Team:Hannover/Description

iGEM Hannover 2020

Project Description

Summary: We develop an inflammatory toxin sensor, which is based on mammalian cells. On this site, we first describe the medical background. Secondly, we desribe our motivation to work on this project. To learn about the biological concept of our sensor, which is based on the NF-κB pathway and uses specific promoters and two reporter proteins for visualization, MagAcreated part:
BBa_K3338000 and Gaussia Luciferasecreated part:
BBa_K3338001, follow us to the third part of this page. Please also refer to the on the bottom of the page.

Medical background

One of the most frequently performed operations in hospitals all over the world is the placement of artificial hip implants and knee joints. Furthermore, the number of implants used, such as dental implants and pacemakers, is continuously increasing [1].

The risk of developing implant-associated inflammation (implantitis) after such an operation is very high. Such inflammations are very painful. They can lead to necrosis of the surrounding tissue and therefore to loss of the implant, when they remain untreated. However, the loss of the implant is not the only risk, because the inflammation can spread throughout the body via the bloodstream and thus lead to sepsis.

Image of biofilm formation on a knee implant
Figure 1: Illustration of biofilm formation on a knee implant. These biofilms can lead to severe inflammation.
In many cases, implantitis is caused by an infectious biofilm. A biofilm is a film of carbohydrates, extracellular matrix and microorganisms such as bacteria. Biofilms form at the interface between a liquid medium and a solid object such as an implant surface. The bacteria either reach the surface of the implants during surgery or they attach themselves to the implants after the operation. Despite all efforts, infections during surgery represent a very huge risk.

Our body's own immune system reacts with various defense mechanisms such as fever to infections in order to kill the bacteria. Some bacteria have developed mechanisms to protect themselves from our immune system. For example, they form cell toxins that damage our cells. Either the bacteria actively release these toxins or they are released when they are attacked and killed by our immune system. This also explains why the tissue around the implant dies during implantitis. The problem with such inflammations is that they are difficult to detect and are usually only detected at a very advanced stage of the disease. By then it is often too late to receive the inserted implant, or a potentially life-threatening sepsis has already developed. Targeted treatment of biofilms is also often difficult. In many cases, the complex composition of the biofilms makes effective medical therapy with antibiotics, for example, difficult [2].

It would therefore be helpful to detect the formation of a biofilm at an early stage. After an early detection we would be able to start treatment as fast as needed. Our goal is to make this possible. We want to develop a sensor that makes biofilms on implants visible. The sensor will be realized by genetically engineered intestinal epithelial cells, which are able to recognize bacterial toxins and produce two specific proteins in response.

Our own motivation

The implant-associated development of biofilms is a lifelong risk for all patients, who undergo such an operation. After the insertion of a foreign object, a biofilm can form at any time, which has immense effects on the person concerned. This is where our team from Hannover comes into play. We would like to help patients to detect implant-associated infections and enable early treatment. We are based in the NIFE (Lower Saxony Center for Biomedical Engineering, Implant Research and Development). Here, different disciplines and their students meet. That is also the way we met and decided to work on this project and register for the IGEM competition. We would like to contribute “our small part” to implant research via synthetic biology. At the NIFE in Hannover, we have the optimal surrounding for this project development and realization. The coming together of different students from different study programs at the Leibniz University Hannover and the Hannover Medical School yields a perfect symbiosis regarding the development of new approaches. We learn from the others and we can explore new subject areas. The interdisciplinary team makes it possible for all participating subject areas to work on topics that have not been covered by the studies so far. All students benefit enormously and grow beyond themselves. The project challenges and encourages all participants. In addition to an enormous benefit for our team and our own development, our project can, by thinking in a future perspective, pave the way for new therapeutics for implant receiving patients. Our joint and goal-oriented work can one day help people all over the world, and this is what motivates us.

