Composite

Part:BBa_K5115061

Designed by: Yi Shi   Group: iGEM24_Fudan   (2024-09-19)


ribozyme+RBS+hoxF-GS-EP+stem-loop

contributed by Fudan iGEM 2023

Introduction

This composite part is composed of hoxF-GS-EP coding sequence (CDS), wrapped by ribozyme-assisted polycistronic co-expression system (pRAP) sequences. By inserting BBa_K4765020 before CDS, the RNA of Twister ribozyme conduct self-cleaving in the mRNA.[1] To protect the mono-cistron mRNA from degradation, a stem-loop structure is placed at the 3' end of CDS.[2] In 2023, we extensively tested various stem-loops using BBa_K4765129. For parts we made this year, this strong protective stem-loop sequence was used.

As for the ribosome binding sequence (RBS) after the ribozyme and before the CDS, we used T7 RBS, from bacteriophage T7 gene 10.[3] It is an intermediate strength RBS according to our 2022 results, which allows us to change it to a weaker J6 RBS or a stronger B0 RBS if needed, enabling flexible protein expression levels between various ribozyme connected parts.

The CDS sequence include hoxF, EP and a linker of GS. The hoxF is a hydrogenase subunit responsible for electron transport. Under anaerobic conditions, NADH is oxidized to NAD+ on the surface of hoxF subunit[4]. The EP sequence encodes an endogenous encapsulation peptide, which plays a crucial role in directing external proteins into bacterial microcompartments like carboxysomes[5]. Linking EP with hoxF, we hope that EP can import the hydrogenase into the carboxysome, creating a stable environment for the enzyme to work.

Usage and Biology

This part can import the hydrogenase into the carboxysome, creating a stable environment for the enzyme to work.

Get details in BBa_K5115067.


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 654
    Illegal BglII site found at 1472
    Illegal BglII site found at 1765
    Illegal XhoI site found at 99
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 533
    Illegal NgoMIV site found at 1071
    Illegal NgoMIV site found at 1281
    Illegal NgoMIV site found at 1593
    Illegal AgeI site found at 2548
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 702
    Illegal BsaI.rc site found at 1206


References

  1. ↑ Eiler, D., Wang, J., & Steitz, T. A. (2014). Structural basis for the fast self-cleavage reaction catalyzed by the twister ribozyme. Proceedings of the National Academy of Sciences, 111(36), 13028–13033.
  2. ↑ Liu, Y., Wu, Z., Wu, D., Gao, N., & Lin, J. (2022). Reconstitution of Multi-Protein Complexes through Ribozyme-Assisted Polycistronic Co-Expression. ACS Synthetic Biology, 12(1), 136–143.
  3. ↑ The T7 phage gene 10 leader RNA, a ribosome-binding site that dramatically enhances the expression of foreign genes in Escherichia coli. Olins PO, Devine CS, Rangwala SH, Kavka KS. Gene, 1988 Dec 15;73(1):227-35.
  4. ↑ Löscher, S., Burgdorf, T., Zebger, I., Hildebrandt, P., Dau, H., Friedrich, B., & Haumann, M. (2006). Bias from H2 Cleavage to Production and Coordination Changes at the Ni−Fe Active Site in the NAD+-Reducing Hydrogenase from Ralstonia eutropha. Biochemistry, 45(38), 11658–11665.
  5. ↑ 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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