Team:TU Darmstadt/description

Description – TUDA iGEM 2021

Description

Introduction

With our project PHIRE BYRD – Phage mediated Immune Response by Recognizing, Defensive sleeper cell we aim to create a modular protection system against pathogens in biological systems.

Biofilms are broadly used, e.g., for water purification, for the protection of plant roots from parasites or for the synthesis of biochemical products.​1,2​ At the same time, all those applications are at risk of hosting pathogens such as Pseudomonas aeruginosa in the respective biofilm.

We consequently wanted to design a defense system that allows the threat-free use of functionalized biofilms!

For this motive, we developed a bacterial multi-functional biofilm containing modified Bacillus subtilis sleeper cells. The sleeper cells remain in a dormant so called “sleeper state” until being activated. The activation of the sleeper cells works like a motion sensor which upon recognizing movements turns on the light. In our case, the cells are not activated by motions but by detecting signaling molecules secreted by the pathogens for intercellular communication. The initial sensing triggers a defense reaction cascade within the sleeper cells. This cascade ends with the production and release of specialized phages programmed to attack one specific type of pathogen to eradicate their target.

Where did the idea for our project come from?

Various former iGEM Teams have already developed and worked with modified biological systems like functionalized biofilms e.g., the iGEM Team of William and Mary 2019 and the iGEM Team of the TU Darmstadt 2020.​3,4​

These iGEM Teams realized the broad spectrum for the potential implementation of those functionalized biofilms.​5,6​ They all have in common that the created biofilm is not supposed to be used in a closed system but in an open environment. For the safe and trouble-free implementation of these improved biological systems, it needs to be ensured that the biofilms are protected from harmful outside effects and do not host species which are harmful to humans and the environment. One such threat is the invasion by pathogens that can result either in peaceful co-existence or in the takeover by the pathogens. In any case, if the pathogens are part of the biofilm, they are shielded from the outside world and therefore protected from standard detection methods as well as conventional microbiological eradication methods, i.e., antibiotics or disinfectants.

Therefore, pathogens entering these useful biofilms must be destroyed!

We had the idea to equip functionalized biofilms with a synthetically created immune system. which is able to detect, identify and eradicate the invading pathogens. Additionally, our defense system is modularly designed to enable protection against further pathogens of interest.

But how does this artificial defense system work?

To answer this question, we need to talk in depth about the different and complex parts that make up our project. In the following section, we present an overview of the various necessary components to make PHIRE BYRD applicable.

To create a modular phage-defense system against pathogens for functionalized biofilms, we first had to grow a biofilm that contains and protects our sleeper cells. This biofilm is supposed to contain multiple differently genetically modified B. subtilis sleeper cells from the strain DK1042​7​, forming a multi-functional B. subtilis biofilm. Our proof of concept experiments show the successful creation of a homogenous co-culture biofilm paving the way towards creating a modular system. This shall ensure the optimal protection of our biofilm and any other functionalized biofilm against any targeted pathogen.

Please find further information on this component of our project here.

One idea regarding the creation of a multi-functional biofilm was the implementation of metabolic dependencies between different B. subtilis strains. This would ensure homogeneous spread of our sleeper cells and also increase biosafety. Born from these deliberations was our software concept: Phoenix, derivative of our project title PHIRE BYRD.
It encompasses two tools for modeling metabolic dependencies: an algorithm based on DOLMN​8,9​, which allows us to generate microbial communities with different metabolic dependencies, and a software framework called FLYCOP​10​, which then evaluates these configurations in the long-term based on their fitness and chooses the best consortium configuration.
Since both of these tools require specific knowledge, we decided to automate the workflow and take care of the pre- and post-processing involved. This increases the accessibility for scientists with limited programming experience, such as many iGEM students, to the computer-aided design of microbial communities by themselves.

Please find further information on this component of our project here. Also keep in mind, that this software was conceptualized by our team, not implemented. We hope, that future iGEM Team can build up on the research we conducted regarding these microbial community models.

An essential aspect and significant goal of our project is the generation of a detection system that recognizes and eliminates pathogens. The intention is to develop a genetic circuit in Bacillus subtilis which activates the production of bacteriophages against the targeted pathogen. Consequently, the production should only take place when the pathogen infiltrates the biofilm, to be precise when pathogen-specific signaling molecules are present.​11,12​ In any other case, the B. subtilis cells remain inactive. Hence, these cells are called sleeper cells.

Please find further information on this component of our project here.

We have modeled our genetic circuit upon being triggered by the quorum sensing (QS) signaling molecules to support the pathogen sensing part of our project. Knowledge of the relationship between the number of QS signaling molecules as input at the cell exterior and the number of proteins, e.g., GFP or RecA730, as output is crucial for the design of our genetic circuit. Therefore, we have developed a deterministic model in which the concerning molecular processes are firstly represented as ordinary differential equations (ODEs) and are later summarized in a simpler and easier way by using mathematical functions based on equations from the Modeling Webinars of the iGEM Engineering Committee. The model was built to especially represent our laboratory experiments in order to improve the design of our genetic circuit. Also, it helped us to gain a deeper understanding of the molecular processes inside our system.

