Team:Hannover/Implementation

iGEM Hannover 2020

Proposed Implementation

Summary: The overall goal of our project InToSens is to design and build a cell-based sensor for the early and minimally-invasive detection of bacterial biofilms on implants using the toolbox of synthetic biology. Within the iGEM competition 2020, we already successfully performed the very first steps of this comprehensive project. These first accomplishments include the cloning and characterization of the basic parts to be used as reporters and expression regulator (see Engineering and see Parts) und within our sensor. In the course of this, we conducted in vitro proof-of-concept experiments (see Proof of Concept for an overview and see Results for more details) in which we introduced the genetic constructs into mammalian cells and demonstrated the expression of our reporter proteins under different conditions. Based on these results, we made a choice regarding which parts to be composited for our final sensor construct. Also, to gain a better understanding of the growth of bacterial biofilms, which our sensor should be able to detect, a comprehensive modelling/software development (see Software) was conducted in collaboration with the iGEM Team Darmstadt. Furthermore, we developed a novel magnetophoresis measuring chamber (see Design and Engineering) for the detection of magnetic particles. In all these aspects, experts (see Human Practices) coming from different fields of research gave us important advices which paved the way for our project to progress in the right direction.

Implementation in the real world

Implementation in the real world In our future vision, our sensor should be applied in clinical use. With this, we aim to prevent the development of chronic inflammation or even sepsis in patients with implants, thereby preventing the doctors’ challenging treatment approaches against advanced biofilms, unfortunately often in vain, high health care system expenses as well as the patients’ suffering and maybe even life!

So how do we imagine to implement our sensor in the real world?

We plan to have our sensor, consisting of genetically modified cells, attached directly to the implant. This is done before implantation takes place so that the sensor gets introduced into the patient during the process of implantation. Shortly after the operation, the basal expression levels of our sensor’s reporter proteins have to be determined. Based on the properties of the reporters, this is done in two ways. On the one hand, MagAcreated part:
BBa_K3338000 expression is examined via MRI. On the other hand, Gaussia Luciferasecreated part:
BBa_K3338001 expression is measured by performing a luciferase assay with the patient’s blood and/or urine samples. Due to close proximity of the sensor to the implant and therefor to the site of biofilm formation, the sensor will be able to actually “sense” an accumulation of bacteria around the implant and increase the synthesis of reporter proteins in response. This again can be measured as stated before once the patient shows first signs of inflammation. In this way, our sensor enables an early and minimally-invasive detection of biofilms, which, at the current state, still poses a great challenge in clinical life.

Taken together, our sensor will bring an advantage in diagnosis of bacterial inflammation both for patients and doctors! As detailed below and in Figure 1, its further development would have to follow a long line! In addition to in vitro experiments, in vivo experiments would also have to be carried out to make a statement about feasibility possible. If the application passes the proof of concept, the clinical research phase would follow. The potential of the method would be evaluated, in a clinical study with a small number of people. In a follow-up study, our sensor would then be applied to a sufficiently large group of patients to allow comparison with the standard intervention and a statement on the potential of the method as a treatment alternative. Afterwards, the approval of the sensor would be possible.

Figure 1: The path from idea to an approved medical device is long, and many research approaches do not make it to clinical application. Therefore, it was essential for us to know the individual phases of development. This figure shows the development phases of our cell-based sensor from the design phase to approval. We have developed a concept and started the proof of concept in vitro experiments thus we are in an advanced stage of the Design Phase. Constantly we are in exchange with experts to re-evaluate our sensor, too.

What has to be done for this implementation?

Of course, we know that there is still a long way to go from what we developed so far until our sensor can be implemented as proposed. So, what has to be done next?

In vitro experiments

With the experiments we already performed for characterization of our sensor’s parts, we demonstrated the expression of both reporter proteins MagAcreated part:
BBa_K3338000 and Gaussia Luciferasecreated part:
BBa_K3338001 in the mammalian epithelial cell line HeLa. Moreover, we showed that Gaussia Luciferasecreated part:
BBa_K3338001 is indeed secreted by the cells as expected and that the enzyme is properly working as detection via luminescence assay proved to be possible. In the case of MagAcreated part:
BBa_K3338000 though, it still needs further characterization especially regarding its functionality to demonstrate its suitability as a reporter protein for our sensor concept. The novel measuring chamber (see Design and Engineering) we built in the course of our project was especially designed for this purpose. Due to time limitations – and also attributable to strict Covid-19 safety regulations – we only managed to perform validation experiments with the measuring chamber, while characterization of MagAcreated part:
BBa_K3338000 expressing cells – namely showing the cells’ magnetizability due to MagAcreated part:
BBa_K3338000 – remain to be done. In a next step, we have planned to examine if MagAcreated part:
BBa_K3338000 expressing cells can indeed be visualized by MRI as reported. For this, the use of a small MRI scanner is scheduled.

Performing lipopolysaccharide (LPS) stimulation experiments (see Proof of Concept and see Results), we already showed that the IL6 promoter’s activity can be induced by bacterial toxins leading to increased expression levels of our reporters. While LPS represents a bacterial endotoxin common for gram negative bacteria such as E.coli [1]., biofilms usually consist of a broad variety of bacteria strains [2].which release an equally broad variety of bacterial toxins. Therefore, it is extremely important to study to what extent the inflammatory toxins produced within the environment of a biofilm induce the expression of the sensor’s reporter proteins. Consequently, an experiment in which our sensor cells are co-cultivated with a bacterial biofilm can yield the appropriate information.

