iGEM 2022 — Team xmu-china

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{% block title %}Notebook{% endblock %}
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## Notebook

February

- Our team was formally founded.

- We looked through the previous excellent projects and learned the basic notion of synthetic biology and iGEM. After comprehensive survey and discussion, we decided our project theme. We were then divided into three groups to implement our project separately. We held meetings and discussed every weekend about everyone’s progress and solved problems together.

3.19

- We celebrated the birthday of our three members together. Happy Birthday, Shuwan & Yue & Yiran!

- It’s the first birthday party of our team this year.

![](https://static.igem.wiki/teams/4195/wiki/web-picture/notebook/birthday.jpg)

April-May

- We learned the fundamental knowledge of molecular cloning and laboratory qualities. After that, we were trained for molecular cloning operations, which helped us to design experiments rationally.

- We optimized and constructed the sequences of the needed genes.

- We completed the original hypothesis testing experiments needed, then consulted professors for advice. With their help, we gradually refined our project.

April-May

- Interview with Prof. Lina Wu
We interviewed Ms. Lina Wu who’s the
associate professor of Xiamen
University. She gave us a lot of advice on the design of OMVs.

- Fisheries Bureau
We contacted the Fisheries Technology Promotion
Station of Fujian Provincial Fisheries Bureau, and conducted an online interview with Mr. Lin who is the deputy director of the laboratory.

May-June

BBa\_K4195135, BBa\_K4195136, BBa\_K4195137 were
constructed
successfully.

6.15

- Our team was officially accepted by iGEM.

6.10-6.26

BBa\_K4195116, BBa\_K4195117 were constructed successfully.

6.27-7.8

BBa\_K4195138 was constructed.

7.5

- Two first-year teams from Fujian Agriculture
and Forestry University (FAFU) contacted us hoping for a visit. We accepted their invitation and held an offline seminar at Xiamen University. After the meeting, we also helped them contact professor Chenyun Guo for an interview.

7.7

- We are all sincerely believed in the power of
prayer: everyday before
doing experiment, we turn facing the South Putuo Temple, and wish today everything will go well.

7.16

- We were invited by GXU-China to take part in an
online symposium held to
share projects among a few teams. Grabbing the chance to discuss the prototype of project with experienced teams, we took part in the symposium.

7.18

- After induction, the mortality of engineered bacteria carrying kill-switch system: 0%

7.9-7.20

- BBa\_K4195120, BBa\_K4195121, BBa\_K4195114,
BBa\_K4195115 were constructed
successfully.

7.29

Give up purifying OMVs using nickel column. Thank you for nothing, high
throughput Ni-based IMAC.

7.21-8.2

- BBa\_K4195164, BBa\_K4195165, BBa\_K4195166 were constructed successfully.

Interviewing Prof. Shaowei Li

We visited Professor Shaowei Li at the National
Research Center for
Infectious Disease Diagnostic Reagents and Vaccine Engineering Technology, and asked him about the problems we encountered in designing the surface display system of *E. coli* and the CRISPR/Cas9 system.

8.3-8.10

- BBa\_K4195164, BBa\_K4195165, BBa\_K4195166 were successfully characterized.

8.5

- Prepare to purify OMVs using ultracentrifugation.
Getting prepared for doing
experiment two o’clock in the morning.

8.8

- [Summer the girl]: I've created the greatest work of art on my NC membrane while the f\*\*king dot-blot SHOWED ME NOTHING!!!

![](https://static.igem.wiki/teams/4195/wiki/web-picture/notebook/nothing.jpg)

8.10

- The negative detection circuits (yes without
input) reports fluorescence.
That's the OPPOSITE results of what was expected.

8.14

- Thanks to South Putuo Temple, for the first time, the results are what I expected. The circuits with input reports STRONGER fluorescence than the circuits without input. Don’t forget to redeem tomorrow.

8.11-8.15

- BBa\_K4195120 was successfully characterized
.

8.15

- OMVs under TEM! We finally made it
[CRYOUT]!

8.16-8.24

- BBa\_K4195121 was successfully characterized
.

8.24

- Pumpkin boy said he was too tired to continue
the RNA EXTRACTION after getting nothing from the qPCR for several times.

8.31

- Discard the method of quantifying OMV by lipid using DiI dye. Embrace the BCA kit.

September

- interview with Prof. Yun Tian
We visited Professor Yun Tian at the School
of Life Sciences, Xiamen University, and asked her about the problems we encountered in the detection of pathogenic bacteria and the induction of engineered bacteria.

9.4

- We went to Xiamen Science and Technology Museum
for our mass education.
Meeting those children with their innocent smile really enlightened my day. I’m energetic again for those shit results (or maybe not).

9.17

- Successfully prove that OMV can envelop plasmid.

9.18

- I’m so exhausted with these fickle circuits. A circuit with the opposite result will still have the opposite result, and a circuit with the right result will not be reversed.

10.12

Wiki Freeze!

10.14

Wiki Freeze Again. Seriously?

10.30

Farewell, My magical friends! Love you all.

read me ↑

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## Engineering

#### Overview

Our strategy of prevention is to neutralize the toxins *via* outer membrane vesicles (OMVs) with the toxin receptors displayed on the surface. In the experimental workflow, we **designed** different parts to **verify** the function of surface display system and the displayed receptors, and finally **demonstrated** that **the receptor displayed both on the surface of engineered bacteria and OMVs can bind to toxins**.

#### Cycle 1

#### Verifying the function of surface display system ClyA

#### Design

ClyA is commonly used as a surface display system which can anchor heterologous protein on the surface of bacteria (*1*). r*Lv*APN1 is a truncated form of *Lv*APN1 (residues 205-591) which has higher affinity to toxins than the receptors on shrimp intestinal epithelial cells (*2*). In order to neutralize toxins, we tried to fuse r*Lv*APN1 to the C-terminal of ClyA for displaying the receptor. We constructed **the fusion protein ClyA-r*Lv*APN1-his** to verify whether r*Lv*APN1 could be displayed on the surface of the engineered bacteria or not at first.

#### Build

There were four basic parts assembled into the vector: promoter ([BBa\_I0500](http://parts.igem.org/Part%3ABBa_I0500)), RBS ([BBa\_B0034](http://parts.igem.org/Part%3ABBa_B0034)), ClyA-r*Lv*APN1-his coding sequence ([BBa\_K4195030](http://parts.igem.org/Part%3ABBa_K4195030)) and terminator ([BBa\_B0015](http://parts.igem.org/Part%3ABBa_B0015)). We got the constructed plasmid [BBa\_K4195131](http://parts.igem.org/Part%3ABBa_K4195131) successfully and transformed it into *E. coli* BL21(DE3), then the positive transformants were selected through colony PCR (Fig. 1) and sequencing.

![](https://static.igem.wiki/teams/4195/wiki/web-picture/engineering-success/bba-k4195131.png)

**Fig. 1 DNA gel electrophoresis of the colony PCR products of [BBa\_K4195131](http://parts.igem.org/Part%3ABBa_K4195131)\_pSB1C3.** Target bands (3879 bp) can be observed at the position between 3000 bp and 5000 bp.

#### Test

[Immunofluorescence (IF) technology](https://2022.igem.org/Team%3AXMU-China/experiments)  was implemented to test whether the display system is functional or not. [BBa\_K4195134](http://parts.igem.org/Part%3ABBa_K4195134) (for r*Lv*APN1-his expression) which has no surface display system was set as negative control, while [BBa\_K4195131](http://parts.igem.org/Part%3ABBa_K4195131) (for ClyA-r*Lv*APN1-his expression) was set as positive control. The arabinose-induced overnight culture of the two groups were then incubated with FITC-labeled anti-His-tag antibody and washed for several times, followed by measuring fluorescence intensity and OD600.

![](https://static.igem.wiki/teams/4195/wiki/web-picture/engineering-success/clya.png)

**Fig. 2 The results of immunofluorescence to characterize the function of ClyA surface display system (*p* = 0.0092).**

The ratio of fluorescence intensity to OD600 of positive control is higher than that of negative control (*p* = 0.0092) (Fig. 2), which indicates that our surface display system works well that the cargo (r*Lv*APN1) is displayed successfully.

#### Learn

This result allowed us to conclude that the **ClyA can display heterologous proteins on the surface of the engineered bacteria**. However, it was **not sufficient** to prove that the binding affinity of displayed receptor to the toxins is still retained.

#### Cycle 2

#### Attempt to verify the function of displayed r*Lv*APN1 through ClyA-r*Lv*APN1-his

#### Design and Build

After successfully demonstrating the function of ClyA system to display r*Lv*APN1, we then turned to the verification of **the function of r*Lv*APN1 after displayed**. Followed by the Cycle 1, we continued to use ClyA-r*Lv*APN1-his ([BBa\_K4195131](http://parts.igem.org/Part%3ABBa_K4195131)) to verify the function of displayed r*Lv*APN1 by testing its ability to bind to toxin PirB.

#### Test

IF technology was implemented again to test whether the displayed r*Lv*APN1 on the engineered bacteria is functional or not. The induced culture with **purified his-PirB** added was set as positive control while which had no toxins added was set as negative control.

Unfortunately, we had **never got** result of significant differences on the ratio of fluorescence intensity to OD600 between positive control and negative control.

#### Learn

After several failures, we checked our design carefully. We found that the problem was the displayed receptor and the toxins purified **were both fused with His-tag (6×His)**, so the FITC-labeled anti-His-tag antibody **can recognize and bind to the bacteria of both positive control and negative control**, resulting in no differences on the ratio of fluorescence intensity to OD600 between the two groups.

#### Cycle 3

#### Verifying the retained function of displayed r*Lv*APN1 through ClyA-r*Lv*APN1

#### Design

After realizing the problem, we constructed the fusion protein **ClyA-r*Lv*APN1 without His-tag** to verify the function of displayed receptor r*Lv*APN1.

#### Build

The coding sequence of ClyA-r*Lv*APN1-his in [BBa\_K4195131](http://parts.igem.org/Part%3ABBa_K4195131) was replaced by ClyA-r*Lv*APN1 to create [BBa\_K4195130](http://parts.igem.org/Part%3ABBa_K4195130). We got the constructed plasmid successfully and transformed it into *E. coli* BL21(DE3), then the positive transformants were selected through colony PCR (Fig. 3) and sequencing.

![](https://static.igem.wiki/teams/4195/wiki/web-picture/engineering-success/bba-k4195130.png)

**Fig. 3 DNA gel electrophoresis of the colony PCR products of [BBa\_K4195130](http://parts.igem.org/Part%3ABBa_K4195130)\_pSB1C3.** Target bands (3816 bp) can be observed at the position between 3000 bp and 5000 bp.

#### Test

1. IF technology was implemented as before to determine **the function of displayed r*Lv*APN1 on the surface of engineered bacteria**. Control settings were the same to the last cycle.

![](https://static.igem.wiki/teams/4195/wiki/web-picture/engineering-success/pirb.png)

**Fig. 4 The results of immunofluorescence to probe the binding event
on the surface of engineered bacteria.** Purified his-PirB was tested to interact with the displayed receptor r*Lv*APN1 (*p* = 0.0213).

The ratio of fluorescence intensity to OD600 of positive control is higher than that of negative control (*p* = 0.0213) (Fig. 4), which indicates that the displayed r*Lv*APN1 can bind to the toxins.

2. Besides, [dot blot](https://2022.igem.org/Team%3AXMU-China/experiments) (*3*, *4*) was further implemented to test **whether the displayed r*Lv*APN1 on OMVs is functional or not**. The OMVs extracted from the culture of engineered bacteria harboring [BBa\_K4195130](http://parts.igem.org/Part%3ABBa_K4195130) was set as positive control while which without r*Lv*APN1 displayed was set as negative control.

![](https://static.igem.wiki/teams/4195/wiki/web-picture/engineering-success/dot-blot-in-engineering-success.png)

**Fig. 5 The imaging results of chemiluminescence (dot blot
analysis) to probe the binding event on the surface of OMVs.**

As with his-PirB added, we observed a strong signal from the OMVs fraction derived from the engineered bacteria harboring [BBa\_K4195130](http://parts.igem.org/Part%3ABBa_K4195130). However, a faint detectable signal was observed from the OMVs fraction derived from the bacteria without r*Lv*APN1 displayed (INPNC-TTPA in this case) (Fig. 5), suggesting that r*Lv*APN1 displayed on OMVs can still bind to toxins PirB specifically. For the faint detectable signal observed from the OMVs franction derived from the bacteria without r*Lv*APN1 displayed, we attributed this to nonspecific adsorption or potential weak cross-reactivity of our FITC-labeled anti-His-tag antibody to INPNC-TTPA.

For the faint detectable signal observed from the OMVs fraction derived from the bacteria without r*Lv*APN1 displayed, we attributed this to the potential cross-reactivity of our FITC-labeled anti-His-tag antibody to INPNC-TTPA. Learn more information about this from our [Proof of Concept](https://2022.igem.org/Team%3AXMU-China/proof-of-concept) page.

#### Learn

The result shows that **the ClyA-mediated displayed r*Lv*APN1 on the surface of engineered bacteria and OMVs can bind to toxins**. This was **the most important step** in proving that our design works.

#### Conclusion

Engineering a biological system cannot be easy, we need to put our heart into it and move towards the success through experiment and feedback step by step. Here we have tried to demonstrate how **we were able to verify that the receptor displayed both on the surface of engineered bacteria and OMVs can bind to toxins** through successive iterations of the **DBTL** (Design-Build-Test-Learn) cycle (Fig. 6).

![](https://static.igem.wiki/teams/4195/wiki/web-picture/engineering-success/engineering-success.png)

**Fig. 6 Summary of the different engineering steps completed before reaching the final proof of concept of the retained function of r*Lv*APN1 displayed.**

#### Reference

1. K. Murase, Cytolysin A (ClyA): A Bacterial Virulence Factor with Potential Applications in Nanopore Technology, Vaccine Development, and Tumor Therapy. *Toxins (Basel).* **14**, 78 (2022).

