Difference between revisions of "Part:BBa K5115015"
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__TOC__
 
__TOC__
 
===Introduction===
 
===Introduction===
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This composite part is composed of hoxW and improved ribozyme-assisted polycistronic co-expression system: pRAP. The hoxW is a subunit of the hydrogenase made up by hox and hyp, get details about hoxW in  [https://parts.igem.org/Part:BBa_K5115020 BBa_K5115020(hox and hyp operon)]. This composite part will finally be used in [https://parts.igem.org/Part:BBa_K5115066 BBa_K5115066(mineral, U module)]and [https://parts.igem.org/Part:BBa_K5115067 BBa_K5115067(mineral, F module)]for further experiment.
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This composite part is composed of hoxW coding sequence (CDS), wrapped by ribozyme-assisted polycistronic co-expression system (pRAP) sequences. By inserting [https://parts.igem.org/Part:BBa_K4765020 BBa_K4765020] before CDS, the RNA of Twister ribozyme conduct self-cleaving in the mRNA.<ref>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.</ref> To protect the mono-cistron mRNA from degradation, a stem-loop structure is placed at the 3' end of CDS.<ref>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.</ref> In 2023, we extensively tested various [https://2023.igem.wiki/fudan/part-collection/#ribozyme-assisted-polycistronic-co-expression stem-loops] using [https://parts.igem.org/Part:BBa_K4765129 BBa_K4765129]. For parts we made this year, this strong protective stem-loop sequence was used.
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As for the ribosome binding sequence (RBS) after the ribozyme and before the CDS, we used [https://parts.igem.org/Part:BBa_K4162006 T7 RBS], from bacteriophage T7 gene 10.<ref>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.</ref> It is an intermediate strength RBS according to [https://2022.igem.wiki/fudan/measurement#optimization our 2022 results], which allows us to change it to a weaker [https://parts.igem.org/Part:BBa_J61100 J6 RBS] or a stronger [https://parts.igem.org/Part:BBa_B0030 B0 RBS] if needed, enabling flexible protein expression levels between various ribozyme connected parts.
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The hoxW is a hydrogenase subunit which assists the enzyme's function and stability.
  
 
===Usage and Biology===
 
===Usage and Biology===
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The ribozyme-assisted polycistronic co-expression system can ensure that each cistron can initiate translation with comparable efficiency. For more information, please check [https://2022.igem.wiki/fudan/parts part wiki of 2022 Fudan iGEM].
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The heterologously expressed codon-optimized hoxW can help with the overall function of Ni-Fe hydrogenase.
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Get details in [https://parts.igem.org/Part:BBa_K5115063 BBa_K5115063].
  
  

Revision as of 07:51, 1 October 2024


ribozyme+RBS+hoxW+stem-loop

contributed by Fudan iGEM 2023

Introduction

This composite part is composed of hoxW 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 hoxW is a hydrogenase subunit which assists the enzyme's function and stability.

Usage and Biology

The heterologously expressed codon-optimized hoxW 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 259
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 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.