Difference between revisions of "Part:BBa K5115014"
 
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<partinfo>BBa_K5115014 short</partinfo>
 
<partinfo>BBa_K5115014 short</partinfo>
  
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<html><img style="float:right;width:128px" src="https://static.igem.wiki/teams/5115/czh/mineral-logo.svg" alt="contributed by Fudan iGEM 2023"></html>
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<html><img style="float:right;width:128px" src="https://static.igem.wiki/teams/5115/czh/mineral-logo.svg" alt="contributed by Fudan iGEM 2024"></html>
 
__TOC__
 
__TOC__
 
===Introduction===
 
===Introduction===
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This composite part is composed of hoxH and improved ribozyme-assisted polycistronic co-expression system: pRAP. The hoH is a subunit of the hydrogenase made up by hox and hyp, get details about hoxH 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 hoxH 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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===Usage and Biology===
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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 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 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<ref>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.</ref>.
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===Usage and Biology===
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The hoxH can join in the overall function of Ni-Fe hydrogenase.
  
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Get details in [https://parts.igem.org/Part:BBa_K5115063 BBa_K5115063].
  
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===Sequence and Features===
 
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<span class='h3bb'>Sequence and Features</span>
 
<span class='h3bb'>Sequence and Features</span>

Latest revision as of 09:08, 2 October 2024

ribozyme+RBS+hoxH+stem-loop

contributed by Fudan iGEM 2024

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

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.