Difference between revisions of "Part:BBa K5115035"
 
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<partinfo>BBa_K5115035 short</partinfo>
 
<partinfo>BBa_K5115035 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 MTA 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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This composite part is composed of MTA 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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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 Metallothioneins (MTA) are intracellular, low molecular, low molecular weight, cysteine-rich proteins. Ubiquitous in eukaryotes, MTA has unique structural characteristics to give potent metal-binding and redox capabilities.<ref>Coyle, P., Philcox, J. C., Carey, L. C., & Rofe, A. M. (2002). Metallothionein: The multipurpose protein. Cellular and Molecular Life Sciences: CMLS, 59(4), 627–647.</ref>
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The Metallothioneins (MTA) are intracellular, low molecular, low molecular weight, cysteine-rich proteins. Ubiquitous in eukaryotes, MTA has unique structural characteristics to give potent metal-binding and redox capabilities<ref>Coyle, P., Philcox, J. C., Carey, L. C., & Rofe, A. M. (2002). Metallothionein: The multipurpose protein. Cellular and Molecular Life Sciences: CMLS, 59(4), 627–647.</ref>.
 
    
 
    
 
===Usage and Biology===
 
===Usage and Biology===
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Get details in [https://parts.igem.org/Part:BBa_K5115050 BBa_K5115050]
 
Get details in [https://parts.igem.org/Part:BBa_K5115050 BBa_K5115050]
  
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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 10:00, 2 October 2024


ribozyme+RBS+MTA+stem-loop

contributed by Fudan iGEM 2024

Introduction

This composite part is composed of MTA 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 Metallothioneins (MTA) are intracellular, low molecular, low molecular weight, cysteine-rich proteins. Ubiquitous in eukaryotes, MTA has unique structural characteristics to give potent metal-binding and redox capabilities[4].

Usage and Biology

The MTA can endowing E.coli with detoxifying capability.

Get details in BBa_K5115050

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 198
  • 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. ↑ Coyle, P., Philcox, J. C., Carey, L. C., & Rofe, A. M. (2002). Metallothionein: The multipurpose protein. Cellular and Molecular Life Sciences: CMLS, 59(4), 627–647.