Poster: UM_Macau

Title: Biofilm Removing E.coli for Aquarium Cleaning (BREAC)

Poster presented by: UM_Macau_2020

¹iGEM Student Team Member, ²iGEM Team Mentor, ³iGEM Team Primary PI, §Faculty Sponsor, Faculty of Health Sciences, University of Macau, Macau, China.

iGEM Student Team Member: Yuzhao Feng, Shuyao Xie, Leong Hoi Cheng, Yuanming He, Hengyi Fu, Ioi Fu, Yu Lun, Chan Tat Leong, Chengzong Hou, Huichao Zhao, Di Sun, Xuemeng Li, Changcheng Lu, Ruiying Ma, Lesi Chen.

iGEM Team Primary PI: Prof. Leo Tsz On Lee, Prof. Ruiyu Xie, Prof. Tzu-Ming Liu.

Faculty sponsor: Faculty of Health Sciences, University of Macau.

Abstract:

Since biofilms grow rapidly, and attach to the inner surface of aquariums and aquatic organisms, this lowers the water quality if not cleaned regularly. However, the removal of biofilms in large aquariums is time-consuming and relies on inefficient labor through scrubbing. To address these concerns, we engineered the bacteria strain of E. coli (BL21) to detect and biodegrade biofilms. The receptor, LuxR, is utilized to drive the expression of enzyme and adhesion molecules. This receptor recognizes the signaling molecule AHL that is secreted by biofilms and bind on the corresponding promoter to regulate gene transcription. With co-expression of enzymes, including DNase, proteases and an adhesive protein, Ag43, our engineered bacteria will bind to the biofilm and degrade it efficiently.

Introduction

Our project Biofilm-Removing E. coli for Aquarium Cleaning (BREAC) is to design a E. coli that can express adhesive protein to stick on biofilm and express digestive enzymes to biodegrade biofilm in aquarium tanks with modeling demonstration. For outreach part, we communicated with the stakeholders and optimized our design according to their feedbacks. We also collaborated with other teams with the same target on biofilm and formed a community called Anti-Biofilm Community (ABC), and we helped each other to better improve our projects. In addition, we did public education through both online account and face-to-face high school talk in local school, raising people’s awareness of biofilm and synthetic biology knowledge.

Fig. 1 Overview of our project.

Significant and impact of BREAC

As time goes, the public's awareness in protecting marine life has improved. Therefore, more and more aquariums and ocean parks are opening up all over the world. However, all aquariums are struggling with a cleaning problem, that is the fight with biofilm. Biofilm grows rapidly and need to be cleaned regularly and frequently. Otherwise, the accumulation of biofilm would lower the water quality which is harmful to aquatic creatures. Our BREAC can help solve this worldwide problem in an efficient and man-power saving way, with biosafety guarantee.

Problem

Biofilm is a layer of bacteria that binds together and they are embedded in the extracellular substance they secrete, including polysaccharides, lipids, proteins and DNA. The rapid growth rate of biofilm in aquariums can cause these problems:

1. Manpower and materials consuming: the bristle of brush can damage the surface, scratching glass and destroying the decoration on the rock.

2. Low efficiency and time-consuming: one cleaner can only clean about 5 m² in one hour, which can be a big problem for large aquariums with large tanks.

3. May be unsafe for cleaners: cleaners suffer from high-water pressure and may get injured by other aquatic creatures in the tanks, such as shark attacks, allergy to jellyfish toxins, etc.

4. May be harmful to aquatic creatures: some fragile creatures may be hurt. For example, corals may be damaged when scraping too hard, and jellyfishes could die from absorbing too much air bubbles generated by cleaners.

Inspiration

Since the removal of biofilms is a large issue for aquariums and ocean parks around the world. Our team was inspired to apply synthetic biology to society in order to develop an efficient biofilm removal model called Biofilm-Removing E. Aquarium Cleaning Coli (BREAC). This is accomplished by the engineering of bacteria to express the adhesive protein to be bound to biofilm, and the digestive enzyme to biodegrade biofilm. In addition, based on the feedbacks from stakeholders. We optimized our concept with two systems: a light-inducing system and a magnetic-removing system. With these two systems, our bacteria can degrade biofilm safely without accumulating in water and reducing the quality of the water.

