Difference between revisions of "Part:BBa K5115002"
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===Usage and Biology===
 
===Usage and Biology===
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We performed codon optimization specifically for the ''Escherichia coli'' K12 strain, resulting in the creation of this part. The Hpn protein binds absorbed nickel ions, enhancing ''E. coli'''s tolerance to elevated nickel concentrations within ''E. coli''.
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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===
 
===Characterization===
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Our experiments demonstrated that expressing Hpn in ''E. coli'' significantly enhanced the bacterium's ability to absorb nickel ions. We hypothesize that this increased absorption is due to Hpn’s strong nickel-binding capacity. However, for optimal nickel uptake, other factors, such as the availability of additional nickel transporters or regulatory proteins, may also be necessary.
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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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Revision as of 09:34, 15 September 2024


EP

contributed by Fudan iGEM 2023

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].

contributed by Fudan iGEM 2023
Figure 1. The biosynthetic pathway of shinorine, porphyra-334, palythine-Ser, and palythine-Thr

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

Nickel tolerance Assay

We.

contributed by Fudan iGEM 2023
Figure 2. Anti-UV Assay.

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.

contributed by Fudan iGEM 2023
Figure 3. Plates displaying transformed E. coli after anti-UV assay.


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 646
  • 1000
    COMPATIBLE WITH RFC[1000]


References

  1. ↑ 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.