3739038 and BBa\_K3739041 into *V. natriegens*. Then we use the anti-GFP antibody to detect secreted GFP in the filtered supernatant of *V. natriegens* liquid culture by western blotting after *V. natriegens* is cultivated for .16 hours. The concentration of his-GFP in the supernatant of BBa\_K3739041 is significantly higher than that in J23100-B0030-GFP. (Fig. 23A, 23B) Furthermore, less expression of BBa\_K3739041 than that of GFP conforms that LMT-his-GFP is secreted rather than from lysed cells. (Fig. 23C) However, the concentration of Aly01-his-GFP is less than that of GFP. (Fig. 23A, 23B) It is probably because that there are less expression of Aly01-his-GFP in *V. natriegens*. (Fig. 23C) Thus, we use another method to prove our hypothesis.

![](https://static.igem.org/mediawiki/parts/c/ce/T--XMU-China--waimi-1.png)

**Fig. 23.** (A) Western Blotting detection of the *V. natriegens* supernatants of J23100-B0030-GFP, BBa\_K3739038 and BBa\_K3739041 after 16 h cultivation in LBv2 liquid culture, 37 ℃. *Sup.* means supernatant. (B) Grayscale analysis of (A). (C) Fluorescence intensity/OD600value of *V. natriegens* strain of J23100-B0030-GFP, BBa\_K3739038 and BBa\_K3739041 in LBv2 liquid culture after 16 h cultivation in 37 ℃.

### Quantitation results

In order to exclude the difference of expression among J23100-B0030-GFP, BBa\_K3739038 and BBa\_K3739041 and quantify secretion effect of signal peptide, we treated *V. natriegens* liquid cultures after 12.5 h cultivation in 37 ℃ with 12.5 µg/mL chloramphenicol. Then we sampled the cultures every 6 minutes and terminated secretion with 20 mM NaN3. As time goes, the his-GFP concentration of culture supernatant were increased in both BBa\_K3739038 and BBa\_K3739041. This is indicated that both LMT and Aly01 signal peptide are able to translocate fused protein to extracellular environment. (Fig. 24A, 24B) We made a grayscale analysis for the modelling of secretion, and the total GFP is detected for confirmation that the translation is completely repressed. (See our [Model pages](https://2021.igem.org/Team%3AXMU-China/Model))

![](https://static.igem.org/mediawiki/parts/a/a1/T--XMU-China--waimi-2.png)

**Fig. 24.** (**A**) Western blotting detection of sampled supernatants treated with chloramphenicol in different times. (**B**) GFP concentration of treated supernatant of BBa\_K3739038 and BBa\_K3739041 in (A)

### Secretion in *E. coli*

Besides conducting protein secretion in *V. natriegens*, Aly01 signal peptide is reported that it is able to conduct protein secretion in *E. coli* as well. 5 We supposed that LMT signal peptide may also have conducting function in *E. coli* Thus, we used filtered culture of BL21(DE3) strain transformed with pET28a(+)-BBa\_K3739043 and pET-28a(+)-BBa\_K3739041 for SDS-PAGE. Then the SDS-PAGE gel is stained by silver staining. Contrasting to BBa\_K3739043, his-GFP is detected in the supernatant of pET-28a(+)-BBa\_K3739041.(Fig. 25) It is indicated that LMT signal peptide can also work for conducting secretion in *E. coli*.

![](https://static.igem.org/mediawiki/parts/f/f8/T--XMU-China--waimi-3.png)

**Fig. 25.** Silver staining result of supernatant of BL21(DE3) transformed with BBa\_K3739043 and BBa\_K3739041, respectively.

Then we used an alternative way to verify the secretion function of signal peptides in *E. coli*. The proteins labeled of tetracysteine motif tag (FlAsH tag, FT in short) could be detected with the biarsenical compound FlAsH-EDT2. 6

To quantify and qualify the secretion effect, FT was added into the C-terminal of BBa\_K3739000, BBa\_K3739006 and BBa\_K3739009. Composite parts BBa\_K3739084, BBa\_K3739085, BBa\_K3739087 were constructed (Fig. 26) on plasmid backbone pSB1C3 or pET-28a(+) and then the recombinant plasmid was transformed into *E. coli* BL21(DE3). Positive colonies selected by colony PCR and sequencing were cultivated for secretion analysis.