The biology

The sensor concept was realized by genetically engineered cells. These are able to recognize bacterial toxins and produce two specific reporter proteins in response. The genetically modified cells in our project react to lipopolysaccharides or LPS for short, which activate the NF-κB-pathway [3]. Via a signaling cascade that starts at the Toll-like receptor over a kinase and different phosphorylation steps, the transcription factor NF-κB is induced to translocate into the nucleus. Here it can specifically influence the expression of certain genes by binding to promoters on the DNA and allowing downstream proteins to be strongly expressed. In our project we tested as promoter parts first the IL6-promotercreated part:
BBa_K3338008, which has specific binding sites for certain inflammatory signals, such as NF-κB or AP-1 [4]. Additionally, two self-designed promoters with binding sites for AP-1 and NF-κB and the CMV-promoterused part:
BBa_I712004 were tested.

Illustration of the NF-κB pathway in mammallianallian cells.
Figure 2: Signaling pathway used for targeted expression of two reporter proteins. Via LPS the NF-κB pathway is activated and translocated to the nucleus. Through this transcription factor two proteins are produced, which can indicate the formation of a biofilm by simple imaging techniques.

We came up with the idea of using the protein MagA created part:
BBa_K3338000 from magnetotactic bacteria as one of the two reporter proteins. MagA created part:
BBa_K3338000 is a transmembrane iron transporter which can yield to accumulation of iron inside the cell via magnetosomes. Thus, the cells become magnetizable because of that protein. This makes it possible to detect the expression of MagA created part:
BBa_K3338000 during an MRI examination [5]. To display a biofilm or the toxins contained in it, the MagA created part:
BBa_K3338000 was cloned behind the respective promoters, which are influenced via the NF-κB Pathway. Therefore, the condition or the appearance of a biofilm on the implant surface can be visualized with relatively low stress to the patient by means of a non-invasive imaging method.

To enhance the sensitivity of our sensor and to express an additional reporter protein, the secretion of a Luciferase is used to detect the biofilm. Thus, analogous to MagA created part:
BBa_K3338000, the gene of Gaussia Luciferasecreated part:
BBa_K3338001 was cloned behind the different promotor parts. If a biofilm is formed, the Luciferase is expressed and secreted. This protein can be easily detected in blood and urine samples through a fast detection method [6].

But how should we combine the expression of both reporter proteins? We decided to compare an internal ribosomal entry site (IREScreated part:
BBa_K3338004) and a P2A created part:
BBa_K3338003 peptide to combine the reporter proteins.

By cloning two sequences in suitable combination, a potentially fatal disease or complete loss of the implant can be stopped. An inflammation that can take a lot of power away from the patient and is also extremely painful is detected at a very early stage and can be treated. All this is made possible by our sensor concept, which makes use of the immense possibilities of synthetic biology.

Bibliography and references

  1. Zimmerli, W., Trampuz, A., & Ochsner, P. E. (2004). Prosthetic-joint infections. The New England journal of medicine, 351(16), 1645–1654.
  2. Trampuz, A., & Widmer, A. F. (2006). Infections associated with orthopedic implants. Current opinion in infectious diseases, 19(4), 349–356.
  3. Sakai, J., Cammarota, E., Wright, J. A., Cicuta, P., Gottschalk, R. A., Li, N., Fraser, I., & Bryant, C. E. (2017). Lipopolysaccharide-induced NF-κB nuclear translocation is primarily dependent on MyD88, but TNFα expression requires TRIF and MyD88. Scientific reports, 7(1), 1428.
  4. Libermann, T. A., & Baltimore, D. (1990). Activation of interleukin-6 gene expression through the NF-κB transcription factor. Molecular and cellular biology, 10(5), 2327–2334.
  5. Uebe, R., Henn, V., & Schüler, D. (2012). The MagAcreated part:
    BBa_K3338000     protein of Magnetospirilla is not involved in bacterial magnetite biomineralization. Journal of bacteriology, 194(5), 1018–1023.
  6. Tannous B. A. (2009). Gaussia Luciferasecreated part:
    BBa_K3338001     reporter assay for monitoring biological processes in culture and in vivo. Nature protocols, 4(4), 582–591.