In our final sensor, the sensor cells are supposed to be encapsulated to keep them at the appropriate site of the implant and to prevent cell migration. Therefore, in a next experiment, the encapsulation of our sensor cells, e.g. using alginate gel, onto a material used for implantable medical devices needs to be conducted. It then needs to be further validated that encapsulation does not alter the inducibility of reporter gene expression. Also, it needs to be examined that the encapsulation is not destroyed by bacteria. For this, the previously proposed co-culture experiment could be repeated with enclosed sensor cells.

In regard to biosafety aspects, our final sensor shall not consist of HeLa cells as used for our proof-of-concept experiments. Generally, cell lines are characterized by immortality and thus the ability of unlimited proliferation potential. While this feature makes them ideal for first in vitro studies as their availability is generally not restricted, it also poses great risks in the context of an introduction into human beings. This is since the cell lines’ immortality is usually based on genetic alterations which involve an increased carcinogenic potential. Though the sensor cells would quite probably be eliminated by the patient’s immune system in case they find a way to escape their encapsulation, we definitely aim to prevent any harm posed by our sensor in the best possible way. Therefore, our final sensor is supposed to consist of genetically engineered human intestinal epithelial cells instead. We considered this cell type since in its native environment, its exposed to a variety of bacteria as for example originating from the intestinal microbiota but also pathogenic bacteria, which the cells interact with in a specialized way. We thus concluded that this cell type can tolerate bacterial toxins to a certain extent. This property makes intestinal epithelial cells a great option for our concept since our sensor also has to stand bacterial toxins without inducing apoptosis right away.

Figure 2: The first proof of concept ("sensing and reporting") is realized. However, further in vitro experiments and in vivo validation would be mandatory.

Preclinical in vivo experiments

Presumed that the before mentioned in vitro experiments yield promising results, as a next step, in vivo experiments would need to be conducted for the implementation of our sensor. These would especially be necessary to study the influence of the organism’s immune system, which cannot be fully mimicked in cell culture experiments, on the sensor. There are several reports on preclinical small animal models for implant-associated biofilm infections, including mice, rats and rabbits [3-6]. which could be adapted for validation of our cell-based sensor. Of course, there are strict regulations concerning any in vivo experiments and all these need to be authorized by an ethical committee before conduction.

Clinical studies

If preclinical in vivo experiments using animal models will lead to promising results, in a last phase of the implementation of the sensor, clinical studies would need to be conducted. These, of course, would also need approval by different committees, e.g. regarding ethical considerations.

Ethical and Safety Reflections

While the toolbox of synthetic biology offers great options to modify living organisms, it also has to face large skepticism in society regarding its ethical acceptance. As for our sensor concept, we aim to utilize genetically engineered mammalian cells, which shall be introduced into human beings, we definitely need to critically reflect our project in the means of ethics and safety. As for now, in Germany, approval of such projects for clinical trials is fraught with many hurdles, especially as it is still unclear how the introduced cells will behave once within the human body. As said before, the risk of developing cancer has to be strictly prevented. This definitely also poses one of the biggest concerns stated by society. Therefore, it could be a great alternative for our sensor to be applied in a modified way, in which the sensor does not stay within the human body for a prolonged time, first. In an interview with one of our experts (see Human Practices), she gave us this guiding advice and also, in this sense, suggested the utilization of the sensor at a urine catheter. We think that, in the future, this could be a great first step to clinical introduction since the sensor would probably experience a higher degree of acceptance within the society and among the patients when applied rather outside the human body.

Bibliography and references

  1. López, D., Vlamakis, H., & Kolter, R. (2010). Biofilms. Cold Spring Harbor perspectives in biology, 2(7), a000398.
  2. Beutler, B., & Rietschel, E. T. (2003). Innate immune sensing and its roots: the story of endotoxin. Nature Reviews Immunology, 3(2), 169-176.
  3. Wang, Y., Cheng, L. I., Helfer, D. R., Ashbaugh, A. G., Miller, R. J., Tzomides, A. J., Thompson, J. M., Ortines, R. V., Tsai, A. S., Liu, H., Dillen, C. A., Archer, N. K., Cohen, T. S., Tkaczyk, C., Stover, C. K., Sellman, B. R., & Miller, L. S. (2017). Mouse model of hematogenous implant-related Staphylococcus aureus biofilm infection reveals therapeutic targets. Proceedings of the National Academy of Sciences of the United States of America, 114(26), E5094–E5102.
  4. Poultsides LA, Papatheodorou LK, Karachalios TS, Khaldi L, Maniatis A, Petinaki E, Malizos KN. Novel model for studying hematogenous infection in an experimental setting of implant-related infection by a community-acquired methicillin-resistant S. aureus strain. J Orthop Res. 2008 Oct;26(10):1355-62.
  5. Shiels, S. M., Bedigrew, K. M., & Wenke, J. C. (2015). Development of a hematogenous implant-related infection in a rat model. BMC musculoskeletal disorders, 16(1), 1-8.
  6. An, Y. H., & Friedman, R. J. (1998). Animal models of orthopedic implant infection. Journal of Investigative Surgery, 11(2), 139-146.