Team:TU Eindhoven/RCA

Rolling Circle Amplification

One way to use the Click Coli system developed by iGEM Eindhoven 2014 is to functionalize the outside of the bacterial cell membrane with DNA molecules. This offers many exciting possibilities for applications. For example, Bertozzi et al. [1] showed that DNA-bound to the outside of cells could be used for 3-dimensional tissue engineering. This technique would allow a vast array of applications where two or more cell types have to communicate with each other to be more finely controlled.

Another application is covering the membrane with functional aptamers, which can be used for targeting specific molecules or diseases [2-5]. Also, Lee et al. showed that DNA can be used to form a hydrogel like material, which has potentially interesting properties when coupled to a cell membrane [6]. All these functionalities have in common that they are almost always synthesized using so called Rolling Circle Amplification. The Eindhoven iGEM 2014 tries to use Rolling Circle Amplification to create a functional coating around the bacterial cell using the Click Coli system and specifically engineered DNA templates.

Rolling Circle Amplification creates a long strand of single strand DNA by “rolling” a circular template along that is used for amplification. Because a circular template is used the strands can, theoretically, be as long as needed. But they will also contain many repeats of the same template. We demonstrated that this can be used to greatly increase the number of binding sites on the bacterial cell membrane.

Bibliography

[1] Gartner, Z. J., & Bertozzi, C. R. (2009). Programmed assembly of 3-dimensional microtissues with defined cellular connectivity. Proceedings of the National Academy of Sciences, 106(12), 4606-4610.

[2] Huang, Y., Cheng, X., Duan, N., Wu, S., Wang, Z., Wei, X., et al. (2015). Selection and characterization of DNA aptamers against Staphylococcus aureus enterotoxin C1. Food Chemistry, 166(1), 623-629.

[3] Cha, T., Cho, S., Kim, Y., & Lee, J. (2014). Rapid aptasensor capable of simply diagnosing prostate cance. Biosensors and Bioelectronics, 62, 31-37.

[4] Chen, H., Hou, Y., Qi, F., Zhang, J., Koh, K., Shen, Z., et al. (2014). Detection of vascular endothelial growth factor based on rolling circle amplification as a means of signal enhancement in surface plasmon resonance. Biosensors and Bioelectronics,61, 83-87.

[5] Hu, R., Zhang, X., Zhao, Z., Zhu, G., Chen, T., Fu, T. and Tan, W. (2014), DNA Nanoflowers for Multiplexed Cellular Imaging and Traceable Targeted Drug Delivery. Angew. Chem. Int. Ed., 53: 5821–5826.

[6] Lee, J. B., Wu, M., Luo, D., Long, R., Chen, L., Rice, E. J., et al. (2012). A mechanical metamaterial made from a DNA hydrogel. Nature Nanotechnology,7(12), 816-820.