2. W. Luangtrakul *et al.*, Cytotoxicity of *Vibrio parahaemolyticus* AHPND toxin on shrimp hemocytes, a newly identified target tissue, involves binding of toxin to aminopeptidase N1 receptor. *PLoS Pathog*. **17**, e1009463 (2021).

3. J. L. Valentine *et al.*, Immunization with Outer Membrane Vesicles Displaying Designer Glycotopes Yields Class-Switched, Glycan-Specific Antibodies. *Cell Chem. Biol.* **23**, 655-665 (2016).

4. T. C. Stevenson *et al.*, Immunization with outer membrane vesicles displaying conserved surface polysaccharide antigen elicits broadly antimicrobial antibodies. *Proc. Natl. Acad. Sci. U. S. A.* **115**, E3106-E3115 (2018).

* [Overview](#Overview)
* [Cycle1](#Cycle1)
* [Cycle2](#Cycle2)
* [Cycle3](#Cycle3)
* [Reference](#reference)

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## Experiments

#### Common Experiment

Molecular Cloning Handbook

#### [Handbook](#collapse0)

[Molecular\_Cloning\_Handbook.pdf](https://static.igem.wiki/teams/4195/wiki/pdf/molecular-cloning-handbook-8-0.pdf)

#### Detection Part

Fiber Filter Paper DNA enrichment

#### [I. Materials & Apparatus](#collapse51)

● guanidine hydrochloride
● Tris (pH 8.0)
● Triton X-100
● Tween-20
● Proteinase K
● Rapid quantification filter paper
● Hole punch (pore diameter 3 mm)
● 1.5 mL centrifuge tube

#### [II. Before Experiment](#collapse52)

(1) Prepare extraction buffer
800 mM guanidine hydrochloride, 50 mM Tris, 0.5% Triton X-100, 1% Tween-20, 40 μg/mL proteinase K.
(2) Prepare wash buffer (10 mM Tris, 0.1% Tween-20)
(3) Prepare circular filter paper
Use the hole punch to punch out the circular filter papers with diameter of 3 mm, 4 mm, 5 mm, 6 mm. Before the experiment, a circular filter paper was placed in each 1.5 mL tube.

#### [III. Protocol](#collapse53)

(1) Add 300 μL DNA solution (1 ng/mL, 5 ng/mL, 10 ng/mL) to the 1.5 mL tube containing a circular filter paper for approximately 1 minute.
∗If the sample is bacteria culture: Dilute the bacteria culture after shaking (10 times, 100 times, 1000 times). Add 100 μL sample to 200 μL extraction buffer and vortex for 10 seconds for cell lysis, then add this cell lysate to the 1.5 mL tube containing a circular filter paper for approximately 1 minute.
(2) Use the pipette to draw out the fluid, then add 200 μL wash buffer to this tube for 1 minute to remove contaminants including amplification inhibitors.
(3) After 1 minute, the circular filter paper containing nucleic acid was then transferred into an amplification reaction.
(4) After amplification, agarose gel electrophoresis was used to determine the effect.

Recombinase Polymerase Amplification

#### [I. Materials & Apparatus](#collapse54)

● Different concentrations of plasmid solution: 0.1 ng/ L, 1 ng/mL, 10 ng/mL, 100 ng/mL, 1000 ng/mL.
● DNA extraction products of filter paper diluted with different multiples (1, 10, 100, 1000, 10000) of bacterial culture.
● TwistAmp® Liquid Basic Kit
● 1.8 mM dNTPs

#### [II. Protocol](#collapse55)

(1) Add 2.4 μL of each primer at 10 μM concentration to 0.2 mL PCR tubes.
(2) Prepare a pre-master mix (per reaction) in the order below.

|  | Name Volume | |
| --- | --- | --- |
|  | 2x Reaction Buffer | 25 µL |
|  | 1.8 mM dNTPs | 9 µL |
|  | ddW | 0.2 µL |
|  | 10x Basic E-mix | 5 µL |

Vortex and spin briefly.
(3) To the pre-master mix, add 2.5 μL 20x Core Reaction Mix to tube lid. Spin briefly. Master mix is now complete.
(4) Add 41.7 μL of master mix to primers prepared in tubes (step 1) and evenly mix.
(5) Add 2.5 μL of 280 mM MgOAc and 1 μL template or cellulose-based paper separately to tube lids. Spin briefly.
(6) Incubate at 37°C for 20-40 minutes.
(7) Separate pieces of DNA using gel electrophoresis.

Cell-free System (myTXTL)

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

● myTXTL Mix (8 μL autolysate and 8.9 μL TX-TL premix)
● DNA template
● Nuclease-free water

#### [II. Protocol](#collapse42)

(1) Preheat water bath to 37 °C.
(2) Completely thaw the myTXTL Mix and DNA on ice completely. Keep reagents on ice till use.
(3) Directly before use, vortex the myTXTL Mix for 2-3 seconds and spin down briefly. If any precipitate is visible hereafter, resuspend master mix solution gently about 10 times to ensure homogeneity. Avoid formation of bubbles and foam.
(4) Add 3.1 uL DNA or other reagents into the myTXTL Mix.
(5) Vortex the reaction mixture for 2-3 seconds gently and centrifuge the assembled myTXTL reaction briefly to collect the entire volume at the bottom of the tube.
(6) Incubate the myTXTL positive control reaction(s) for 3~6 h at 37 °C.
(7) Stop the myTXTL positive control reaction by inserting the tube(s) into ice.

Preparation of myTXTL Mix

#### [Preparation of myTXTL Mix](#collapse-p)

[Preparation-of-myTXTL-Mix.pdf](https://static.igem.wiki/teams/4195/wiki/pdf/preparation-of-mytxtl-mix.pdf)

*In vivo* Detection (AmpC)

#### [I. Materials & Apparatus](#collapse43)

● TECAN® Infinite M200 Pro Microplate Reader
● 96-well microtiter plates
● 1x PBS
● LB liquid medium
● Chloramphenicol
● Kanamycin
● BeyoLyticTM Bacterial Active Protein Extraction Reagent
● Nitrocefin

#### [II. Before Experiment](#collapse44)

Prepare two groups of 50-mL conical flask, breathable sterile sealing film covers the bottleneck. 10 mL of LB liquid medium with corresponding antibiotics was added to each flask.

#### [III. Protocol](#collapse45)

(1) Inoculate 100 µL bacteria glycerol stock into 10 mL LB liquid medium with corresponding antibiotics, 37 ℃, 200 rpm overnight.
(2) Inoculate 100 µL bacteria culture of experimental and control group into 10mL liquid LB culture medium with corresponding antibiotics. Incubate at 37°C for 4–6 h until the OD600=0.6-0.8.
(3) The bacterial culture was induced with 1‰ IPTG and cultured at 37°C for 1 h.
(4) Add 200 µL bacteria culture to the 96-well plate and OD600 was measured by a microplate reader.
(5) Add liquid to the 96-well plate in the following order according to the following table:

|  | Name Volume | |
| --- | --- | --- |
|  | 1 x PBS | 100 µL |
|  | ddW | 20 µL |
|  | bacteria culture | 20 µL |
|  | BeyoLyticTM Bacterial Active Protein Extraction Reagent | 50 µL |
|  | Nitrocefin (0.5 mg/ mL) | 10 µL |

(6) The relative absorbance unit (RAU) was measured by a microplate reader (490 nm), every 2 minutes for 20 minutes.

#### [IV. Notes](#collapse46)

The relative absorbance unit (RAU) at different time points for a sample culture was determined by the blank corrected output signal divided by OD600, and after subtracting its triplicate-averaged counterpart of the negative control cultures (reporter-free) at the same time. Unless indicated otherwise, each reporter within different sensors was tested with three biological replicates. All the data shown are mean values with standard deviation as error bars.

*In vivo* Detection (NanoLuc)

#### [I. Materials & Apparatus](#collapse47)

● TECAN® Infinite M200 Pro Microplate Reader
● 96-well microtiter plates
● 1x PBS
● LB liquid medium
● Chloramphenicol
● Kanamycin
● BeyoLyticTM Bacterial Active Protein Extraction Reagent
● Nano-Glo® Luciferase Assay Substrate
● Nano-Glo® Luciferase Assay Buffer

#### [II. Before Experiment](#collapse48)

(1) Prepare two groups of 50 mL conical flask, ventilate sterile sealing film covers the bottleneck. 10 mL of LB liquid medium with corresponding antibiotics was added to each flask.
(2) Nano-Glo® Luciferase Assay Reagent Preparation:
Mix Nano-Glo® Luciferase Assay Substrate with 50-fold Nano-Glo® Luciferase Assay Buffer.

#### [III. Protocol](#collapse49)

(1) Inoculate 100 µL bacteria glycerol stock into 10 mL LB liquid medium with corresponding antibiotics, 37°C, 200 rpm overnight.
(2) Inoculate 100 µL bacteria culture of experimental and control group into 10 mL liquid LB culture medium with corresponding antibiotics. Incubate at 37°C for 4–6 hours until the OD600 = 0.6-0.8.
(3) The bacterial culture was induced with 1‰ IPTG and cultured at 37°C for 1 h.
(4) Add 200 µL bacteria culture to the 96-well plate and OD600 was measured by a microplate reader.
(5) Add liquid to the 96-well plate according to the following table:

|  | Name Volume | |
| --- | --- | --- |
|  | 1 x PBS | 190 µL |
|  | bacteria culture | 1 µL |
|  | Bacterial lysate | 5 µL |
|  | Nano-Glo® Luciferase Assay Reagent | 4 µL |

Vortex briefly.
(6) The relative luminescence unit (RLU) was measured by microplate reader (460 nm), every 5 minutes for 30 minutes.

#### [IV. Notes](#collapse50)

The relative luminescence unit (RLU) at different time points for a sample culture was determined by the blank corrected output signal divided by OD600. Unless indicated otherwise, each reporter within different sensors was tested with three biological replicates. All the data shown are mean values with standard deviation as error bars.

#### OMVs Part

OMV purification

#### [I. Materials & Apparatus](#collapse56)

● LB liquid medium with 34 μg/mL chloramphenicol
● Shaking incubator
● OPTIMA＆trade; XE-90 ultracentrifuge
● Transmission electron microscope
● 0.45-μm and 0.22-μm filter

#### [II. Before Experiment](#collapse57)

Use sterile water to prepare the resuspension buffer: 50 mM HEPES-0.85% NaCl buffer, pH = 7.4. Then filter it through 0.22-μm filter.

#### [III. Protocol](#collapse58)

(1) Inoculate 200 µL bacteria glycerol stock into 10 mL LB liquid medium with 34 μg/mL chloramphenicol, 37°C and 200 rpm overnight.
(2) Inoculate 1 mL bacteria culture into flasks contained 50 mL LB liquid medium with 34 μg/mL chloramphenicol, incubate the culture at 37°C and 200 rpm until the OD600 reaches 0.6-0.8.
(3) If necessary, add appropriate amount of inducer into bacteria culture, and then incubate the culture at 28°C and 200 rpm for 16 hours. Or directly incubate the culture at 37°C and 200 rpm for 12 hours.
(4) After incubation, 50 mL of the culture is centrifuged at 10000 *×g* for 15 minutes at 4°C to remove cells.
(5) Then, the supernatant was filtered through the 0.45-μm filter once then 0.22-μm filter for a second time.
(6) The cell-free supernatant is used to extract the OMV *via* ultracentrifugation at 140,000 *×g* for 2 hours at 4°C.
(7) The OMV pellets (may not be easily visible) are suspended in 100 μL resuspension buffer, then stored at -20°C.
(8) The diluted OMV samples were spotted onto LB agar plate with 34 μg/mL chloramphenicol to verify that there is no bacteria component.
(9) The OMV samples are negatively stained for TEM observation.

#### [IV. Notes](#collapse59)

When the culture supernatant is filtered, be very careful to prevent bacterial contamination!

OMV quantitation

#### [I. Materials & Apparatus](#collapse60)

● TECAN® Infinite M200 Pro microplate reader
● 96-well plates
● BCA Protein Assay Kit (Beyotime)

#### [II. Protocol](#collapse61)

(1) OMVs are extracted according to the OMV purification protocols.
(2) The OMVs are lysed by 1% (w/v) SDS (final concentration) at room temperature for 10 minutes.
(3) Take 4 μL lysate, measure protein concentration of each group’s OMV according to the user’s manual of BCA Protein Assay Kit.
(4) According to the standard curve, calculate the protein concentration and normalize it to OD600.

#### [III. Notes](#collapse62)

Measure the OD600 of each group when they are taken out from the shaking incubator as soon as possible. Subsequently, centrifuge at 10,000 *×g* for 15 minutes at 4°C to separate the bacterial precipitation and supernatant in order to obtain a more convincing data.

Verify whether the outer membrane vesicle contains plasmid

#### [I. Materials & Apparatus](#collapse63)

● DNase I
● Electrophoresis apparatus
● Dual LED blue/white light transilluminator
● PCR thermal cycler machine
● Agarose gel

#### [II. Protocol](#collapse64)

(1) Treat the extracted vesicles with DNase I at 37°C for 30 minutes.
(2) Deactivate DNase I at 75°C for 15 minutes.
(3) Denature in 100℃ water bath for 10 minutes.
(4) Pipet 3 μL of treated solution for PCR amplification.
(5) Apply the PCR products to agarose gel electrophoresis.
(6) Check whether there are correct strips on the gel illuminated by transilluminator.

Verify the efficiency of outer membrane vesicles (OMVs) mediated transformation

#### [I. Materials & Apparatus](#collapse66)

● LB agar plate
● Arabinose
● Purified OMVs

#### [II. Protocol](#collapse67)

(1) Incubate the culture of the experimental group and the control group respectively with the OMVs in different proportions at 37 °C.
(2) Dilute each group to 10-2 and 10-4 concentration respectively.
(3) Apply each diluted culture to agar spot test.
(4) Select single colonies for colony PCR and then apply the PCR products to agarose gel electrophoresis to verify whether the plasmid is transferred during incubation.
(5) Select the colonies with the correct band for culture.
(6) Each culture is applied to agar point test on the medium with arabinose induction and the medium without arabinose induction respectively to verify the killing effect of the plasmid.