Cleaner is scraping off biofilm.

Solution

Fig. 1 Overview of our project.

This graph shows how our BREAC works. After engineering the E. coli, it will be applied into the aquarium from the top of it. The engineered bacteria move with the water flow. When they detect AHL which is secreted by biofilm, they will stick on the biofilm on the surfaces. With the following expression of digestive enzymes, BREAC can degrade the biofilm and then leave the surfaces. Following the water flow, they move to the filter which allows water inside to leave the tank. The magnets we put ahead of entrance of filter can attract the used BREAC. Lastly, the magnets with used BREAC on them can be removed by divers.

Design

Antigen 43
Ag43 is known as a self-recognizing outer membrane adhesion protein, enable our bacteria to bind on biofilm. Its gene is located downstream of LuxR promoter (PLuxR), and will be translated once LuxR senses the biofilm quorum molecule AHL.
Enzymes

Three enzymes are used for biofilm degradation, Alkaline serine protein, Dispersin B and DNase TA. All of them are well constructed at the downstream of PLuxR. Activate the synthesis and secretion by AHL.

Overall original design

Building Library

We use 19 basic part and 4 new composite parts into the experiment
All parts listed in Table 1 were confirmed the basal part that we use with sequencing

We organize the basal parts that we use from the above and form the composite parts:

New Parts
We optimize the new part (BBa_K3630029) to enhance the ferritin magnetic power and efficient from iGEM UM_Macau 2019’s part BBa_K3033014)

Result and discussion

We have successfully cloned the construct LPDsp-T7LR. As we use quorum sensing to detect the presence of biofilm in the aquariums, we checked the expression of LuxR in the transformed bacteria to ensure the AHL-LuxR detection system can work properly and activates the expression of our enzymes and adhesion protein. Before further testing the degradation efficiency of our constructs, we tested the degradation of biofilm by proteinase K for optimizing the future functional assay.

Future work

We will determine the most suitable enzyme from the three enzymes Dspb, DNaseTA and Alkaline serine proteinase by testing their efficiency of degradation.
We will further add plasmids for protein adhesion, light sensitive and magnetic collection for better control and detect their functions in both light and dark conditions.
We will use the zebrafish to detect its harm to fish.
We will apply this engineered bacteria into the aquarium tank and determine the minimum colony number for the highest efficiency of degradation.

Collaboration & Partnership

1.Conference of China iGEMer Community (CCiC)

Fig. 1 The cover of the video we made for CCiC

This year, presentation in CCiC is delivered by videos through online meeting. In the team presentation session, the host played the video produced by each team. After the video, each team had five minutes for comments and online communication. We did trouble-shot with other teams, communicated with others. We learned a lot from them and also the judges and improved our future work.
The content our video includes project introduction, interviews, bacterial membrane formation process and experimental design, etc.

2.Anti-Biofilm Community

Fig. 2 ABC Members: WHU_China_iGEM, UM_Macau_iGEM, DUT_China_iGEM, THU_China_iGEM, SUST_China_iGEM and a high school team HKHCY_iGEM

We held three online meetings with other teams in ABC, sharing the concept and information of our project with other teams and discussing how we could collaborate with each other.
In the first troubleshooting meeting, we received some experimental suggestions on our project and we also gave advice to other teams’ project. In the second meeting we discussed about completion of inducers, addressed new issue of modeling and planed for inviting overseas teams. The last ABC meeting established the idea of “A Handbook of ABC” and designed the content of it.

3.ABC Booklet
The content consists of two parts, one is team projects’ introduction and the other part is in various form designed by each team for the same goal of introducing knowledge of biofilm.

Fig.3 The cover of ABC booklet to promote “Biofilm”.

Fig. 4 The catalog of ABC booklet.

