Part:BBa_K2686001

Encapsulin protein

This is a BioBrick containing the sequence for Thermotoga maritima encapsulin, a bacterial protein nanocompartment which self assembles to form a 60-mer.

Usage and Biology

Encapsulins are versatile proteins found in a variety of different bacteria (Giessen and Silver, 2017). In the case of this specific part derived from Thermotoga maritima, it can be used among other things to deliver cargo, both on the outer surface of the nanoparticle by fusing a peptide in between the 139/140 Amino Acids or the protein's C terminus as well as using a tag binding to Encapsulin's interior surface (Cassidy-Amstutz et al., 2016).

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 77
  Illegal BglII site found at 441
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal SapI.rc site found at 375
  Illegal SapI.rc site found at 406
  

Characterization

A variety of different characterization techniques were used to assess the properties of the encapsulin protein cage.

Expression & Purification

A cell free expression system was used to synthesize the encapsulin proteins in vitro. The PURE cell free system is a robust way to express proteins (Shimizu et al., 2001), and was used by last year's EPFL iGEM team Aptasense.

After having tested a variety of purification procedures, heat purification at 70C for 20 minutes followed by cooling on ice for 15 minutes and a subsequent centrifugation at 12000g for 10 minutes was found to be the most efficient way of isolating the encapsulin.

Assembly

The self assembly of the encapsulin 60-mer was first examined using SDS PAGE, where a high band is expected to form due to the high molecular weight and size of the 1.3MDa complex.

DLS Measurements

DLS measurement of Encapsulin BBa_K2686001 using a Zetasizer Nano ZS from Malvern Analytical determining the average particle size using volumes. The refractive index chosen for the particles was the "protein" presetting and the refractive index of the medium was approximated to be that of water. This plot shows a peak at 21.037 nm which corresponds to the encapsulin protein cage within the literature (Putri et al., 2017; Moon et al. 2014).

DLS measurement of Encapsulin BBa_K2686001 using a Zetasizer Nano ZS from Malvern Analytical determining the average particle size using signal intensity. The refractive index chosen for the particles was the "protein" presetting and the refractive index of the medium was approximated to be that of water. This plot shows a peak at 32.674 nm

DLS measurement of Encapsulin BBa_K2686001 using a Zetasizer Nano ZS from Malvern Analytical determining the average particle size using the amount of counts. The refractive index chosen for the particles was the "protein" presetting and the refractive index of the medium was approximated to be that of water. This plot shows a peak at 32.674 nm

References

Cassidy-Amstutz, C., Oltrogge, L., Going, C., Lee, A., Teng, P., Quintanilla, D., East-Seletsky, A., Williams, E. and Savage, D. (2016). Identification of a Minimal Peptide Tag for in Vivo and in Vitro Loading of Encapsulin. Biochemistry, 55(24), pp.3461-3468.

Giessen, T. and Silver, P. (2017). Widespread distribution of encapsulin nanocompartments reveals functional diversity. Nature Microbiology, 2, p.17029.

Putri, R., Allende-Ballestero, C., Luque, D., Klem, R., Rousou, K., Liu, A., Traulsen, C., Rurup, W., Koay, M., Castón, J. and Cornelissen, J. (2017). Structural Characterization of Native and Modified Encapsulins as Nanoplatforms for in Vitro Catalysis and Cellular Uptake. ACS Nano, 11(12), pp.12796-12804.

Shimizu, Y., Inoue, A., Tomari, Y., Suzuki, T., Yokogawa, T., Nishikawa, K. and Ueda, T. (2001). Cell-free translation reconstituted with purified components. Nature Biotechnology, 19(8), pp.751-755.