#### Protein-protein interaction (PPI) Part

Immunofluorescence (IF) for verification of surface display system functionality

#### [I. Materials & Apparatus](#collapse68)

● LB liquid medium with 0.1 mg/mL chloramphenicol
● PBS buffer
● Microplate reader
● Centrifuge
● FITC anti-6xHis Antibody (Abcam)
● 5% defatted milk

#### [II. Protocol](#collapse71)

(1) Bacteria culture was inoculated into LB liquid mediums at 2% (v/v) and kept at 37 °C, 200 rpm and last 12-16 h.
(2) Bacteria culture from step 1 was inoculated into LB liquid mediums at 2% (v/v) and kept at 37 °C, 200 rpm and last 2-4 h.
(3) When the OD600 of bacteria culture from step (1) is 0.6-0.8, add 0.2% (v/v) arabinose and keep at 37 °C, 200 rpm and last 2-4 h.
(4) Pellet 1 mL bacteria culture from step (3) in a clean 1.5-mL microcentrifuge tube. Centrifuge at 6500 rpm for 3 minutes at room temperature. Decant or aspirate medium and discard.
(5) Add 1 mL PBS buffer, evenly mix the sample. And centrifuge at 6500 rpm for 3 minutes at room temperature. Decant or aspirate medium and discard.
(6) Repeat step (5) twice.
(7) Add 500 μL - 1 mL 5% defatted milk, evenly mix the sample, incubated at 37°C for 0.5-1 h.
(8) Centrifuge at 6500 rpm for 3 minutes at room temperature. Decant or aspirate medium and discard.
(9) Repeat step (5) three times.
(The following operations should be performed in darkness)
(10) Add 490 uL PBS buffer, evenly mix the sample. Add FITC anti-6xHis Antibody（1: 1000）(Abcam), incubate at 37°C for 1 h.
(11) Repeat step (8) once, step (5) three times.
(12) Inoculate 200 μL bacteria culture from step (11) into 96-well microtiter plates, use microplate reader reads the fluorescence intensity (Absorption wavelength: 492 nm, Emission wavelength: 518 nm) and OD600.

IF for verification protein-protein interaction (PPI)

#### [I. Materials & Apparatus](#collapse72)

● LB liquid medium with 0.1 mg/mL chloramphenicol
● TBST buffer
● Microplate reader
● Centrifuge
● FITC anti-6xHis Antibody (Abcam)
● 5% defatted milk

#### [II. Protocol](#collapse73)

(1) Bacteria culture was inoculated into LB liquid mediums at 2% (v/v) and incubated at 37 °C, 200 rpm, 12-16 h.
(2) Bacteria culture from step 1 was inoculated into LB liquid mediums at 2% (v/v) and incubated at 37 °C, 200 rpm, last 2-4 h.
(3) When the OD600 of bacteria culture from step 1 is 0.6-0.8, add 0.2% (v/v) arabinose and keep at 37 °C, 200 rpm and last 2-4 h.
(4) Pellet 1mL bacteria culture from step 3 in a clean 1.5-mL microcentrifuge tube. Centrifuge at 6500 rpm for 3 minutes at room temperature. Decant or aspirate medium and discard.
(5) Add 1mL TBST buffer, evenly mix the sample. And centrifuge at 6500 rpm for 3 minutes at room temperature. Decant or aspirate medium and discard.
(6) Repeat step (5) twice.
(7) Add 500μL - 1mL 5% defatted milk, evenly mix the sample, incubated at 37°C for 0.5-1 h.
(8) Centrifuge at 6500 rpm for 3 minutes at room temperature. Decant or aspirate medium and discard.
(9) Repeat step (5) three times.
(10) Add 500 μL-1 mL TBST, evenly mix the sample. Add appropriate concentration of protein culture to positive control while negative control not. Incubated at 4°C for 10 h.
(11) Repeat step (8) once, repeat step (5) three times.
(The following operations should be performed in darkness)
(12) Add 490 uL TBST buffer, evenly mix the sample. Add FITC anti-6xHis Antibody（1: 2500）(Abcam), incubate at 37°C for 1 h.
(13) Repeat step (8) once, repeat step (5) three times.
(14) Inoculate 200 μL bacteria culture from step (13) into 96-well microtiter plates, use microplate reader reads the fluorescence intensity (Absorption wavelength: 492 nm, Emission wavelength: 518 nm) and OD600.

Dot Blot

#### [I. Materials & Apparatus](#collapse74)

● Wesrern Blot Imager
● Rotating shaker
● Incubation box
● Tweezers
● Scissors
● 2B pencil
● OMVs
● Protein
● 1x TBST
● Non-fat dry milk
● Anti-His Mouse Monoclonal Antibody
● Goat Anti-Mouse IgG (H+L), HRP Conjugate
*EasySee*® Western Blot Kit

#### [II. Before Experiment](#collapse75)

(1) Wipe clean with tweezers and scissors.
(2) Prepare soluton:
TBST(1L) : Tris-HCl(1M) 50 ml, NaCl 8 g, KCl 0.2 g, Ttwain 0.5 ml
5% milk, 1x TBST : Dissolve 5 g non-fat dry milk in 100 mL 1x TBST and filter.

#### [III. Protocol](#collapse76)

(1) Label the nitrocellulose blotting membrane for the ease to locate your individual sample after blotting.
(2) Place the labeled membrane in an incubation box.
(3) Pipette 1.5 uL of each sample onto separate pre-determined locations on the blot, four dots for each sample. The samples should be absorbed to the membrane immediately without any beading on the surface.
(4) Block the membrane in 5% milk, 1x TBST for 1 hour on a rotating shaker.
(5) While blocking, dilute the protein in 5% milk, 1x TBST.
(6) Decant blocking solution, rinse the membrane with 1x TBST, wash the membrane 3 times with 1x TBST buffer, each time 5 minutes with shaking on a rotating shaker.
(7) Incubate the membrane with the diluted protein for 2 hours on a rotating shaker.
(8) Discard the diluted protein and wash with 5% milk, 1x TBST for 5 minutes on a shaking incubator for 3 times.
(9) While washing, dilute the primary antibody in 5% milk, 1x TBST.
(10) After washing, incubate the membrane with the diluted primary antibody. Incubate for 1 hour on a shaking incubator.
(11) Discard the diluted primary antibody and wash with 5% milk, 1x TBST for 5 minutes on a shaking incubator for 3 times.
(12) While washing, dilute the second antibody in 5% milk, 1x TBST.
(13) After washing, incubate the membrane with the diluted second antibody. Incubate for 1 hour on a shaking incubator.
(14) Discard the diluted primary antibody and wash with 1x TBST for 5 minutes on a shaking incubator for 3 times.
(15) Proceed with incubation using *EasySee*® Western Blot Kit for 1 min (using freshly prepared working solution) . Wrap the membrane in a plastic wrap and remove bubbles and wrinkles. Place the membrane in a chemilunescence instrument to capture the image.

#### Others

Verify the function of endolysin

#### [I. Materials & Apparatus](#collapse77)

● LBv2 medium
● LB medium
● PBS
● Ultrasonic crusher
● Bradford
● 96-well plate
● NaOH powder

#### [II. Protocol](#collapse78)

**Expression and purification of Lysqdvp001 gene (his-edl060)**
(1) Incubate *E. coli* BL21(DE3) with I0500-B0034-his-edl060-B0015\_ pUC57-Simple plasmid in LB medium with 1 μL/mL ampicillin at 37°C until the OD600= 0.6.
(2) Pipet 4 mL of the culture and incubate in 400 mL medium with ampicillin for 2-4 hours at 37 °C.
(3) Add arabinose with the final concentration of 0.2%. Then add the same amount of arabinose after 1 hour, and then repeat inducing every 2 hours until the induction time overall reaches 12 hours.
(4) Pour the culture into 50 mL centrifuge tubes.
(5) Centrifugate at 6500 rpm for 15 minutes, then resuspend the precipitate in 1% PBS solution.
(6) Repeat step (5) twice.
(7) Froze the precipitate overnight in the refrigerator at -80 °C.
(8) Suspend the frozen precipitate with 1% PBS solution.
(9) Apply the solution to ultrasonic disruption.
(10) Filtrate the lysate supernatant with 0.45-micron pore size filter membrane.
(11) Use AKTA to purify edl060 protein.
(12) Measure the protein concentration with Bradford method.
(13) Dialyze the protein.
(14) Freeze the dialyzed protein in a -80°C refrigerator.
(15) Freeze dry the solution to concentrate.
(16) Measure the concentration of edl060 protein with Bradford method.
**Verification of the killing effect of endolysin**
(1) Incubate *E. coli* BL21(DE3) and *Vibrio alginolyticus* ATCC33787 in LB medium, *Vibrio natriegens* strain *VnDX* in LBv2 medium.
(2) Pipette 1 mL of each culture. Incubate with 1 mL 150 mM EDTA for 30 minutes.
(3) Centrifuge the solution at 6500 rpm for 10 minutes to remove EDTA, and remove the supernatant. Then add 1mL corresponding medium and resuspend the precipitation.
(4) Repeat step (3) twice.
(5) Dilute each solution, and the dilution concentration is 10-3, 10-4, 10-5, 10-6.
(6) The solution of each dilution concentration shall be treated as follows:
a. each with different diluted concentration shall be treated with isochoric sterile water and then be applied to agar spot test;
b. each with different dilution concentration gradient shall be treated with 150 mM EDTA for 30 minutes and then be applied to agar spot test;
c. each with dilution concentration of 10-5 shall be treated with endolysin edl060 for 5 minutes, 10 minutes, 15 minutes and 20 minutes, and then apply each group to agar spot test;
d. each with different dilution concentration shall be treated with 10% NaOH for 20 minutes and be applied to agar spot test.
(7) Incubate overnight at 37 °C.
(8) Observe the results of agar spot test.

Comparison of the rhamnose-inducible promoter induction effects of *L*-rhamnose and *L*-mannose

#### [I. Materials & Apparatus](#collapse79)

● LB liquid medium with 40 μg/mL kanamycin
● 100 mg/mL *L*-rhamnose and *L*-mannose stock solution
● Shaking incubator
● TECAN＆reg; Infinite M200 Pro microplate reader
● 96-well plates
● Corning＆reg; costar 96-well black flat-bottom plates

#### [II. Protocol](#collapse80)

(1) Inoculate 200 µL bacteria glycerol stock into 10 mL LB liquid medium with 40 μg/mL kanamycin (final concentration), 37°C and 200 rpm overnight.
(2) Inoculate 200 µL bacteria culture into flasks contained 10 mL LB liquid medium with 40 μg/mL kanamycin, then the culture was incubated at 37°C and 200 rpm until the OD600 reaches 0.6-0.8.
(3) Add the *L*-rhamnose and *L*-mannose stock solution to different groups respectively to reach different final concentrations 0, 0.1, 0.4, and 0.7 mg/mL.
(4) After cultured at 37 °C, 200 rpm for 12 hours, take 200 µL of culture for detecting OD600 and fluorescence intensity.

* [Common](#Common)
* [Detection](#Detection)
* [OMVs](#OMVs)
* [PPI](#PPI)
* [Others](#Others)

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## Result

#### Overview

Our solution for **AHPND (Acute Hepatopancreatic Necrosis Disease)** caused by *Vibrio parahaemolyticus* (*Vp* for short) can be divided into three sections: detecting the pathogen, treating the disease and preventing the outbreak of infection. We organized our experimental results in the order of Detection, Treatment and Prevention. Besides, we demonstrated the feasibility of the kill switch, which is also an important part for the sake of biosafety and biocontainment of OMEGA. The fundamental aspects of our projects are composed of all the results we obtained.

#### Detection

Since the AHPND-causing pathogen *Vibrio parahaemolyticus* can secrete toxins PirA and PirB, we decided to choose the toxin encoding genes, *pirA* and *pirB*, as the target for developing our detection system in OMEGA. In order to overcome the limitations of the whole-cell biosensor (*1*-*4*) in which the commonly used input, quorum-sensing signals, are so complicated among *Vibrio* spp. (*5*, *6*) and to bypass the extensive use of technology like [lateral flow assay (LFA)](https://2020.igem.org/Team%3AVilnius-Lithuania), the cell-free system combining post-transcriptional regulation was considered as the strategy to detect the pathogen-derived nucleic acids with leveraging the wisdom of synthetic biology. The total workflow of the detection system can be divided into three sections: nucleic acids enrichment, recombinase polymerase amplification (RPA), *in vitro* transcription, and translation. As described in detail in [Design](https://2022.igem.wiki/xmu-china/design), the ribozyme-enabled detection of RNA (RENDR) (*7*) plays a significant role in signal output, and therefore we constructed an array of expression circuits to test the performance of different split modes combing different reporters *in vivo* in particular, before the real implementation on the cell-free system.

#### Highlight

 Confirm the toxin genes can be enriched from the samples and amplified *via* RPA

 Verify the performance of various guides with three reporters *in vivo* characterization

#### Introduction

Firstly, to guarantee the specificity and accuracy of the RENDR system, the conserved regions of *pirA* and *pirB* were analyzed by using the Nucleotide BLAST of NCBI. And the transcripts of the **conserved regions** of *pirA* and *pirB* were set as input signals. While the RNA guide sequences (guides, for short) linked with split ribozymes are carefully designed to be complementary to the RNA input signals. Secondly, the NUPACK (*8*) web-server was used to calculate the free energy differences between the seed complex and three single-stranded RNAs, which was compared with the free energy differences of the circuit used in the reference (*7*). Two input-guided pairs for each toxin gene, whose free energy differences were **greater than or equal to** that in the reference were selected and named “pirA(B)\_g1” and “pirA(B)\_g2”, respectively. Additionally, we adopted **two ribozymes split modes** for constructing the detection circuits (split site at 15 nt (named “α”) and split site at 402 (named “β”)), which are reported to be highly functional. And therefore, the gene circuits expressing guides were all written in the following style, T7-pirA\_g1α-T7t, which means that the guides are designed to target *pirA* with the pairing type g1 and ribozyme-split mode α under the control of T7-transcriptional system.