![](https://static.igem.org/mediawiki/parts/6/63/T--XMU-China--K3739084-085-087.png)

**Fig. 26.** Colony PCR results of BBa\_K3739084, BBa\_K3739085, BBa\_K3739087

After cultivation for 5 hours at 37℃ and induction for 2 hours with 0.1 mM IPTG, the cell supernatant was separated by centrifugation. The supernatant along with 2 /micro;M FlAsH-EDT 2 and 1 mM DTT were added to wells in a 96-well plate. Following incubation in the dark for 1 h at 37 °C, fluorescence was measured by 503 nm excitation and 528 nm emission, and each value was normalized by OD 600. The result shows that the fluorescence intensity/OD600 of the group Aly01 and LMT is significantly higher than native control group, which proves that these 2 signal peptides can function well. (Fig. 27)

![](https://static.igem.org/mediawiki/parts/0/00/T--XMU-China--FT_Qualify.png)

**Fig. 27.** Fluorescence intensity/OD600 of cell supernatant after incubation in the dark for 1h.

Moreover, to promote our understanding on secretion process mediated by signal peptides, we built a model about relationship between the amount of secreted recombinant protein and time. (See our [Model pages](https://2021.igem.org/Team%3AXMU-China/Model)) In order to get the value of *PT* (the total amount of recombinant protein per unit volume of reactor), P\_M *PM* (the amount of the recombinant protein in the medium compartment in unit volume) and γ(extracellular secretion rate constant), a sets of characterization experiments were done.

BBa\_K3739087 were constructed on plasmid backbone pET-28a(+) and then the recombinant plasmid was transformed into *E. coli* BL21(DE3). After cultivation for 5 hours and induction for 3 hours with 0.1 mM IPTG, 34 mg/L chloroamphenicol was added to bog down the synthesis of protein in the engineered bacteria in order to make the experimental operations more convenient. Then, the cell supernatant was collected in 0, 6, 12, 18, 24, 30, 40 min to test the amount of secreted recombinant protein by fluorescence detection of FlAsH-EDT2 and FT. (Fig. 28)

![](https://static.igem.org/mediawiki/parts/0/0b/T--XMU-China--waimi-6.png)

**Fig. 28.** The fitting of equations and experiments results.

Kill switch system

Our kill switch system is mainly composed of an inducible promoter system (EL222-pBLind system) and a gene encoding toxin (*blrA*), both of which are blue-light activated for normally functioning (see our [Design](https://2021.igem.org/Team%3AXMU-China/Design) page). The toxin gene *blrA* is under the control of promoter pBLind that can be activated by EL222 upon blue-light illumination7. Besides, when exposed to blue-light, the toxin BlrA will be triggered to cause ROS (reactive oxygen species) formation then kill the engineered bacteria8.

Therefore, the engineered bacteria will survive and function as we designed when in the cooling water system of the nuclear power plant, where the environment is relative dark and the kill switch is in OFF state. Once the engineered bacteria escape from working environment and enter the regions with LED strips set in advance (see our [Proposed Implementation](https://2021.igem.org/Team%3AXMU-China/Implementation) page), the blue-light irradiation will turn on the kill switch system for achieving biosafety and biocontainment of our SALVAGE project.

## What work we have done

The cytotoxicity of BlrA was verified.

The function of EL222 system that can be induced by blue-light was verified.

### Cytotoxicity of BlrA

For direct viewing the real-time expression level of BlrA, a reporter GFP was fused to the C-terminal of BlrA *via* 3×GS linker for achieving co-expression. Thus, we could know the approximate expression level of BlrA of different sampling time-points by measuring GFP fluorescence intensity. The *blrA-gfp* was cloned into pET-28a(+) for simplifying the inducing operations when verifying the cytotoxicity of BlrA.

*In vivo* assessment of BlrA-mediated phototoxicity in our engineered bacteria (*V. natriegens*) was implemented and colony forming units (CFUs) were counted for indicating the cell viability. In the experiment, a small part of bacteria culture was taken out to measure OD600 and GFP fluorescence intensity by every 2 hours after inoculation, and the samples were diluted then spread evenly over the surface of nutrient agar plates. When OD600 reached ∼0.8 (at 4th hour), IPTG was added and blue-light irradiation was turned on at the same time to induce the expression of BlrA and activate its phototoxicity, respectively.