Please find further information on this component of our project here.

To control the biosynthesis of bacteriophages, we developed a genetic switch. It allows us to manipulate the native switch of lambda bacteriophages to shift between their lysogenic and their lytic cycle​13​.

This switch consists of two components. The first ensures that the phage stays in its lysogenic cycle by constitutively producing the lambda repressor cI.​14​ The second component is inactive until it is triggered by quorum sensing molecules of the targeted pathogen. Upon its activation, RecA730​15​ is expressed within the bacteriophage, the regulator cI is cleaved and the transition of the bacteriophage into its lytic cycle is activated and thus the overproduction of the bacteriophages in the sleeper cell is started.

Please find further information on this component of our project here.

Since our project aims to make biotechnological applications safer, an essential part of our project was  the biocontainment of our functionalized biofilm. Our sleeper cells classify as genetically modified organisms.​16​ Thus, we wanted to ensure that no viable cells leave the biofilm. Therefore, we further conceptualized and optimized the B. subtilis kill-switch design by last year’s  iGEM Team of TU Darmstadt.​4​

The expression of an essential gene, rpsB​17​, is indirectly linked to the availability of the quorum sensing molecule ComX.​18​ Since this molecule is part of the intracellular communication within a biofilm​18​, only sleeper cells staying inside will be supplied with enough ComX to continue the expression of rpsB. Vice versa, we want to ensure the death of cells escaping the biofilm.


Please find further information on this component of our project here.

The core objective of Human Practices is the communication and discussion of science with people around the world. This year, we focused on science communication, education for children and high school students by producing a podcast and a Science Slam , and by evaluating our project from various ethical perspectives.

We would like to emphasize some of the highlights of this year’s HP work: We participated in a workshop by the science communication experts of “Science Birds”. Through this interactive workshop we learned tools, tips and tricks to be expert ambassadors of our research. We furthermore designed radio plays for Tonie-Boxes to catch young children’s interest in science in an innovative way, and conducted a workshop for high school students. In the workshop, we discussed future study and career opportunities of the students, but also had a general view on biotechnology and aspects of the thriving world of synthetic biology.

Another important aspect was assessing our project from different perspectives. Therefore, we talked to various experts in the field of synthetic biology and biosafety and included their insights into our project design. We also conducted a survey to learn about people’s opinion outside of the scientific community.

However, these are by far not the only things we have done. Please find further information on this component of our project here.

Figure 1. This is an overview of all the parts of the project and how they are connected. We created a bacterial multi-functional biofilm containing modified Bacillus subtilis sleeper cells. The sleeper cells remain in a dormant so called “sleeper state” until being activated by the detection of quorum sensing (QS) molecules secreted by the pathogens. The initial sensing triggers a defense reaction cascade within the sleeper cells ending with the production and release of specialized phages programmed to attack and eradicate one type of pathogen. For the implementation of our phage-defense system in the environment, we had to ensure that our modified Bacillus subtilis cells cannot survive outside the biofilm. Therefore, we developed a kill-switch.

Conclusion

From start to finish, our project was inspired by the vision of iGEM: Building a better world with synthetic biology.

While there might be always room and hunger for new emerging technologies, systems and techniques, the evaluation of the safety and security of those new inventions is urgently needed. This was shown by the results of our public survey, in which only 5% of the participants answered the question of what word they associated with synthetic biology with safe. Actually, most participants [44%] connect the word synthetic biology with risks and danger.

This confirmed our belief that ensuring the risk-free implementation of synthetic biology tools, e.g., functionalized biofilms, is of great importance. In addition, to address the issue of safety in the useful tools of synthetic biology, we wanted to make sure that the public’s fears related to synthetic biology are seen and accordingly acted upon. Hence, we focused to ensure that our system can be facilitated easily and safely by people with and without a scientific background. That way, we contribute to the public acceptance and support of synthetic biology and also helped improving the communication between the science community and society.

To enable the desired modular defense application of our system, we incorporated certain features into our project. Each of our genetically modified B. subtilis sleeper cells only carries the genome of one highly specialized bacteriophage targeting one specific pathogen. This way, future users can generate their desired B. subtilis sleeper cells that responds to the presence of a specific pathogen with the expression of phages. These can then be co-cultivated alongside the functionalized and to be protected biofilm cells to ensure its safe application.

As the potential applications for functional biofilms steadily increase, the need to ensure their safe implementation is mandatory, showing the necessity of PHIRE BYRD.

References

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igem@bio.tu-darmstadt.de

We are the 2021 iGEM Team of the TU Darmstadt. This year we focused on creating a self-protecting biofilm based on Bacillus subtilis "sleeper cells".

Eppendorf hilgenberg Zymo Research New England Biolabs Inc.
IDT Integrated DNA Technologies Snapgene Biebertaler Blutegelzucht Promega
DWK Life Sciences Science Birds Twist Bioscience Microsynth SEQLAB
TU Darmstadt
Supertext Brand
Carl Roth Sparkasse
Quantum Design Europe