Another important part that should be mentioned is the reporter, which determines the feature of the output signal (*9*). We have attempted to apply **three different types of reporters** for constructing the detection system, including fluorescence (GFP), colorimetry (AmpC), and bioluminescence (NanoLuc luciferase), which were all tested for characterization *in vivo*. While for the implementation *in vitro* and related examination in our [home-built hardware](https://2022.igem.wiki/xmu-china/hardware), only colorimetric and bioluminescent reporters were tested in accordance with our [Design](https://2022.igem.wiki/xmu-china/design). The following figure (Fig. 1) presents the different elements composing of the toxin gene sensing circuit.

![15](https://static.igem.wiki/teams/4195/wiki/result/detection/fig1.png)

**Fig. 1 Illustration of the elements involved in the design of the guides circuit of the Detection part.**

#### Results

Toxin genes can be extracted from water, enriched on fiber filter paper, and amplified *via* RPA

#### [Show Details](#collapse-d1)

Fiber filter paper enriches DNA

Filter paper can enrich DNA from plasmid extract

To test the enrichment effect of fiber filter paper, [BBa\_K4195179](http://parts.igem.org/Part%3ABBa_K4195179)\_pSB1C3 (expressing the conserved region of *pirA*) or [BBa\_K4195180](http://parts.igem.org/Part%3ABBa_K4195180)\_pSB1C3 (expressing the conserved region of *pirB*) were used. 300 μL plasmid solution with different concentrations (1 ng/mL, 5 ng/mL, and 10 ng/mL) were added into the 1.5-mL tube containing a circular filter paper with different sizes (diameter was 3 mm, 4 mm, 5 mm, and 6 mm). After being incubated for 1 min, the paper was washed with wash buffer and directly amplified using PCR. In this case, deionized water and plasmid solution with the same concentration were used as negative and positive controls, respectively. More details of the reagents we used are shown in our [Experiments](https://2022.igem.wiki/xmu-china/experiments) page.

As shown in DNA gel electrophoresis of the PCR products (Fig. 2), target bands (567 bp and 569 bp) can be observed at the position between 750 bp and 500 bp. The result proved that **the filter paper is able to enrich the plasmid**. Based on the result, we select the 3 mm filter paper, which had the best enrichment effect even at the plasmid concentration of 1 ng/mL, for further experiments.

![15](https://static.igem.wiki/teams/4195/wiki/result/detection/fig2.png)

**Fig. 2 DNA gel electrophoresis of the fiber paper enriched PCR products from plasmid extract.**

Filter paper can enrich DNA from bacteria lysate

The overnight culture of colonies containing plasmid [BBa\_K4195179](http://parts.igem.org/Part%3ABBa_K4195179)\_pSB1C3 or [BBa\_K4195180](http://parts.igem.org/Part%3ABBa_K4195180)\_pSB1C3 was diluted in 10-fold and 100-fold. The bacterial culture was lysed by extraction buffer, and the plasmids are enriched by the filter paper placed into the bacteria lysate. After enrichment for a certain time, the paper was washed with wash buffer and directly amplified using PCR. Deionized water and plasmid solution were used as negative and positive controls, respectively.

As shown in DNA gel electrophoresis of the PCR products (Fig. 3), enrichment of DNA was accomplished from bacteria lysate dilutions. The ability of the filter paper to enrich DNA had been verified through PCR and DNA gel electrophoresis.

![15](https://static.igem.wiki/teams/4195/wiki/result/detection/fig3.png)

**Fig. 3 DNA gel electrophoresis of the fiber paper enriched PCR products from bacteria lysate.**

RPA: Recombinase Polymerase Amplification

To enhance the input signals and reduce background interference, **Recombinase Polymerase Amplification (RPA)** is implemented to amplify the target toxin genes specifically. After obtaining the enriched nucleic acid sample, the next step is to verify whether this enrichment method can be used for subsequent RPA systems. To obtain the single-stranded RNA (ssRNA) which can be recognized by RENDR, the sequence of **the T7 promoter is added to the upstream of the forward primer** to finally enable the transcription of amplified genes by T7 RNA polymerase.

RPA can distinguish the level of target gene

The activation of protein expression in cell-free system is quantitative with respect to the abundance of target RNA, and increased levels of target will result in increased activation and accumulation of signal outputs. RPA, as with many isothermal amplification methods, undergoes asynchronous amplification with the potential to saturate, which will prevent accurate quantitation. With the intention of the quantitation of target level, **amplification time for target gene was optimized**. Plasmid (input) with different concentrations (10 ng/mL, 100 ng/mL, 1000 ng/mL and 10000 ng/μL) were amplified using RPA, which was performed through the **TwistDx kit** with different amplification times (40 min, 30 min, 20 min and 10 min). As shown in Fig. 4, we found that **as the amplification time decreased**, **the differences of the target bands’ gray scales** among different input concentrations **can be obviously distinguished**, especially the 10 min-amplification (Fig. 4d).

![15](https://static.igem.wiki/teams/4195/wiki/result/detection/fig4.png)

**Fig. 4 Amplification time optimization. a** RPA reactions with different target levels were run at 37 °C for **40** minutes. **b** RPA reactions with different target levels were run at 37 °C for **30** minutes. **c** RPA reactions with different target levels were run at 37 °C for **20** minutes. **d** RPA reactions with different target levels were run at 37 °C for **10** minutes.

RPA can work in the home-built hardware

**We have designed and built a [thermostatic box](https://2022.igem.wiki/xmu-china/hardware#D) to conduct RPA reactions**. After being incubated at 37 °C for 30 min using a functional incubation reaction chamber **in our hardware**, RPA products were verified through DNA gel electrophoresis. The products of RPA reaction incubated at 37 °C in a constant temperature incubator were set as positive control. As shown in Fig. 5, target bands can be observed in all lanes of the reaction in home-built hardware, which suggests that RPA can work well in the hardware we designed.

![15](https://static.igem.wiki/teams/4195/wiki/result/detection/fig5.png)

**Fig. 5 RPA reactions in the home-built hardware or a constant temperature incubator.** Lane 1, 2: RPA products of the home-built hardware, lane 3, 4: RPA products of the 37 °C constant temperature incubator.

*In vivo* Verification

#### [Show Details](#collapse-d3)

We hope to achieve the detection of target toxin genes collected
from the water sample using RENDR-based system. The RENDR system designed for *pirA/B* is
firstly verified *in vivo*, with GFP, *β*-lactamase (AmpC), and NanoLuc luciferase being chosen as the reporters. RNA input signals were transformed into fluorescent, colorimetric, and bioluminescent signals, to verify the feasibility of the detection system (Fig. 6).

![15](https://static.igem.wiki/teams/4195/wiki/result/detection/fig6.png)

**Fig. 6 Schematic illustration of RENDR, in which the light bulb represents different output signals contributed by different reporters.**

Ribozymes have the ability to catalyze RNA splicing in gene expression *in vivo*

Molecular cloning

The circuit used in the reference (*7*) was set as a control circuit. The sequence of self-splicing ribozyme is inserted within the coding sequence (CDS) of a super folder green fluorescence protein (sfGFP) gene such that **the translation of the full-length protein is disrupted in the absence of splicing**. Upon transcription and splice, the ribozyme removes itself from the flanking exons and forms a functional sfGFP mRNA, which is then translated to produce a fluorescent protein as output. The circuit was assembled into the vector pSB1C3 by standard BioBrick assembly. The constructed plasmids were transformed into *E. coli* BL21(DE3), then the positive transformants were selected by chloramphenicol and confirmed by colony PCR (Fig. 7) and sequencing.

![15](https://static.igem.wiki/teams/4195/wiki/result/detection/fig7.png)

**Fig. 7 DNA gel electrophoresis of the colony PCR products of [BBa\_K4195155](http://parts.igem.org/Part%3ABBa_K4195155) in *E. coli* BL21(DE3).** Target bands (1595 bp) can be observed at the position around 1500 bp.

Fluorescence intensity measurement

Colonies harboring the correct plasmid were cultivated and induced. The expression behavior of sfGFP was observed under blue-light gel imager, in which the green fluorescence intensity of the group sfG(R)FP after induction was even stronger than that of the positive control (EYFP) (Fig. 8). Thus, we demonstrated that ribozymes have the ability to catalyze RNA splicing in gene expression *in vivo*.

![15](https://static.igem.wiki/teams/4195/wiki/result/detection/fig8.png)

**Fig. 8 The image of different groups for testing the ribozyme’s activity *in vivo***. Bacteria harboring [BBa\_K914003](http://parts.igem.org/Part%3ABBa_K914003) (pRha) and pT7-B0034-*eyfp*-B0015\_pSB1C3 (pT7-EYFP) was set as negative control and positive control, respectively. sfG(R)FP represents the CDS of sfGFP inserted with ribozyme sequence.

RENDR can regulate the translation of bacterial messenger RNA in response to the presence or absence of any RNA input *in vivo*.

*pirA* toxin gene detection: molecular cloning

For *in vivo* verification of toxin *pirA*, we designed [BBa\_K4195141](http://parts.igem.org/Part%3ABBa_K4195141), [BBa\_K4195142](http://parts.igem.org/Part%3ABBa_K4195142), [BBa\_K4195145](http://parts.igem.org/Part%3ABBa_K4195145), [BBa\_K4195156](http://parts.igem.org/Part%3ABBa_K4195156), [BBa\_K4195157](http://parts.igem.org/Part%3ABBa_K4195157), [BBa\_K4195160](http://parts.igem.org/Part%3ABBa_K4195160), [BBa\_K4195161](http://parts.igem.org/Part%3ABBa_K4195161), [BBa\_K4195168](http://parts.igem.org/Part%3ABBa_K4195168), [BBa\_K4195169](http://parts.igem.org/Part%3ABBa_K4195169), [BBa\_K4195172](http://parts.igem.org/Part%3ABBa_K4195172), [BBa\_K4195173](http://parts.igem.org/Part%3ABBa_K4195173) (different guides with different reporter genes). These parts were all separately assembled into the vector pSB3K3 by standard BioBrick assembly. Each constructed plasmid and plasmid [BBa\_K4195179](http://parts.igem.org/Part%3ABBa_K4195179)\_pSB1C3 (input of *pirA*) were co-transformed into *E. coli* BL21(DE3). Then the positive transformants were selected by kanamycin and chloramphenicol. Other related parts are listed as followings: [BBa\_K4195151](http://parts.igem.org/Part%3ABBa_K4195151), [BBa\_K4195183](http://parts.igem.org/Part%3ABBa_K4195183), [BBa\_K4195184](http://parts.igem.org/Part%3ABBa_K4195184), [BBa\_K4195185](http://parts.igem.org/Part%3ABBa_K4195185), [BBa\_K4195186](http://parts.igem.org/Part%3ABBa_K4195186), which were assembled into pSB1C3 plasmid backbone, respectively. The constructed plasmids were transformed into *E. coli* BL21(DE3), then the positive transformants were selected by chloramphenicol and confirmed by colony PCR and sequencing. The results of colony PCR were recorded on the corresponding parts page.

*pirA* toxin gene detection: RENDR with a GFP output

Colonies harboring the correct plasmid(s) were cultivated and
induced. The expression behavior of GFP is represented by normalized RFUGFP (RFUGFP/OD600), using a microplate reader. The circuit,
ori-g1α with input sequence ([BBa\_K4195176](http://parts.igem.org/Part%3ABBa_K4195176)) used in the reference (7) was used as the positive control. After
evaluating the RFUGFP/OD600 value in each group (Fig. 9), **pirA\_g2
α** and **pirA\_g2β** exhibited better performances in both single plasmid
system and dual plasmid system, especially the group of pirA\_g2α in dual plasmid system (Fig. 9b).

![15](https://static.igem.wiki/teams/4195/wiki/result/detection/fig9.png)

**Fig. 9 The results of reporter GFP for *pirA* toxin gene detection in RENDR-based system. a** Single plasmid-based characterization. **b** Dual plasmid-based characterization. RFU: relative fluorescence unit.

*pirA* toxin gene detection: RENDR with a *β*-lactamase (AmpC) output

Colonies harboring the correct plasmid(s) were cultivated and
induced. The expression behavior of *β*-lactamase (AmpC) is represented by normalized RAUAmpC (RAUAmpC/OD600), after measuring the absorbance in 490 nm as time progressed using a microplate reader. After evaluating the RAUAmpC/OD600 value in each group (Fig. 10), **pirA\_g2α** (Fig. 10b) and **pirA\_g2β** (Fig. 10d) exhibited better performances **in dual plasmid system**, which could be selected as the candidates for following experiments if necessary.

![15](https://static.igem.wiki/teams/4195/wiki/result/detection/fig10.png)

**Fig. 10 The results of reporter AmpC for *pirA* toxin gene detection in RENDR-based system as time progressed of (a) pirA\_g1α, (b) pirA\_g1β, (c) pirA\_g2α and (d) pirA\_g2β.** RAU: relative absorbance unit.

*pirA* toxin gene detection: RENDR with a NanoLuc output

Colonies harboring the correct plasmid(s) were cultivated and induced. The
expression behavior of NanoLuc is represented by normalized RLUNanoLuc (RLUNanoLuc/OD600), after measuring the bioluminescence as time progressed using a microplate reader. After evaluating the RLUNanoLuc/OD600 value in each group (Fig. 11), **pirA\_g1α** (Fig. 11a) and **pirA\_g1β** (Fig. 11c) **in dual plasmid system** exhibited better performances, which could be selected as the candidates for following experiments if necessary.

![15](https://static.igem.wiki/teams/4195/wiki/result/detection/fig11.png)

**Fig. 11 The results of reporter NanoLuc luciferase for *pirA* toxin gene detection in RENDR-based system as time progressed of (a) pirA\_g1α, (b) pirA\_g1β, (c) pirA\_g2α and (d) pirA\_g2β.** RLU: relative luminescence unit.

Thus, based on the results from Fig. 9, Fig. 10, and Fig. 11, we concluded that **pirA\_g1α** and **pirA\_g2β** showed better performances *in vivo*, which may be further used in the cell-free system *in vitro*.

