Part:BBa_K5115002
EP
Contents
Introduction
The EP sequence encodes an endogenous encapsulation peptide, which plays a crucial role in directing external proteins into bacterial microcompartments like carboxysomes. This targeting mechanism is essential for protein encapsulation within these structures, aiding in the assembly of a functional, proteinaceous shell that sequesters enzymes or other proteins, ensuring efficient catalysis or protection from environmental stress [1].
Usage and Biology
We aim to utilize EP to tether hydrogenase and the StayGold fluorescent protein genes to the α-carboxysome, enabling their co-localization and expression within E. coli. This system will allow for the assembly of a functional nanoreactor, where the carboxysome shell provides a protective microenvironment for the hydrogenase enzyme, enhancing its catalytic activity and stability. The StayGold fluorescent protein will serve as a marker, enabling visualization of the compartmentalization process in vivo.
Characterization
Fluorescence microscopy results
Our experiments validated the role of EP by first fusing it with the StayGold fluorescent protein to assess its effectiveness in directing protein localization to the α-carboxysome. The fluorescence microscopy results confirmed successful incorporation of StayGold into the carboxysome shell, indicating that EP effectively directs proteins into the compartment. Subsequently, we incorporated EP with core assembly proteins of hydrogenase to examine nickel nanoparticle formation. Analysis of nickel particle distribution within E. coli revealed that EP facilitated the assembly of the α-carboxysome shell around the hydrogenase, leading to the formation of well-defined nickel nanoparticles. These results demonstrate that EP not only directs protein localization but also supports the functional assembly of the carboxysome, enhancing the stability and activity of encapsulated enzymes.
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| Figure 4. Fluorescence images of E. coli expressing stayGold fused with EP, without or with cso-S3.
Images were captured using spinning disk confocal with a 150x objective lens, as described on our Experiments page. Bacteria in A-C only express stayGold fused with EP BBa_K5115057, while bacteria in D simultaneously express BBa_K5115057 and BBa_K5115065. 1 mM IPTG was added to A,B only. Images without scale bar are 5x5 µm square, unless specifically indicated below. (A) The entire image field is shown (41.27x41.27 µm square), with brightfield image on the left, and green fluorescence image on the right. (B) Four regions in (A) are enlarged, showing uniform distribution of green fluorescence. (C) Although no IPTG was added, leaky expression from the promoter is sufficient to fill bacteria with green. (D) With all carboxysome components expressed, stayGold fused with EP concentrated to the carboxysome. Leaky expression from the promoter is sufficient to drive 1 or 2 carboxysome formed within each bacteria. |
Sequence and Features
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 646
- 1000COMPATIBLE WITH RFC[1000]
References
- ↑ Li, T., Jiang, Q., Huang, J., Aitchison, C. M., Huang, F., Yang, M., Dykes, G. F., He, H. L., Wang, Q., Sprick, R. S., Cooper, A. I., & Liu, L. N. (2020). Reprogramming bacterial protein organelles as a nanoreactor for hydrogen production. Nature communications, 11(1), 5448.
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