Difference between revisions of "Part:BBa K5115018"
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===Usage and Biology===
 
===Usage and Biology===
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The heterologously expressed codon-optimized hypB can help with the overall function of Ni-Fe hydrogenase.
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The hypB can help with the overall function of Ni-Fe hydrogenase.
  
 
Get details in [https://parts.igem.org/Part:BBa_K5115063 BBa_K5115063].
 
Get details in [https://parts.igem.org/Part:BBa_K5115063 BBa_K5115063].

Revision as of 08:28, 1 October 2024


ribozyme+RBS+hypB+stem-loop

contributed by Fudan iGEM 2023

Introduction

This composite part is composed of hypB 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 hypB is a hydrogenase subunit which cooperates with hypA to precisely guide and insert the nickel ions into the hydrogenase catalytic center[4].

Usage and Biology

The hypB can help with the overall function of Ni-Fe hydrogenase.

Get details in BBa_K5115063.


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal XhoI site found at 411
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 100
    Illegal NgoMIV site found at 946
    Illegal AgeI site found at 898
  • 1000
    COMPATIBLE WITH RFC[1000]


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. ↑ Anne K. Jones, Oliver Lenz, Angelika Strack, Thorsten Buhrke, and, & Friedrich*, B. (2004, October 2). NiFe Hydrogenase Active Site Biosynthesis: Identification of Hyp Protein Complexes in Ralstonia eutropha† (world) [Research-article]. ACS Publications; American Chemical Society.