Difference between revisions of "Part:BBa K5115014"
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===Usage and Biology===
 
===Usage and Biology===
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The heterologously expressed codon-optimized hoxH can join in the overall function of Ni-Fe hydrogenase.
+
The hoxH can join in 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 08:26, 1 October 2024

ribozyme+RBS+hoxH+stem-loop

contributed by Fudan iGEM 2023

Introduction

This composite part is composed of hoxH 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 hoxH is the most central component of our hydrogenase. On its surface, oxidation and reduction of hydrogen gas happens alternately according to different oxygen status[4].

Usage and Biology

The hoxH can join in 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
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 371
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 1491


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. ↑ Chan, K.-H., Lee, K.-M., & Wong, K.-B. (2012). Interaction between Hydrogenase Maturation Factors HypA and HypB Is Required for [NiFe]-Hydrogenase Maturation. PLOS ONE, 7(2), e32592.