*pirB* toxin gene detection: Molecular cloning

For toxin *pirB*, we designed [BBa\_K4195143](http://parts.igem.org/Part%3ABBa_K4195143), [BBa\_K4195144](http://parts.igem.org/Part%3ABBa_K4195144), [BBa\_K4195158](http://parts.igem.org/Part%3ABBa_K4195158), [BBa\_K4195170](http://parts.igem.org/Part%3ABBa_K4195170), [BBa\_K4195171](http://parts.igem.org/Part%3ABBa_K4195171), [BBa\_K4195174](http://parts.igem.org/Part%3ABBa_K4195174), [BBa\_K4195175](http://parts.igem.org/Part%3ABBa_K4195175). These parts were respectively assembled into the vector pSB3K3 by standard BioBrick assembly. The result of colony PCR was recorded on the corresponding parts page. Each constructed plasmid and plasmid [BBa\_K4195180](http://parts.igem.org/Part%3ABBa_K4195180)\_pSB1C3 (input of *pirB*) were transformed into *E. coli* BL21(DE3). The positive transformants were selected by kanamycin and chloramphenicol. Other related parts are listed as following: [BBa\_K4195149](http://parts.igem.org/Part%3ABBa_K4195149), [BBa\_K4195150](http://parts.igem.org/Part%3ABBa_K4195150), [BBa\_K4195164](http://parts.igem.org/Part%3ABBa_K4195164), [BBa\_K4195165](http://parts.igem.org/Part%3ABBa_K4195165), [BBa\_K4195166](http://parts.igem.org/Part%3ABBa_K4195166), [BBa\_K4195187](http://parts.igem.org/Part%3ABBa_K4195187), [BBa\_K4195188](http://parts.igem.org/Part%3ABBa_K4195188), [BBa\_K4195189](http://parts.igem.org/Part%3ABBa_K4195189), [BBa\_K4195190](http://parts.igem.org/Part%3ABBa_K4195190).These parts were respectively assembled into the pSB1C3 plasmid backbone. The constructed plasmids were transformed into *E. coli* BL21(DE3), then the positive transformants were selected by chloramphenicol and confirmed by colony PCR and sequencing. The result of colony PCR was recorded on the corresponding parts page.

*pirB* toxin gene detection: RENDR with a GFP output

Colonies harboring the correct plasmid(s) were cultivated and induced. The expression behavior of GFP is represented by normalized RFUNanoLuc (RFUNanoLuc/OD600), after measuring the bioluminescence as time progressed using a microplate reader. The circuit, ori-g1α with input sequence ([BBa\_K4195176](http://parts.igem.org/Part%3ABBa_K4195176)) used in the reference (7) was used as the positive control. After evaluating the RFUGFP/OD600 value in each group (Fig. 12), **pirB\_g2α in both system** and **pirB\_g1α/β in single plasmid system**  exhibited better performances, which could be selected as the candidates for following experiments if necessary.

![15](https://static.igem.wiki/teams/4195/wiki/result/detection/fig12.png)

**Fig. 12 The results of reporter GFP for *pirB* toxin gene detection in RENDR-based system. a** Single plasmid-based characterization. **b** Dual plasmid-based characterization. RFU: relative fluorescence unit.

*pirB* toxin gene detection: RENDR with a *β*-lactamase (AmpC) output

Colonies harboring the correct plasmid(s) were cultivated and induced. The
expression behavior of *β*-lactamase (AmpC) is represented by normalized RAUAmpC
(RAUAmpC/OD600), after measuring the absorbance in 490 nm as time progressed
using a microplate reader. After evaluating the RAUAmpC/OD600 value in each group (Fig. 13), **piB\_g1α in dual plasmid system**(Fig. 13a), **pirB\_g1β** (Fig. 13c) and **pirB\_g2β** (Fig. 13d) exhibited better performances, which could be selected as the candidates for following experiments if necessary.

![15](https://static.igem.wiki/teams/4195/wiki/result/detection/fig13.png)

Fig. 13 The results of reporter AmpC for *pirB* toxin gene detection in RENDR-based system as time progressed of (a) pirB\_g1α, (b) pirB\_g1β, (c) pirB\_g2α and (d) pirB\_g2β. RAU: relative absorbance unit.

*pirB* toxin gene detection: RENDR with a NanoLuc output

Colonies harboring the correct plasmid(s) were cultivated and induced. The
expression behavior of NanoLuc is represented by normalized RLUNanoLuc (RLUNanoLuc/OD600), after measuring the bioluminescence as time progressed using a microplate reader. After evaluating the RLUNanoLuc/OD600 value in each group (Fig. 14), **pirB\_g2α in dual plasmid system** (Fig. 14b) and **pirB\_g1β in single plasmid system** (Fig. 14c) exhibited better performances, which could be selected as the candidates for following experiments if necessary.

![15](https://static.igem.wiki/teams/4195/wiki/result/detection/fig11.png)

**Fig. 14 The results of reporter NanoLuc luciferase for pirB toxin gene detection in RENDR-based system as time progressed of (a) pirB\_g1α, (b) pirB\_g1β, (c) pirB\_g2α and (d) pirB\_g2β.** RLU: relative luminescence unit.

Thus, based on the results from Fig. 12, Fig. 13, and Fig. 14, we concluded that **pirB\_g1β**, **pirB\_g1α**, and **pirB\_g2α** showed better performances *in vivo*, which could be further used in the cell-free system.

#### Conclusion

![15](https://static.igem.wiki/teams/4195/wiki/result/detection/fig-15.png)

**Fig. 15 The heat map of the fold change (with input/without input) of output signals of different guides with diverse reporters.**

Through experiments, we have verified the capability of our hardware to operate the RPA amplification, and the feasibility of the RENDR-based detection system to sense the toxin gene input *in vivo* (Fig. 15). Actually, some guides circuits showed a certain level of leakage in absence of input and the output signals were even lower upon adding the input. This might be attributed to the concentration differences of transcripts of guides and input, which is related to the direction of reaction equilibrium of self-splicing. This was the reason why the tests of some circuits were implemented in dual plasmid system, since the copy number of pSB3K3 that harbors the guides is smaller than that of pSB1C3 for harboring input sequence. As for some guides circuits that showed awful behaviors all the time, we speculated that it may be caused by the unfavorable structure of the transcripts for ribozyme self-splicing. Based on the *in vivo* characterization, we continued to perform the tests in [cell-free expression](https://2022.igem.wiki/xmu-china/proof-of-concept#Detection) system, which is closer to the [Implementation](https://2022.igem.wiki/xmu-china/implementation) form of our project.

#### Future prospects

Many experiments were carried out to achieve the detection targeted to the toxic gene of *pirA* and *pirB* within a limited time. However, the cause of some inverse results upon adding the input sequences should be further investigated. And the combinations that the constitutively expressed guides sequences with the input under the control of an orthogonal promoter may be tested for decreasing the scramble of RNA polymerases.

#### Treatment

Our strategy for treating AHPND is to kill the pathogenic *Vibrio parahaemolyticus* by a specific plasmid-encoded endolysin (*10*), which can be delivered *via* the engineered outer membrane vesicles (OMVs). For selectively targeting, we determined to decorate the surface of OMVs with two ligands, TTPA and TTPB, which were identified from the phage OWB (*11*) of *V. parahaemolyticus* that could bind to Vp0980 receptor on V. parahaemolyticus. In this case, two surface display systems, ClyA (*12*) and INPNC (*13*) were chosen to anchor the ligands on the surface membrane of engineered bacteria, thus resulting in the surface-modified OMVs. In addition, we also implemented RNA interference (RNAi) technology to silence *ompA* and *lpp* and overexpression of *mepS* for hypervesiculation of the engineered bacteria.

#### Highlight

 Confirm the targeted binding of TTPA to Vp0980

 Verify the function of RNAi and MepS for hypervesiculation of the engineered bacteria

 Achieve the killing of endolysin to *Vibrio alginolyticus*

#### Introduction

Successful implementation of the surface display system in bacteria should be carefully verified through two steps, the first is to ensure that the cargo protein can be anchored on the bacterial surface, and the second is the verification of cargo protein’s normal function as its free state. Many methods have been developed to characterize the successful location of cargo protein when fused with the anchor, including flow cytometry (*11*, *14*-*17*), western blot (*14*, *15*, *18*-*20*), enzyme linked immunosorbent assay (ELISA) (*18*), fluorescence microscope imaging (*11*, *14*, *21*) and so on. Among these characterization means, immuno-based technology was the most implemented due to the specificity and accessibility of antibodies. Therefore, given the laboratory resources and the complexity of operations, we finally decided to use the immunofluorescence (IF) technology for testing whether the cargo proteins are displayed or not and further probing the binding event of the protein-protein interaction (PPI) pairs on the surface of bacteria (Fig. 16). This approach was implemented both in the experiments of the Treatment part and Prevention part.

![15](https://static.igem.wiki/teams/4195/wiki/result/treatment/a1.png)

**Fig. 16 Illustration of the immunofluorescence (IF) technology applied in the experiments of the Treatment part and Prevention part.**

As for OMVs, [common extraction and characterization methods](https://2022.igem.wiki/xmu-china/experiments) were
used,
including ultracentrifugation, transmission electron microscope (TEM) imaging, and bicinchoninic acid (BCA) assay. Through those experiments, we could characterize the effect of RNAi and overexpression of the *mepS* gene.

While for the test of endolysin (Lysqdvp001), the killing efficiency was explored by performing the spot assay after incubating the purified endolysin with the *V. parahaemolyticus*-alternative strain *V. alginolyticus*.

#### Results

Promoters induction performance verification

#### [Show Details](#collapse-tr1)

*L*-rhamnose-inducible promoter induction performance verification

We improved the original promoter ([BBa\_K914003](http://parts.igem.org/Part%3ABBa_K914003)) and compared the expression level between two genetic circuits (Fig. 17). Then, the effects of different inducers on the improved *L*-rhamnose-inducible promoter were also investigated. As shown in Fig. 18, the improved *L*-rhamnose-inducible promoter under the induction of *L*-rhamnose possessed the advantage of a stronger induction effect and lower leakage, which we will choose finally.

![15](https://static.igem.wiki/teams/4195/wiki/result/treatment/shl1-fig2.png)

**Fig. 17 The comparison of normalized fluorescence intensity
between [BBa\_K4195109](http://parts.igem.org/Part%3ABBa_K4195109)
and [BBa\_K4195191](http://parts.igem.org/Part%3ABBa_K4195191).**

![15](https://static.igem.wiki/teams/4195/wiki/result/treatment/shl2-fig3.png)

**Fig. 18 Characterization of the *eyfp* expression and bacterial growth in different inducer concentrations (a: *L*-rhamnose, b: *L*-mannose).**

Ligands can be anchored onto the bacterial surface

#### [Show Details](#collapse-tr2)

ClyA/INPNC-TTPB-his and ClyA/INPNC-TTPA-his

Molecular cloning

![15](https://static.igem.wiki/teams/4195/wiki/result/treatment/fig-19.png)

**Fig. 19 Graphic description of the expression gene circuits for display cassette designed in the Treatment part.**

In order to verify whether ClyA or INPNC can display heterologous proteins on the surface of the engineered bacteria or not, a His-tag (6×His) was fused to the C-terminal of the two ligands (TTPA and TTPB). We used both [BBa\_I0500](http://parts.igem.org/Part%3ABBa_I0500) (*araC*/pBAD) and [BBa\_B0034](http://parts.igem.org/Part%3ABBa_B0034) to construct the expression system and obtained the four composite parts [BBa\_K4195100](http://parts.igem.org/Part%3ABBa_K4195100) (for INPNC-TTPB-his), [BBa\_K4195101](http://parts.igem.org/Part%3ABBa_K4195101) (for ClyA-TTPB-his), [BBa\_K4195120](http://parts.igem.org/Part%3ABBa_K4195120) (for ClyA-TTPA-his) and [BBa\_K4195122](http://parts.igem.org/Part%3ABBa_K4195122) (for INPNC-TTPA-his) (Fig. 19), which are respectively assembled into the vector pSB1C3 by standard BioBrick assembly. The constructed plasmid was transformed into *E. coli* BL21(DE3), then the positive transformants were selected by chloramphenicol and confirmed by colony PCR (Fig. 20) and sequencing.

![15](https://static.igem.wiki/teams/4195/wiki/result/treatment/fig-20.png)

**Fig. 20 DNA gel electrophoresis of the colony PCR products.** **a** [BBa\_K4195100](http://parts.igem.org/Part%3ABBa_K4195100)\_pSB1C3 in *E. coli* BL21(DE3). Target band (5020 bp) can be observed at the position around 5000 bp. **b** [BBa\_K4195101](http://parts.igem.org/Part%3ABBa_K4195101)\_pSB1C3 in *E. coli* BL21(DE3). Target band (5002 bp) can be observed at the position around 5000 bp. **c** [BBa\_K4195120](http://parts.igem.org/Part%3ABBa_K4195120)\_pSB1C3 in *E. coli* BL21(DE3). Target band (3220 bp) can be observed at the position around 3000 bp. **d** [BBa\_K4195122](http://parts.igem.org/Part%3ABBa_K4195122)\_pSB1C3 in *E. coli* BL21(DE3). Target bands (3238 bp) can be observed at the position around 3000 bp.

Immunofluorescence (IF)

The bacterial culture was cultivated overnight after *L*-arabinose induction. Then the FITC-labeled anti-His-Tag antibody was used to target the fused His-tag (6×His) displayed *via* ClyA and INPNC, followed by measuring the fluorescence intensity and OD600 of the culture.

![15](https://static.igem.wiki/teams/4195/wiki/result/treatment/fig-21.png)

**Fig. 21 The results of immunofluorescence to characterize the function of the two surface display systems.** **a** Fluorescence intensity/OD600 of *E. coli* whether TTPB fused to anchors or not. The left is for [BBa\_K4195100](http://parts.igem.org/Part%3ABBa_K4195100) (*p* = 0.00277) and the right is for [BBa\_K4195101](http://parts.igem.org/Part%3ABBa_K4195101) (*p* = 0.0019). **b** Fluorescence intensity/OD600 of *E. coli* whether TTPA fused to anchors or not. The left is for [BBa\_K4195120](http://parts.igem.org/Part%3ABBa_K4195120) (*p* = 0.0126) and the right is for [BBa\_K4195122](http://parts.igem.org/Part%3ABBa_K4195122) (*p* = 0.0024).

