Team:TU Eindhoven/Project Description

Project Description

Problem

The iGEM team TU Eindhoven 2014 sees high potential in the usage of genetically modified bacteria as innovative solutions in a wide range of fields such as energy, health and the environment. However, the actual application of the micro-organisms often does not only give rise to regulatory issues, but also to issues concerning the resilience of these bacteria. For example, the application of bacteria in clinical settings - for instance as drug delivery systems in cancer therapies [1] or as devices for local production of contrast agents for MRI visualization (TU Eindhoven 2013 iGEM team) - is not that straightforward, since the human immune system will neutralize and eventually remove the bacteria. Also, the bioreactor industry would greatly benefit from more resilient bacteria. The efficiency of these bioreactors would increase, if the bacteria are able to survive under higher pressures and temperatures (Figure 1). Moreover, in numerous iGEM projects actual application of the bacteria in the intended way, would result in inactivation or removal before the bacteria can fulfill its engineered purpose. For these reasons, the iGEM project of team TU Eindhoven 2014 focuses on the limited ability of genetically engineered bacteria to survive under harsch conditions, since this is a fundamental problem in their future application. Therefore, the aim is to develop and characterize a universal tool, which could be a starting point for a solution to this fundamental problem.

Click Coli

In order to increase the resilience, the bacteria could be encapsulated in a biocompatible gel or coated with a specific substance. Therefore, the “Plug and Play” system named Click Coli has been designed and developed. This system enables the formation of a covalent bond between any dibenzocyclooctyne (DBCO) functionalized molecule and the outer membrane of E. coli. This way a functional coating can be attached to the E. coli’s membrane. The DBCO functionalized molecules are able to bind covalently with protein anchors, which are called Clickable Outer Membrane Proteins. Each Clickable Outer Membrane Protein (COMP) contains an azidophenylalanine, which can bind very specifically and effectively to DBCO in a so called SPAAC Click Reaction. Since in principle most organic molecules can be DBCO functionalized, the Click Coli is a versatile tool providing numerous possibilities for engineering E. coli on the outer membrane.

The TU Eindhoven 2014 team has designed two Clickable Outer Membrane Proteins (COMPx and COMPy) and has developed and characterized the system of Click Coli. In order to explore the possibilities of Click Coli, DBCO functionalized polyethylene glycol (PEG) polymers were clicked on the cell’s membrane. Since PEG is already clinically used for its antifouling properties, a starting point for an antifouling coating was made [2]. This would enable the use of genetically modified E. coli in the human body for healthcare purposes.

Rolling Circle Amplification

The amount of possible applications of the Click Coli system could be increased by functionalizing the outer membrane with DNA molecules. This enables the process of Rolling Circle Amplification on the outer membrane. This process involves the synthesis of long single stranded DNA molecules from a short circular ssDNA template by using a single DNA primer. As the Click Coli system allows the process of Rolling Circle Amplification to take place from the outer cell membrane, this results in numerous opportunities in engineering the outer membrane. For example, highly specific targeting structures could be formed and it would also allow the synthesis of materials from the outer membrane.

Microfluidics

In order to gain more control over the conditions of the click reaction, microfluidic devices have been designed, developed and tested. Microfluidics is a very useful technique when it comes to the encapsulation of single cells. First a droplet device was made in order to investigate the features required for a cell encapsulation device. The aim of the cell encapsulation device is to function as an ideal working space with regard to the application of the Click Coli system. Modeling has been a useful support in estimating certain parameters required for the microfluidic devices.

Zwitterionic Antifouling Approach

When genetically engineered bacteria are given the ability to produce their own antifouling coating - and ideally tune this coating based on its permeability - a wide range of therapeutic applications would be possible. For this reason, an effort has been made to design a Zwitterionic Antifouling Protein (ZAP) . Once these proteins are displayed on the outside of the cell membrane, the mutual zwitterionic interactions would assist in evasion of the natural immune response of the human body and allow the genetically engineered bacteria to fulfill their therapeutically approved function.

Bibliography

[1] Patyar, S., Joshi, R., Byrav, D. P., Prakash, A., Medhi, B., & Das, B. (2010). Bacteria in cancer therapy: a novel experimental strategy. Journal of Biomedical Science, 17(1), 21.

[2] FDA (U.S. Food and Drug Administration), Center for Drug Evaluation and Research, Polyethylene Glycerol, see http://www.accessdata.fda.gov/scripts/cder/drugsatfda/index.cfm?fuseaction=Search.SearchAction&SearchType=BasicSearch&SearchTerm=polyethylene+glycol