![](https://static.igem.org/mediawiki/2021/e/e0/T--XMU-China--result--fig29.png)

**Fig. 29. The cytotoxicity of BlrA-GFP.** (**A**) The RFUGFP/OD600 of different groups were calculated as time progressed. (**B**) The growth curves were measured as time progressed. (**C**) Calculated CFUs of the groups with/without IPTG added. (**D**) Calculated CFUs of the groups with IPTG added under different illumination circumstances. Gray areas represent dark state while the blue areas represent illumination state (if need). IPTG was added (if need) at the 4th hour after inoculation.

BlrA was expressed after inducing since the GFP fluorescence intensity (relative fluorescent unit, RFU) normalized to OD600 (RFUGFP/OD600) increased fast after 4 hours (Fig. 1A). And after IPTG inducement, the CFUs (counted at the time of 24 hours after plating) of the group exposed to blue-light showed a significant declined tendency while in contrast the CFUs of the group with no IPTG added increased as time progressed, which indicated that **BlrA functioned as a toxin and resulted in cell death** (Fig. 1C). However, the decrease of CFUs of the group with IPTG inducing while incubated in darkness was also observed (Fig. 1D). This implied that BlrA-GFP might have low or no light-response property, compared to simple BlrA.

## Discussion for BlrA

BlrA, namely YtvA, is an FMN-binding fluorescence protein from *Bacillus subtilis*, which contains the photosensitive light-oxygen-voltage (LOV) domain and can cause lethal ROS formation in cells under the circumstance of rich-oxygen 9. However, our results showed above did not support this. Based on the phenomena we observed, we attributed this difference to the structural change of BlrA due to the fusion of GFP in the C-terminal of BlrA. Generally, conformational change and exposure of LOV domain might occur for BlrA during blue-light illumination, which results in ROS formation in a photosensitive manner. While fused with GFP, the LOV domain of BlrA might exposes consistently since the BlrA’s folding is influenced. Therefore, BlrA-GFP always shows cytotoxicity, whether being exposed to blue-light irradiation or not.

Additionally, the growth (OD600) of bacteria harboring *blrA-gfp* seemed not affected by the cytotoxicity after toxin gene was induced to express (Fig. 1B). However, this result supported that the killing mechanism of BlrA was not a cell-lysis manner but others. Due to potential time-lag of bacterial lysis and time-need for BlrA’s functioning, the OD600 could maintain in a relative stable level or increase slowly, albeit that cell death might has happened.

In conclusion, we showed that BlrA-GFP had cytotoxicity and could be used in the kill switch system.

## The performance of EL222-pBLind system can be induced by blue-light

In order to obtain a photo-response system induced by blue-light, two genetic circuits of *J23106-EL222-pBLind-sfGFP* and *pBLind-EL222-pBLind-sfGFP* were constructed to verified the performance of EL222-pBLind in *Vibrio natriegens*. As a reporter protein, fluorescence intensity from GFP can represent the expression intensity of downstream gene in the two circuits with the condition of blue-light illumination and dark conditions respectively. Positive colonies were selected by kanamycin preliminarily and then verified by colony PCR (BBa\_K3739064), regular colony PCR (BBa\_K3739065) (Fig. 30) and sequencing.

![](https://static.igem.org/mediawiki/2021/c/cb/T--XMU-China--result--fig30.png)

**Fig. 30. The result of regular and colony PCR. Plasmid pET-28a(+). BBa\_K3739064 (left) and BBa\_K3739065 (right).**

In the experiment, the two bacteria with different circuits were respectively set up in the uninduced (light avoidance) group and induced (blue light exposure) group. Samples were taken every two hours after inoculation, OD600 and fluorescence intensity were measured and recorded for 8 times. At the same time, the induced group of the two circuits illuminated by blue-light irradiation from time of 4.5 h after inoculation.

![](https://static.igem.org/mediawiki/2021/d/d8/T--XMU-China--result--fig31.png)

**Fig. 31. EL222-pBLind system can be induced by blue-light.** (**A**) The normalized fluorescence intensity (RFUsfGFP/OD 600) was measured as time progressed. J: *J23106-EL222-pBLind-GFP*, P: *pBLind-EL222-pBLind-GFP*, I: illumination, D: darkness. (**B**) The dynamic range of the two systems was compared by the RFUsfGFP/OD 600 at the last time-point of measurement. Gray areas represent dark state while the blue areas represent illumination state.