The results showed that the ratio of fluorescence intensity to OD600 of positive control (bacteria harboring surface display system) is higher than that of negative control (bacteria without surface display system) (Fig. 21), which indicates that both of the two ligands can be successfully located on the surface of engineered bacteria.

The function of ligands can be retained after displayed on the bacterial surface

#### [Show Details](#collapse-tr3)

Vp0980 is a membrane protein

Molecular cloning and purification of Vp0980

In order to purify Vp0980, a His-tag (6×His) was fused to the N-terminal of it. *L*-arabinose-inducible expression system was also constructed and we obtained the composite part [BBa\_K4195110](http://parts.igem.org/Part%3ABBa_K4195110) at pSB1C3. The plasmid was transformed into *E. coli* Shuffle T7, then the positive transformants were selected by chloramphenicol and confirmed by colony PCR (Fig. 22a) and sequencing.

![15](https://static.igem.wiki/teams/4195/wiki/result/treatment/fig-22.png)

**Fig. 22 DNA gel electrophoresis of the colony PCR products of [BBa\_K4195110](http://parts.igem.org/Part%3ABBa_K4195110)\_pSB1C3 (a) and [BBa\_K4195006](http://parts.igem.org/Part%3ABBa_K4195006)\_pET-28a(+) (b). a** Target bands (2218 bp) can be observed at the position between 3000 bp and 2000 bp. **b** Target band (716 bp) can be observed at the position around 750 bp.

Colonies with correct sequence were cultivated and induced by *L*-arabinose to express the protein. Protein purification was carried out through GE AKTA Prime Plus FPLC System, which was verified by sodium dodecyl sulfate (SDS)-12% (wt/vol) polyacrylamide gel electrophoresis. We found that Vp0980 was expressed so poorly (data not shown) that we did not obtain much target protein. Therefore, we turned to reconstruct this part on the common expression vector pET-28a(+) through Gibson assembly and transformed the plasmid into *E. coli* DH5α and *E. coli* Origami 2(DE3). The positive transformants were selected by kanamycin and confirmed by colony PCR (Fig. 22b) and sequencing.

Because the Vp0980 is a receptor which has many transmembrane domains, we tried to purify the protein with 1% Triton X-100 and 1 mmol/L PMSF (both final concentrations) added to the buffer before ultrasonication, which is a necessary and crucial step for protein purification. As shown in the gel image of his-Vp0980 (Fig. 23), the target protein (20.0 kDa) can be observed at the position around 20 kDa on the purified protein lanes (FR), although shown with many other protein bands together.

![15](https://static.igem.wiki/teams/4195/wiki/result/treatment/shl5-fig-10.png)

**Fig. 23 SDS-PAGE analysis of his-Vp0980 protein.** Target bands (20 kDa) can be observed at the position 20 kDa.

We attributed that no obvious protein target band in LS because to that the content of proteins located on the bacterial membrane is minimal.

Immunofluorescence (IF) performed for confirming localization

For further verifying that Vp0980 was localized in membrane, after being induced by 2% arabinose, the *E. coli* was incubated with FITC-labeled anti-His-tag Antibody at 37 °C for 1 h. The microplate reader was used to measure the value of fluorescence intensity and OD600. The fluorescence intensity/OD600 value in the group of his-Vp0980 (Fig. 24) was stronger than that of I0500\_pSB1C3 (negative control), which demonstrated that his-Vp0980 is located on the outer membrane of *E. coli*.

![15](https://static.igem.wiki/teams/4195/wiki/result/treatment/shl6-fig11.png)

**Fig. 24** Immunofluorescence (IF) technology was used to probe the localization of Vp0980 (*p* = 0.0232).

Displayed ligands can bind to Vp0980

IF experiments

![15](https://static.igem.wiki/teams/4195/wiki/result/treatment/fig-25-new.png)

**Fig. 25 IF experiments were performed to verify whether the ligands displayed can bind to Vp0980 or not. a** [BBa\_K4195121](http://parts.igem.org/Part%3ABBa_K4195121). **b** [BBa\_K4195123](http://parts.igem.org/Part%3ABBa_K4195123). **c** [BBa\_K4195103](http://parts.igem.org/Part%3ABBa_K4195103). **d** [BBa\_K4195102](http://parts.igem.org/Part%3ABBa_K4195102).

Immunofluorescence was used as well to demonstrate the interaction of his-Vp0980 with ClyA/INPNC-TTPA or ClyA/INPNC-TTPB, in which the group without adding his-Vp0980 was set as negative group. After being induced by 2% arabinose, the *E. coli* (ClyA/INPNC -TTPA or ClyA/INPNC-TTPB) was incubated with his-Vp0980 at 37 °C for 1 h, then incubated with FITC-labeled anti-His-Tag Antibody at 37 °C for 1 h. The microplate reader was used to measure the value of fluorescence intensity and OD600. We found that the TTPA, no matter displayed by ClyA or INPNC, could bind to the purified Vp0980 (Fig. 25a & 25b). However, TTPB showed no ligand activity when displayed on the surface of engineered bacteria (Fig. 25c & 25d). We may attributed it to the potential cross-reactivity of our anti-His-Tag antibody, since the signals of both groups with or without purified Vp0980 added were much higher than that of other groups we have tested. Therefore, we could confirm that the TTPA might be further implemented for real applications.

Evaluate the binding affinity of ligands and Vp0980

Molecular cloning and purification of ligands

In order to further quantify the binding affinity of ligands and Vp0980, a myc-tag was fused to the C-terminal of the ligands. We constructed these parts on the common expression vector pET-28a(+) through Gibson assembly and transformed the plasmids into *E. coli* BL21(DE3). The positive transformants were selected by kanamycin and confirmed by colony PCR (Fig. 26) and sequencing.Colonies with correct sequence were cultivated and induced by IPTG to express the protein. Protein purification was carried out through GE AKTA Prime Plus FPLC System, which was verified by sodium dodecyl sulfate (SDS)-12% (wt/vol) polyacrylamide gel electrophoresis (Fig. 26).

![15](https://static.igem.wiki/teams/4195/wiki/result/treatment/fig-26-shl.png)

**Fig. 26 DNA gel electrophoresis of the colony PCR products and SDS-PAGE analysis of the target proteins of [BBa\_K4195089](http://parts.igem.org/Part%3ABBa_K4195089)\_pET-28a(+) (a,b) and [BBa\_K4195090](http://parts.igem.org/Part%3ABBa_K4195090)\_pET-28a(+) (c,d). a** Target bands (764 bp) can be observed at the position between 750 bp and 1000 bp. **b** Target band (23.6 kDa) can be observed at the position between 20 kDa and 30 kDa. **c** Target bands (2546 bp) can be observed at the position between 2000 bp and 3000 bp. **d** Target band (87.6 kDa) can be observed at the position around 80 kDa.

Dot blot analysis

Dot blot analysis was used to determined the interaction between Vp0980 and its ligands. Firstly, the protein Vp0980 with his-tag was directly spotted onto the nitrocellulose (NC) membrane. Then the NC membrane was incubated with purified ligands with myc-tag and anti-myc-tag antibody in turn and finally probed by the HRP-conjugated secondary antibody. BSA was also directly spotted onto the NC membrane as the negative control. By reading the gray scale value of experimental groups from chemiluminescence imaging results, we quantitatively calculated the binding affinity (*K*d) of ligands and Vp0980 (Fig. 27).

![15](https://static.igem.wiki/teams/4195/wiki/result/treatment/dot-blot-together.png)

**Fig. 27 The binding affinity (*K*d) of Vp0980 and its ligands (a TTPA. b TTPB.) obtained from dot blot analysis.**

OMVs massive release verification

#### [Show Detail](#collapse-tr4)

OMVs can be purified via ultracentrifugation

Molecular cloning

The basic part [BBa\_I0500](http://parts.igem.org/Part%3ABBa_I0500) was assembled into pSB1C3 plasmid backbone for the purification of OMVs. The plasmid constructed was transformed into *E. coli* DH5α and *E. coli* BL21(DE3), and the positive colonies were confirmed by chloramphenicol, colony PCR (Fig. 28), and sequencing. The target band (1524 bp) can be observed at the position around 1500 bp.

![15](https://static.igem.wiki/teams/4195/wiki/result/treatment/shl-7fig13.png)

**Fig. 28 The result of colony PCR. Plasmid pSB1C3.**

OMVs purification and transmission electron microscope observation

TThe colony with the corrected sequence was cultivated for OMVs purification. After being collected from the supernatant of culture *via* ultracentrifugation, the OMVs samples were negatively stained for TEM observation (Fig. 29) to confirm that we purified the OMVs successfully.

![15](https://static.igem.wiki/teams/4195/wiki/result/treatment/shl-8-fig-14.png)

**Fig. 29 Transmission electron microscopy observation of OMVs.**

Construction of hypervesiculation *E. coli* strain

siRNA design

siRNA sequences were designed through siRCon which is developed by [Team Bielefeld-CeBiTec](https://2018.igem.org/Team%3ABielefeld-CeBiTec/Software). OmpA 5’UTR and Hfq binding sequence are added to the flanks of siRNA sequences to enhance the binding ability to target sequence as well as increase the half-life of siRNA. Promoter ([BBa\_I0500](http://parts.igem.org/Part%3ABBa_I0500)) and terminator ([BBa\_B0015](http://parts.igem.org/Part%3ABBa_B0015)) were used to construct composite parts (Fig. 30).

![15](https://static.igem.wiki/teams/4195/wiki/result/treatment/shl-fig15.png)

**Fig. 30 The composite parts about siRNA.**

Molecular cloning

All related siRNA basic parts and mepS ([BBa\_K4195004](http://parts.igem.org/Part%3ABBa_K4195004)) were assembled into the pSB1C3 plasmid backbone, which was transformed into *E. coli* DH5α and *E. coli* BL21(DE3). Positive colonies were confirmed by chloramphenicol, colony PCR (Fig. 31), and sequencing.

![15](https://static.igem.wiki/teams/4195/wiki/result/treatment/fig-31.png)

**Fig. 31 The result of colony PCR. Plasmid pSB1C3. a** the target band (1890 bp) of *ompA 0.83*. **b** the target band (1890 bp) of *ompA 0.88*. **c** the target band (1890 bp) of *lpp 0.88*. **d** the target band (1890 bp) of *lpp 1.0*. **e** the target band (2254 bp) of *mepS*.

OMVs purification and BCA assay

OMVs expressed from the gene circuits were purified *via* ultracentrifugation to verify the successful construction of hypervesiculation *E. coli*, in which [BBa\_I0500](http://parts.igem.org/Part%3ABBa_I0500)\_pSB1C3 was used as control. Based on the standard curve developed from BCA Kit, the concentration of OMVs can be quantized to exhibit the effect of RNA interference (RNAi) in different gene circuits. As shown in Fig. 32, compared with that to *lpp*, the interference to *ompA* did not promote the generation of hypervesiculation. The lower OD600 also revealed that RNA interference to *lpp* can promote the production of OMVs, leading to the destruction of cytomembrane stability.

![15](https://static.igem.wiki/teams/4195/wiki/result/treatment/fig-32.png)

**Fig. 32 Comparison of normalized protein concentration and OD600 between the RNAi circuits and negative control. a** Verification of [BBa\_K4195104](http://parts.igem.org/Part%3ABBa_K4195104). **b** Verification of [BBa\_K4195105](http://parts.igem.org/Part%3ABBa_K4195105). **c** Verification of [BBa\_K4195106](http://parts.igem.org/Part%3ABBa_K4195106). **d** Verification of [BBa\_K4195107](http://parts.igem.org/Part%3ABBa_K4195107).

As shown in Fig. 33, Compared with the negative control ([BBa\_I0500](http://parts.igem.org/Part%3ABBa_I0500)\_pSB1C3), MepS also resulted in a higher concentration of OMVs and lower OD600 values, which verified its effective function of hypervesiculation.

![15](https://static.igem.wiki/teams/4195/wiki/result/treatment/shl-11-fig-18.png)

**Fig. 33 Comparison of normalized protein concentration and OD600 between [BBa\_K4195108](http://parts.igem.org/Part%3ABBa_K4195108) and negative control.**

Furthermore, about MepS ([BBa\_K4195108](http://parts.igem.org/Part%3ABBa_K4195108)), we also measured the dynamic tendency of **normalized protein concentration** to **time after induction**, which indicated the massive secretion of OMVs over time (Fig. 34).

![15](https://static.igem.wiki/teams/4195/wiki/result/treatment/shl-12-fig-19.png)

**Fig. 34 Normalized protein concentration-time curves of [BBa\_K4195108](http://parts.igem.org/Part%3ABBa_K4195108) under the induction of *L*-arabinose.**

Lysqdvp001 can kill *Vibrio alginolyticus* specially

#### [Show Detail](#collapse-tr5)

The killing effect of Lysqdvp001

Molecular cloning and protein purification

In order to purify Lysqdvp001, a His-tag (6×His) was fused to the N-terminal of Lysqdvp001. [BBa\_I0500](https://parts.igem.org/wiki/index.php?title=Part:BBa_I0500) and [BBa\_B0015](https://parts.igem.org/wiki/index.php?title=Part:BBa_B0015) were used to construct the expression system, which was assembled on the expression vector pUC57-Simple by the standard assembly. The constructed plasmids were transformed into *E. coli* DH5α and BL21(DE3), then the positive transformants were selected by ampicillin and confirmed by colony PCR (Fig. 35) and sequencing.

![10](https://static.igem.wiki/teams/4195/wiki/result/treatment/endolysin-1-bba-k4195114-his-edl060-colony-pcr-bl21-de3.png)

**Fig. 35 The result of colony PCR. Plasmid pUC57-Simple.**

Colonies with correct sequence were cultivated and induced by *L*-arabinose to express the protein. Protein purification was carried out through AKTA Prime Plus FPLC System (GE, America), which was verified by sodium dodecyl sulfate (SDS)-12% (wt/vol) polyacrylamide gel electrophoresis (PAGE). As shown in the gel image of the his-Edl060 (Fig. 36), the target protein (26.0 kDa) could be observed at positions between 25 kDa and 30 kDa on the purified protein lanes (FR).

