Group: UM_Macau 2020
Author: Leong Chan Tat, Shuyao Xie, Yuzhao Feng
Summary: We introduced a new part iBox-PAK4 to improve the part ferritin from UM_Macau team 2019, enhancing the magnetization capability of the FtnA protein.
Inspired by the magnetization and control system from UM_Macau team 2019, we designed our bacteria cleaning system for aquariums and make an improvement on their part. They overexpressed ferritin in E. coli to enhance bacterial iron storage capabilities, which allow them to control E. coli movement via magnetization.
However, the magnetization capability of Ferritin is poorly limited. We have found literature which proves that tagging this iBox-PAK4 would be enhancing the magnetization capability of the FtnA protein. Ferritin is a ubiquitous iron storage protein. Thus, a larger protein assembly that contains more mineralized iron will generate larger response to magnetic forces.
Inka-PAK4, was originally described by Baskaran et. Al, spontaneously forms needle-like crystals when expressed in mammalian cells.
In this literature, researchers found that inkabox with PAK4cat undergoes conformational changes which allows the spontaneous crystallization of the complex, producing long rod-shaped crystals with a unit cell that has a hexagonal arrangement of subunits around a hollow channel (Fig. 1). Engineered ferritin-containing protein crystals, which named ftn-PAK4, exert magnetic forces that are 9 orders of magnitude larger than those in previous reports.
Figure 1: Schematic of how ferritin subunits fit inside of the crystals’ hollow channel
For bacterial expression, Robert C. Robinson has used pGEX4T1 (GE), pET28a (Novagen) and pSY5 (His-tagged) as expression vectors for Inka1 and PAK4. Furthermore, the inka-PAK4 and GFP-inka-PAK4 plasmids have been transfected into HEK293T cells and have produced protein crystals successfully.
Therefore, we linked this iBox-PAK4 encoding gene downstream of FtnA in our construct (Fig. 2). This hopefully enables our BREAC to better respond to magnetic forces applied to it and helps with their collection.
Figure 2: Optimized construct with iBox-PAK4
- Thomas, L, Li., Zegao, W., He, Y., Qun, X, O., Varanasi, V, J., Ming, D, D., Bai L., Paşca, Sergiu, P., and Bian, X, C., (2019). Engineering a Genetically Encoded Magnetic Protein Crystal https://pubs.acs.org/doi/10.1021/acs.nanolett.9b02266
- Matsumoto, Y., Chen, R., Anikeeva, P., Jasanoff, A., (2015). Engineering intracellular biomineralization and biosensing by a magnetic protein https://www.nature.com/articles/ncomms9721
- Baskaran, Y., Ang, K, C., Anekal, P, V., W, L, Chan, W, L., Grimes, J, M., Manser, E., Robinson, R, C. (2015). An in cellulo-derived structure of PAK4 in complex with its inhibitor Inka1 https://www.nature.com/articles/ncomms9681