Education and public engagement

1.High School Talk

We give a lecture talk in the local Sum Yuk High School on September 23rd about ‘Microbes in Daily Life’ aiming to raise the awareness of microbiology in teenagers. The talk is elaborated by three aspects, microbes in gut, microbes in food, and microbes in water, which gets an active response.

Fig. 1 Group photos with students in Sum Yuk High School

2.Social account

We also create our own WeChat Official account and Instagram account which posts more than 20 articles receiving more than 2000 reads. In these articles, we specially create a series of articles in the arena of synthetic biology in order to make the public know more about this area. For the online survey about biofilm, our team totally receive more than 200 replies and get fully analyzed. At the beginning, our survey shows most people know little about biofilm and this changed a lot for our article readers after our synthetic biology series have been posted.

Fig. 2 The social media account and some posts screen shot

Human practice

Biofilm in aquarium can harm the health of organisms inside. After our visit to a neighbourhood aquarium, we found that the current cleaning method of biofilm in the aquarium is not only time and energy consuming, but also threaten both human and aquatic creatures’ health. With an urgent need of improvement, our team launch the iGEM2020 project on BREAC, the biofilm cleaning E. coli of Aquarium Cleaning.

To get more information of the most suitable biofilm removing methods, we interviewed the staff of the Chimelong Ocean Kingdom. We learnt from the staff, Mr East, that they would like the new technology to be simple-handling and easy-learning, more importantly, it must be safe to the creatures living inside the display tank. Therefore, we engineered the E. coli to express magnetism which made it to be removed by magnetic force.

Fig1a. Visit to Chimelong Ocean Kingdom

Fig1b. Online interview with experts from Ocean Park Hong Kong

To further explore the biosafety of our product and related knowledge, we conducted an interview with Dr Zhang, an expert from the Ocean Park Hong Kong. Dr Zhang claimed there are some useful biofilms stay in the biofilter connected to the tank. As the biofilter is in dark-shaded area, we came up an idea of a light-inducible system.

Fig 2.
Three-way-dialogue of human practice

Through interviews, conversations, and events, we successfully established dialogues between the aquarium and our team, our team to the public, the public to the aquarium. All the suggestions given to us can help us to improve our design, so that we can further solve the stakeholders’ problem.

Optimization

Two systems are engineered to optimize our original mechanism. Light inducible system makes it possible to control our bacteria with light, so that they would not degrade the useful biofilm on the biofilter in the dark place. The magnetization system can help to remove bacteria efficiently, avoiding the accumulation of bacteria in the water. Both systems greatly improve the biosafety of our engineered bacteria.

1.Light inducible system

The expression of LacI will be activated under darkness, thus the LuxR gene expression will be repressed in the dark since the upstream Lac operon will be inhibited by LacI. When this system is under illuminated environment, LacI will be inhibited. In this case, LuxR will normally function to activate downstream reactions.

2.Magnetization system

Gene FtnA encodes ferritin, a protein that can store and crystalize iron ions, allowing the bacteria to be magnetizable. In this case, the bacteria can be easily removed by applying magnetic field, or just by applying the magnet.

Fig. 1Overall optimized design

Modeling

Based on the cellular automata model and NetLogo language, we established a simulation dynamic model to measure the cleaning efficiency and economic value of our engineering bacteria, and finally presented a simulation program that can be changed according to actual needs.

Fig. 1 data and control desk

In the figure below you can see the Initial situation (respect number of group of engineering bacteria is 50) and final result of the simulation process (respect our expectation of our product).

Among them:

The blue ball represents engineering bacteria;

The gray surface represents the surface covered by the bacterial membrane;

The red surface represents the bacterial film removed by the engineered bacteria;

The actual flow rules of engineering bacteria, removal rules and a brief simulation process demonstration can be found on our wiki page.

Fig 2.1 Initial situation

Fig 2.2 final result

Acknowledgements

University of Macau

Faculty of Health Science_UM

UM_Macau 2019 team members

Chimelong Ocean Kingdom

Ocean Park Hong Kong

Dr. Zhang Ying

Sponsors:

University of Macau

Faculty of Health Science_UM

Integrated DNA Technologies

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