![10](https://static.igem.wiki/teams/4195/wiki/result/treatment/endolysin-2bba-k4195114.png)

**Fig. 36 SDS-PAGE analysis of protein in the lysate of *E. coli* BL21(DE3) and the elution samples.** Target bands (26.0 kDa) can be observed at positions between 25 kDa and 30 kDa.

Spot assay

Three kinds of bacteria (*E. coli*, *Vibrio alginolyticus*, and *Vibrio natriegens*) were diluted from 1/10-3 to 1/10-5 and treated with EDTA and sterilized water (negative control, Fig. 37a), alkaline (positive control, Fig. 37b), respectively. As shown in Fig. 37c, three kinds of the bacterium were diluted to 1/10-5 and treated with EDTA and purified endolysin Lysqdvp001 for 5 min, 10 min, 15 min, and 20 min. After being treated, these bacteria samples were used for spot assay respectively. The result demonstrated that Lysqdvp001 showed not any killing effect on other bacteria, but was **highly specific** to the *Vibrio alginolyticus* (*Vibrio parahaemolyticus*-alternative strain), which was consistent with the killing results that were treated by alkaline lysis.

![10](https://static.igem.wiki/teams/4195/wiki/result/treatment/fig-37.png)

**Fig. 37 The result of killing effect verification.** Grouped by different treatment: **a** EDTA and sterilized water. **b** Alkaline Lysis. **c** EDTA and Lysqdvp001.

#### Conclusion

The efficiency of the inducible promoters [BBa\_I0500](http://parts.igem.org/Part%3ABBa_I0500) and [BBa\_K914003](http://parts.igem.org/Part%3ABBa_K914003) was verified, which resulted in high expression with induction and low leakage without induction. We successfully proved that the two ligands (TTPA and TTPB) can be displayed by two surface display systems (ClyA and INPNC) on the surface of engineered bacteria. Besides, the function of TTPA to bind Vp0980 was verified after the ligand was displayed.The practicability of the plasmid transfer killing method was verified as effective. The RNAi and MepS methods were verified effective to promote OMVs secretion.

The experiments on the *L*-rhamnose-induced promoter can be divided into mainly two parts. Adding a regulatory element in front of the *L*-rhamnose-induced promoter made its inducing efficiency stronger. Then, a comparative experiment was conducted on the improved promoter with different inducers (*L*-rhamnose and *L*-mannose), which demonstrated that *L*-rhamnose is a better inducer.

In the surface-display system, we display the tail tubular protein TTPA/TTPB of *Vibrio parahaemolyticus* bacteriophage on the surface of OMVs by which to target *Vibrio parahaemolyticus* more efficiently. ClyA and INPNC are employed to build a surface display system, through which TTPA/TTPB can be displayed on the surface of OMVs.

For the plasmid transfer killing system, experiments designed for it were
partially finished. We verified the killing effect of Lysqdvp001 expressed by edl060. Results showed that the endolysin has a high killing effect on *Vibrio alginolyticus* (similar to *Vibrio parahaemolyticus*), which created the precondition for the
[Proof of Concept](https://2022.igem.wiki/xmu-china/proof-of-concept) verification of Lysqdvp001 for *Vibrio alginolyticus* killing.

As for the RNAi method, it is proved that the interference to *ompA* has no obvious effect while the interference to lpp triggered the massive secretion of OMVs. At the same time, MepS method also triggered massive secretion of OMVs. With these results, a kinetic model to characterize an enzymology dynamic curve was developed successfully.

#### Future prospects

The experiments have been done, and the design has been proven. Given the fact
that *Vibrio alginolyticus* is similar to *Vibrio parahaemolyticus*, we expect to express
the *vp0980* gene in *Vibrio alginolyticus* so that we can simulate the natural environment
more realistically. The TTPA or TTPB displayed OMVs containing plasmid (loading the gene *edl060* encoding endolysin Lysqdvp001) are wished to incubate with *Vibrio alginolyticus*
expressing Vp0980 to verify the feasibility and effectiveness of our complete design.

Despite the essential experiments have been complete to verified our concept in design, there were still many experiments need to be finished to make project much better. However, time is up. For example, due to the time limit, it is a pity that we cannot verify the combination ability of TTPA/TTPB with Vp0980 successfully. We are looking forward to proving it using dot blot or isothermal titration calorimetry (ITC) in the future.

#### Prevention

Our strategy for preventing AHPND mainly caused by the VPAHPND toxins PirA and PirB is to neutralize the toxins *via* the receptors that are surface-displayed on OMVs released from the engineered bacteria. To build the fortifications against toxins PirA and PirB, two surface display systems (ClyA and INPNC) were chosen to anchor two receptors (rFET and r*Lv*APN1) in our [Design](https://2022.igem.wiki/xmu-china/design). At first, we have constructed four expression circuits for the anchor-receptor fusion proteins and verified that the function of receptors is retained on the surface of engineered bacteria. Besides, the interaction between toxins and receptors has been detected *in vitro*.

#### Highlight

 Confirm the display of the receptor rFET and r*Lv*APN1

 Verify the retained function of displayed receptors

 Detect the interaction between r*Lv*APN1 and PirA *in vitro*

#### Introduction

In order to verify whether two receptors can be displayed by the two surface display systems or not and whether the receptors after displayed can still bind the toxins or not, we chose immunofluorescence (IF) technology as our characterization method, which has been mentioned above. Besides, isothermal titration calorimetry (ITC) (*1*, *2*) was used to detect the interaction between receptors and toxins *in vitro*, which gives out the binding affinities of those protein-protein interaction (PPI) pairs.

#### Results

Toxin receptors can be anchored onto the bacterial surface

#### [Show Details](#collapse-pr1)

ClyA/INPNC-rFET-his and ClyA/INPNC-rLvAPN1-his

Molecular cloning

![10](https://static.igem.wiki/teams/4195/wiki/result/pre/pre-fig-1.png)

**Fig. 38 Graphic description of the expression gene circuits for display cassette designed in the Prevention part.**

In order to verify whether ClyA or INPNC can display heterologous proteins on the surface of the engineered bacteria or not, a His-tag (6×His) was fused to the C-terminal of the two receptors. We used both [BBa\_I0500](https://parts.igem.org/wiki/index.php?title=Part:BBa_I0500) (*araC*/pBAD) and [BBa\_B0034](https://parts.igem.org/wiki/index.php?title=Part:BBa_B0034) to construct the expression system and obtained the four composite parts [BBa\_K4195126](https://parts.igem.org/wiki/index.php?title=Part:BBa_K4195126) (for INPNC-rFET-his), [BBa\_K4195127](https://parts.igem.org/wiki/index.php?title=Part:BBa_K4195127) (for ClyA-rFET-his), [BBa\_K4195131](https://parts.igem.org/wiki/index.php?title=Part:BBa_K4195131) (for ClyA-r*Lv*APN1-his) and [BBa\_K4195133](https://parts.igem.org/wiki/index.php?title=Part:BBa_K4195133) (for INPNC-r*Lv*APN1-his) (Fig. 38), which are respectively assembled into the vector pSB1C3 by standard BioBrick assembly. The constructed plasmid was transformed into *E. coli* BL21(DE3), then the positive transformants were selected by chloramphenicol and confirmed by colony PCR (Fig. 39) and sequencing.

![10](https://static.igem.wiki/teams/4195/wiki/result/pre/pre-fig-2.png)

**Fig. 39 DNA gel electrophoresis of the colony PCR products. a** [BBa\_K4195127](https://parts.igem.org/wiki/index.php?title=Part:BBa_K4195127) in *E. coli* BL21(DE3). Target bands (2484 bp) can be observed at the position between 3000 bp and 2000 bp. **b** [BBa\_K4195126](https://parts.igem.org/wiki/index.php?title=Part:BBa_K4195126) in *E. coli* BL21(DE3). Target bands (2505 bp) can be observed at the position between 3000 bp and 2000 bp. **c** [BBa\_K4195131](https://parts.igem.org/wiki/index.php?title=Part:BBa_K4195131) in *E. coli* BL21(DE3). Target bands (3879 bp) can be observed at the position between 5000 bp and 3000 bp. **d** [BBa\_K4195133](https://parts.igem.org/wiki/index.php?title=Part:BBa_K4195133) in *E. coli* BL21(DE3). Target bands (3900 bp) can be observed at the position between 5000 bp and 3000 bp.

Immunofluorescence (IF)

The bacterial culture was cultivated overnight after *L*-arabinose induction. Then the FITC-labeled anti-His-Tag antibody was used to target the fused His-tag (6×His) displayed *via* ClyA and INPNC, followed by measuring the fluorescence intensity and OD600 of the culture.

![10](https://static.igem.wiki/teams/4195/wiki/result/pre/pre-fig-3.png)

**Fig. 40 The results of immunofluorescence to characterize the function of the two surface display systems. a** Fluorescence intensity/OD600 of *E. coli* whether rFET is fused to anchors or not. The left is for [BBa\_K4195127](https://parts.igem.org/wiki/index.php?title=Part:BBa_K4195127) (*p* = 0.0024) and the right is for [BBa\_K4195126](https://parts.igem.org/wiki/index.php?title=Part:BBa_K4195126) (*p* = 0.0199). b Fluorescence intensity/OD600 of *E. coli* whether r*Lv*APN1 is fused to anchors or not. The left is for [BBa\_K4195131](https://parts.igem.org/wiki/index.php?title=Part:BBa_K4195131) (*p* = 0.0092) and the right is for [BBa\_K4195133](https://parts.igem.org/wiki/index.php?title=Part:BBa_K4195133) (*p* = 0.0406).

The results showed that the ratio of fluorescence intensity to OD600 of positive control (bacteria harboring surface display system) is higher than that of negative control (bacteria without surface display system) (Fig. 40), which indicates that both of the two receptors can be successfully located on the surface of engineered bacteria.

Receptors can bind to the toxins with different affinities in vitro

#### [Show Details](#collapse-pr2)

Molecular cloning and protein purification of the toxins

In order to purify PirA and PirB, a His-tag (6×His) was fused to the C-terminal of PirA and the N-terminal of PirB. Likewise, *L*-arabinose-inducible expression system was built to obtain the composite part [BBa\_K4195137](https://parts.igem.org/wiki/index.php?title=Part:BBa_K4195137) (for PirA-his) and [BBa\_K4195138](https://parts.igem.org/wiki/index.php?title=Part:BBa_K4195138) (for his-PirB) at pSB1C3. The constructed plasmid was transformed into *E. coli* BL21(DE3), then the positive transformants were selected by chloramphenicol and confirmed by colony PCR (Fig. 41) and sequencing.

![10](https://static.igem.wiki/teams/4195/wiki/result/pre/pre-fig-4.png)

**Fig. 41 DNA gel electrophoresis of the colony PCR products. a** [BBa\_K4195137](https://parts.igem.org/wiki/index.php?title=Part:BBa_K4195137) in *E. coli* BL21(DE3). Target bands (2086 bp) can be observed at the position between 3000 bp and 2000 bp. **b** [BBa\_K4195138](https://parts.igem.org/wiki/index.php?title=Part:BBa_K4195138) in *E. coli* BL21(DE3). Target bands (3070 bp) can be observed at the position around 3000 bp.

After being cultivated and induced by arabinose, GE AKTA Prime Plus FPLC System was employed to get purified protein from the lysate supernatant. Purified protein was verified by sodium dodecyl sulfate (SDS)-12% (wt/vol) polyacrylamide gel electrophoresis (SDS-PAGE) and Coomassie blue staining.

After the first time of purification, we got PirA-his successfully. As shown in the gel image of PirA-his (Fig. 42), the target protein (14.4 kDa) can be observed at the position between 25 kDa and 14 kDa on the purified protein lanes (FR).

![10](https://static.igem.wiki/teams/4195/wiki/result/pre/pre-fig-5.png)

**Fig. 42 SDS-PAGE analysis of PirA-his protein in lysate of *E. coli* BL21(DE3) and the elution samples.** Target bands (14.4 kDa) can be observed at the position between 25 kDa and 14 kDa.

However, we found that PirB was expressed so poorly (data not shown) that we did not obtain the target protein. Therefore, we turned to construct this part on the common expression vector pET-28a(+) by Gibson assembly, then transformed the plasmid into *E. coli* BL21(DE3). The positive transformants were selected by kanamycin and confirmed by colony PCR (Fig. 43) and sequencing.

![10](https://static.igem.wiki/teams/4195/wiki/result/pre/pre-fig-6.png)

**Fig. 43 DNA gel electrophoresis of the colony PCR products of [BBa\_K4195036](https://parts.igem.org/wiki/index.php?title=Part:BBa_K4195036)\_pET-28a(+).** Target bands (1539 bp) can be observed at the position between 2000 bp and 1500 bp.

Unfortunately, after the second round of purification, we found that the expression of PirB remained low as the first time (data not shown). Under this circumstance, we co-transformed another plasmid that could express molecular chaperones GroES and GroEL with his-PirB\_pET-28a(+) into *E. coli* BL21(DE3) to try to improve the expression efficiency. As shown in the gel image of his-PirB (Fig. 44), the target protein (51.9 kDa) can be observed at the position between 60 kDa and 50 kDa on the purified protein lanes (FR), although there were some unwanted bands shown together.

![10](https://static.igem.wiki/teams/4195/wiki/result/pre/pre-fig-7.png)

**Fig. 44 SDS-PAGE analysis of his-PirB protein in lysate of *E. coli* BL21(DE3) and the elution samples.** Target bands (51.9 kDa) can be observed at the position between 60 kDa and 50 kDa.

The interaction between rFET and toxin PirA and PirB

Molecular cloning and protein purification of rFET

In order to purify rFET, a His-tag (6×His) was fused to the C-terminal of it. *L*-arabinose-inducible expression system was also constructed and we obtained the composite part [BBa\_K4195112](https://parts.igem.org/wiki/index.php?title=Part:BBa_K4195112) at pSB1C3. The plasmid was transformed into *E. coli* Shuffle T7, then the positive transformants were selected by chloramphenicol and confirmed by colony PCR (Fig. 45a) and sequencing.

