Difference between revisions of "Part:BBa K2686002"
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− | == | + | ==Characterization== |
The construct was tested inside a pet14 vector under a T7 promoter and a T7 terminator. | The construct was tested inside a pet14 vector under a T7 promoter and a T7 terminator. | ||
+ | |||
+ | ===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 [[2017.igem.org/Team:EPFL/Description/Lysates| 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=== | ===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. | 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. | ||
− | |||
[[File:Encapsulins SDS.png|thumb|center|upright=3|SDS PAGE of the different encapsulin proteins expressed by iGEM EPFL 2018. | [[File:Encapsulins SDS.png|thumb|center|upright=3|SDS PAGE of the different encapsulin proteins expressed by iGEM EPFL 2018. | ||
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===References=== | ===References=== | ||
Moon, H., Lee, J., Min, J. and Kang, S. (2014). Developing Genetically Engineered Encapsulin Protein Cage Nanoparticles as a Targeted Delivery Nanoplatform. Biomacromolecules, 15(10), pp.3794-3801. | Moon, H., Lee, J., Min, J. and Kang, S. (2014). Developing Genetically Engineered Encapsulin Protein Cage Nanoparticles as a Targeted Delivery Nanoplatform. Biomacromolecules, 15(10), pp.3794-3801. | ||
+ | |||
+ | 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. |
Revision as of 21:32, 15 October 2018
Encapsulin protein with HexaHistidine insert
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
The part can be used to deliver cargo, both on the outer surface of the nanoparticle by fusing a peptide in between the 139/140 Amino Acids as well as the protein's C terminus. Cargo proteins can also be loaded inside the nano-cage using a tag binding to Encapsulin's interior surface. The protein is modified with an additonal amino acid sequence (GGGGGGHHHHHHGGGGG) between positions 43/44 granting it better stability and high heat resistance (Moon et al., 2014).
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 77
Illegal BglII site found at 492 - 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal SapI.rc site found at 426
Illegal SapI.rc site found at 457
Characterization
The construct was tested inside a pet14 vector under a T7 promoter and a T7 terminator.
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.
![](/wiki/images/4/4e/Encapsulins_SDS.png)
References
Moon, H., Lee, J., Min, J. and Kang, S. (2014). Developing Genetically Engineered Encapsulin Protein Cage Nanoparticles as a Targeted Delivery Nanoplatform. Biomacromolecules, 15(10), pp.3794-3801.
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.