The RFUsfGFP/OD 600 value of the induced groups increased more than that of the uninduced groups over time after exposed to blue-light, indicating that the EL222 system harbors blue-light inducement property (Fig. 31A). By comparing the two groups of circuit *J23106-EL222-pBLind-sfGFP* alone, it was found that the leakage of circuit *J23106-EL222-pBLind-sfGFP* was already high without blue-light inducement, and the expression of sfGFP was not significantly increased after blue-light irradiation (Fig. 31A, pink lines).

At the same time, for the circuit *pBLind-EL222-pBLind-sfGFP*, since EL222 was almost not expressed in the uninduced condition, the sfGFP leakage of the circuit was relative low in the dark condition. In the induced group, a small amount of EL222 dimerized and bound to the promoter pBLind, greatly increasing the expression level of EL222, and then forming a positive feedback to enhance the expression level of downstream sfGFP, resulting in a rapid increase of RFUsfGFP/OD 600 value (Fig. 31A, blue lines). The higher dynamic range of circuit *pBLind-EL222-pBLind-sfGFP* was obtained, compared to *J23106-EL222-pBLind-sfGFP* (Fig. 31B). Similar phenomenon was observed when the two circuits were implemented in *E. coli* (see our [Improve page](https://2021.igem.org/Team%3AXMU-China/Improve)).

In conclusion, the blue-light induced system based on transcription factor EL222 and its regulated promoter pBLind has excellent blue-light photo-sensitivity, which can keep the expression level of downstream genes in a relative low level in dark conditions. Once activated by blue-light illumination, a large number of downstream genes can be expressed via positive feedback regulation mechanism. Therefore, we hypothesized that the blue light-induced priming subsystem of *pBLind-EL222-pBLind*-effector protein was more suitable for the kill switch system (see our [Proof of Concept page](https://2021.igem.org/Team%3AXMU-China/Proof_Of_Concept)).

Reference

1. Alderkamp, A.-C.; Buma, A. G. J.; van Rijssel, M., The carbohydrates of Phaeocystis and their degradation in the microbial food web. Biogeochemistry 2007, 83 (1-3), 99-118.

2. Matias de Oliveira, D.; Rodrigues Mota, T.; Marchiosi, R.; Ferrarese-Filho, O.; Dantas dos Santos, W., Plant cell wall composition and enzymatic deconstruction. AIMS Bioengineering 2018, 5 (1), 63-77.

3. Hu M, Zhang C, Mu Y, Shen Q, Feng Y. 2010. Indole affects biofilm formation in bacteria. Indian J. Microbiol. 50: 362-368.

4. Eichmann, J.; Oberpaul, M.; Weidner, T.; Gerlach, D.; Czermak, P., Selection of High Producers From Combinatorial Libraries for the Production of Recombinant Proteins in Escherichia coli and Vibrio natriegens. Frontiers in Bioengineering and Biotechnology 2019, 7.

5. Meng, Q.; Tian, X.; Jiang, B.; Zhou, L.; Chen, J.; Zhang, T., Characterization and enhanced extracellular overexpression of a new salt‐activated alginate lyase. Journal of the Science of Food and Agriculture 2021.

6. Haitjema, C. H.; Boock, J. T.; Natarajan, A.; Dominguez, M. A.; Gardner, J. G.; Keating, D. H.; Withers, S. T.; Delisa, M. P., Universal Genetic Assay for Engineering Extracellular Protein Expression. ACS Synthetic Biology 2014, 3 (2), 74-82.

7. Jayaraman, P.; Devarajan, K.; Chua, T. K.; Zhang, H.; Gunawan, E.; Poh, C. L., Blue light-mediated transcriptional activation and repression of gene expression in bacteria. Nucleic Acids Res 2016, 44 (14), 6994-7005.

8. Endres, S.; Wingen, M.; Torra, J.; Ruiz-Gonzalez, R.; Polen, T.; Bosio, G.; Bitzenhofer, N. L.; Hilgers, F.; Gensch, T.; Nonell, S.; Jaeger, K. E.; Drepper, T., An optogenetic toolbox of LOV-based photosensitizers for light-driven killing of bacteria. Sci Rep 2018, 8 (1), 15021.

9. Drepper, T.; Eggert, T.; Circolone, F.; Heck, A.; Krauss, U.; Guterl, J. K.; Wendorff, M.; Losi, A.; Gartner, W.; Jaeger, K. E., Reporter proteins for in vivo fluorescence without oxygen. Nat Biotechnol 2007, 25 (4), 443-5.

Xiamen University, Fujian, China

No.422, Siming South Road, Fujian, P.R.China 361005