![10](https://static.igem.wiki/teams/4195/wiki/result/pre/pre-fig-8.png)

**Fig. 45 DNA gel electrophoresis of the colony PCR products of [BBa\_K4195112](https://parts.igem.org/wiki/index.php?title=Part:BBa_K4195112) (a) and [BBa\_K4195009](https://parts.igem.org/wiki/index.php?title=Part:BBa_K4195009)(+) (b). a** Target bands (1801 bp) can be observed at the position around 2000 bp. **b** Target bands (215 bp) can be observed at the position between 250 bp and 100 bp.

After the first time of purification, we found that rFET was expressed so poorly (data not shown) to obtain the target protein. Like his-PirB, we constructed this part on the expression vector pET-28a(+) by Gibson assembly, then transformed the plasmid into *E. coli* BL21(DE3). The positive transformants were selected by kanamycin and confirmed by colony PCR (Fig. 45b) and sequencing.

It was so regret that we could not confirmed that we obtained the purified rFET during the whole season, since the rFET-his is so small (37 aa, 4.1 kDa) and the target bands cannot be observed obviously even though the concentration of separating gel has been increased to 20% for SDS-PAGE. Thus, we were not obsessed with purifying this protein for *in vitro* bio-chemical test and turned to the characterization on the surface of bacteria.

The interaction between r*Lv*APN1 and toxin PirA and PirB

Molecular cloning and protein purification of r*Lv*APN1

![10](https://static.igem.wiki/teams/4195/wiki/result/pre/pre-fig-9.png)

Fig. 46 DNA gel electrophoresis of the colony PCR products of [BBa\_K4195134](https://parts.igem.org/wiki/index.php?title=Part:BBa_K4195134) (a) and [BBa\_K4195038](https://parts.igem.org/wiki/index.php?title=Part:BBa_K4195038)(+) (b). a Target bands (2925 bp) can be observed at the position around 3000 bp. **b** Target bands (1386 bp) can be observed at the position between 1500 bp and 1000 bp.

r*Lv*APN1-his ([BBa\_K4195038](https://parts.igem.org/wiki/index.php?title=Part:BBa_K4195038)) was designed for protein purification and we obtained the composite part [BBa\_K4195134](https://parts.igem.org/wiki/index.php?title=Part:BBa_K4195134) at pSB1C3, which was also induced by *L*-arabinose (Fig. 46a). However, this expression system did not work as expected and we reconstructed this part at pET-28a(+) like before (Fig. 46b). Finally, as shown in the gel image of r*Lv*APN1-his (Fig. 47), the bands of target protein (45.5 kDa) can be observed at the position between 50 kDa and 35 kDa on the purified protein lanes (FR). Besides, with the help of CUG-China for offering the operation of western blot, we could confirm that the r*Lv*APN1-his was expressed indeed (Fig. 47b), after struggling to obtain the purified protein for several rounds mentioned above (Learn more from our [Partnership](https://2022.igem.wiki/xmu-china/partnership) page).

![10](https://static.igem.wiki/teams/4195/wiki/result/pre/pre-fig-10.png)

**Fig. 47 Confirmation of the purification for rLvAPN1-his.** a SDS-PAGE analysis of r*Lv*APN1-his in lysate of *E. coli* BL21(DE3) and the elution samples, in which target bands (45.5 kDa) can be observed at the position between 50 kDa and 35 kDa. **b** Western blot analysis of r*Lv*APN1-his in lysate supernatant, in which target bands (45.5 kDa) can be observed at the position between 43 kDa and 55 kDa.

Isothermal titration calorimetry (ITC)

ITC was used to detect the interaction of r*Lv*APN1 to PirA. Assuming a one-site binding model, the apparent dissociation constants (*Kd*) of r*Lv*APN1 to PirA, as calculated from the ITC curves, were 5.9 μM (Fig. 48), which is similar to the reported binding affinity (3.2 μM) that determined through ELISA. The result indicates that r*Lv*APN1 can bind directly to toxin PirA. However, due to the need of large sample amount of ITC, we could not obtain a satisfying titration curve of r*Lv*APN1 to PirB because of the few amount of soluble components of this toxin.

![10](https://static.igem.wiki/teams/4195/wiki/result/pre/pre-itc.png)

**Fig. 48 The binding affinity of r*Lv*APN1 to PirA determined by ITC.** One-site binding model was applied to curve fitting.

The function of receptors can be retained after displayed on the bacterial surface

#### [Show Details](#collapse-pr3)

Molecular cloning

In order to verify whether two receptors on the surface of the engineered bacteria can bind toxins, we constructed four composite parts [BBa\_K4195125](https://parts.igem.org/wiki/index.php?title=Part:BBa_K4195125) (for INPNC-rFET), [BBa\_K4195128](https://parts.igem.org/wiki/index.php?title=Part:BBa_K4195128) (for ClyA-rFET), [BBa\_K4195130](https://parts.igem.org/wiki/index.php?title=Part:BBa_K4195130) (for ClyA-r*Lv*APN1), and [BBa\_K4195132](https://parts.igem.org/wiki/index.php?title=Part:BBa_K4195132) (for INPNC-r*Lv*APN1) at pSB1C3, which are all under the control of *L*-arabinose-inducible system. The plasmid was transformed into *E. coli* BL21(DE3), then the positive transformants were selected by chloramphenicol and confirmed by colony PCR (Fig. 49) and sequencing.

![10](https://static.igem.wiki/teams/4195/wiki/result/pre/pre-fig-12.png)

**Fig. 49 DNA gel electrophoresis of the colony PCR products.a** [BBa\_K4195125](https://parts.igem.org/wiki/index.php?title=Part:BBa_K4195125) in *E. coli* BL21(DE3). Target bands (2484 bp) can be observed at the position between 3000 bp and 2000 bp. **b** [BBa\_K4195128](https://parts.igem.org/wiki/index.php?title=Part:BBa_K4195128) in *E. coli* BL21(DE3). Target bands (2466 bp) can be observed at the position between 3000 bp and 2000 bp. **c** [BBa\_K4195130](https://parts.igem.org/wiki/index.php?title=Part:BBa_K4195130) in *E. coli* BL21(DE3). Target bands (3816 bp) can be observed at the position between 5000 bp and 3000 bp. **d** [BBa\_K4195132](https://parts.igem.org/wiki/index.php?title=Part:BBa_K4195132) in *E. coli* BL21(DE3). Target bands (3834 bp) can be observed at the position between 5000 bp and 3000 bp.

The interaction between displayed rFET and toxin PirA and PirB

Immunofluorescence (IF)

After induction, *E. coli* harboring [BBa\_K4195125](https://parts.igem.org/wiki/index.php?title=Part:BBa_K4195125) and [BBa\_K4195128](https://parts.igem.org/wiki/index.php?title=Part:BBa_K4195128) were cultivated overnight. The bacterial culture with his-PirB added was set as positive control while which had no toxins added was set as negative control. Then IF experiment was performed again to test whether the receptor activity is retained or not after displayed on the bacterial surface.

The ratio of fluorescence intensity to OD600 of the positive control (culture was incubated with toxins) is higher than that of negative control (culture was incubated with 1×TBST) (Fig. 50). The results proved that our surface display system works well and the rFET can still bind PirA-his and his-PirB, even on the surface of bacteria.

![10](https://static.igem.wiki/teams/4195/wiki/result/pre/pre-fig-13.png)

**Fig. 50 Ability of binding his-PirB on the surface of *E. coli* BL21(DE3). a** Fluorescence intensity/OD600 of *E. coli* harboring [BBa\_K4195128](https://parts.igem.org/wiki/index.php?title=Part:BBa_K4195128) which was incubated with PirB or not (*p* = 0.0002). **b** Fluorescence intensity/OD600 of *E. coli* harboring [BBa\_K4195125](https://parts.igem.org/wiki/index.php?title=Part:BBa_K4195125) which was incubated with PirB or not (*p* = 0.0426).

The interaction between displayed rLvAPN1 and toxin PirA and PirB

Immunofluorescence (IF)

After induction, *E. coli* harboring BBa\_K4195130 and BBa\_K4195132 were cultivated overnight. The bacteria culture with PirA-his or his-PirB added was set as positive control while which had no toxins added was set as negative control. IF was implemented once again to test whether the receptor activity was retained or not after displayed on the bacterial surface.

The ratio of fluorescence intensity to OD600 of positive control (culture was incubated with toxins) is higher than that of negative control (culture was incubated with 1×TBST) (Fig. 51), which indicates that our surface display system works well and the displayed r*Lv*APN1 can still bind PirA-his and his-PirB.

![10](https://static.igem.wiki/teams/4195/wiki/result/pre/pre-fig-14.png)

**Fig. 51 Ability of binding PirA-his and his-PirB on the surface of *E. coli* BL21(DE3). a** Fluorescence intensity/OD600 of *E. coli* harboring [BBa\_K4195130](https://parts.igem.org/wiki/index.php?title=Part:BBa_K4195130) which was incubated with toxins or not (*p* = 0.0082 for PirA-his, *p* = 0.0213 for his-PirB). **b** Fluorescence intensity/OD600 of *E. coli* harboring [BBa\_K4195132](https://parts.igem.org/wiki/index.php?title=Part:BBa_K4195132) which was incubated with toxins or not (*p* = 0.0014 for PirA-his, *p* = 0.0329 for his-PirB).

#### Conclusion

We successfully proved that the two receptors (rFET and r*Lv*APN1) can be displayed by two surface display systems (ClyA and INPNC) on the surface of engineered bacteria. Together with the results of Treatment part, it is convincing that the surface display system we chose can function as expected to locate the cargo proteins onto the cell surface. Besides, the function of rFET to bind the toxin PirB and r*Lv*APN1 to bind both PirA and PirB was verified after the receptor was displayed. As for the binding affinity of displayed rFET to PirA, we attributed this to the potential weak interaction between the two proteins, which may be similar to the situation that the interaction between r*Lv*APN1 and PirA (*Kd* = 3.2 μM) is weaker than that between r*Lv*APN1 and PirB (*Kd* = 0.5 μM). Because the concentration of some protein purified can hardly meet the need of ITC, we only obtained the binding affinity of r*Lv*APN1 to PirA (*Kd* = 5.9 μM) in this *in vitro* characterization. In summary, the outcome we have gotten set the fundamental for the verification at the level of OMVs (more details shown in our [Proof of Concept](https://2022.igem.wiki/xmu-china/proof-of-concept) page), since OMVs are originated from the bacterial surface (*3*).

#### Future prospects

Given the length of rFET (31 aa), such protein should be considered as polypeptide and obtained through solid-phase peptide synthesis (SPPS). Alternative characterization method, such as ESI-MS or MALDI-MS, can be applied to verification rather than SDS-PAGE. We hope this will be realized thus the *in vitro* protein-protein interaction could be probed.

To achieve the virulence loss of *V. parahaemolyticus* for further prevention of AHPND, we also designed a CRISPR/Cas system to destroy both *pirA* and *pirB* gene (Learn more in our [Design](https://2022.igem.wiki/xmu-china/design) page). However, due to the time limit, we have not cloned the type I-F *cas* cluster from the *V. parahaemolyticus*-alternative strain *V. alginolyticus* (BSL1) and constructed the complete circuits as designed. We are looking forward to proving its effectiveness in the future.

#### Kill Switch

The kill switch system is mainly composed of an inducible promoter (pBAD/araC) and a *mazF* gene (express toxin protein of MazF). Both promoters could be induced by *L*-arabinose to express toxin protein. Our GMOs will be mixed in the fodder and put into the water environment of prawn ponds, then colonize directly in prawns’ intestines. The kill switch is essential for preventing the leakage of GMOs. Before harvesting and marketing mature prawns, we must ensure that the GMOs colonized in prawn intestines are killed. Besides, GMOs, that remain in the tailwater of prawn ponds, must be killed before the tailwater is drained into the environment out of the prawn ponds. We planned to throw *L*-arabinose into the tailwater, so that MazF can be expressed and kill them, guaranteeing biosafety by cleaving the gnome of GMOs.

Cytotoxicity of MazF

Molecular cloning

We use pBAD/*araC* ([BBa\_I0500](https://parts.igem.org/wiki/index.php?title=Part:BBa_I0500)), RBS ([BBa\_B0034](https://parts.igem.org/wiki/index.php?title=Part:BBa_B0034)), terminator ([BBa\_B0015](https://parts.igem.org/wiki/index.php?title=Part:BBa_B0015)), mazF ([BBa\_K1096002](https://parts.igem.org/wiki/index.php?title=Part:BBa_K1096002)) to construct the composite part [BBa\_K3332083](https://parts.igem.org/wiki/index.php?title=Part:BBa_K3332083), which were assembled on pSB1C3 backbone by standard assembly. The constructed plasmids were transformed into *E. coli* DH5α and BL21(DE3), then the positive transformants were selected by chloramphenicol and confirmed by colony PCR (Fig. 52) and sequencing.

![10](https://static.igem.wiki/teams/4195/wiki/result/treatment/kill-switch-2-bba-k3332083.png)

**Fig. 52 DNA gel electrophoresis of the colony PCR products** ([BBa\_K3332083](https://parts.igem.org/wiki/index.php?title=Part:BBa_K3332083)).

Killing effect test

CFU assay was implemented to characterize the effect of toxin MazF (Fig. 53a). We observed that the number of colonies on the plate decreased significantly after induction, while there was no significant change in the non-induced group (Fig. 53b). This demonstrated that toxin MazF could be lethal to the bacteria.

![10](https://static.igem.wiki/teams/4195/wiki/result/treatment/fig24.png)

**Fig. 53 CFU assay for characterizing the function of MazF. A** Solid medium plate of CFU assay. **b** survival Ratio of different groups against time (h).

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* [Overview](#Overview)
* [Detection](#Detection)
* [Treatment](#Treatment)
* [Prevention](#Prevention)
* [Kill Switch](#kill)
* [Reference](#Reference)

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