Difference between revisions of "Part:BBa K2686001"

Revision as of 14:45, 15 October 2018

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.

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 1.3 MDalton complex. Multiple gels were made, some containing the denatured cell free reaction mixture, others containing the same but after a variety of purification procedures.

DLS Measurements

DLS measurement of Encapsulin BBa_K2686001 using a Zetasizer Nano ZS from Malvern Anaytical 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)

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.

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 ====DLS Measurements====
-[[File:EncapsulinVolume.png File:EncapsulinIntensity.png|thumb|center|upright=.5|DLS measurement of Encapsulin [[parts.igem.org/Part:BBa_K2686001|BBa_K2686001]] using a Zetasizer Nano ZS from Malvern Anaytical 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)]]
+[[File:EncapsulinVolume.png|thumb|center|upright=1|DLS measurement of Encapsulin [[parts.igem.org/Part:BBa_K2686001|BBa_K2686001]] using a Zetasizer Nano ZS from Malvern Anaytical 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)]]
 ===References===