Difference between revisions of "Part:BBa K5115012"
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<partinfo>BBa_K5115012 short</partinfo> | <partinfo>BBa_K5115012 short</partinfo> | ||
| − | <html><img style="float:right;width:128px" src="https://static.igem.wiki/teams/5115/czh/mineral-logo.svg" alt="contributed by Fudan iGEM | + | <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=== | ||
| − | This composite part is composed of hoxF 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 | + | This composite part is composed of hoxF 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. |
| − | 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 | + | 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. |
| − | The hoxU is a hydrogenase subunit, conducting electrons between hoxH and hoxF | + | The hoxU is a hydrogenase subunit, conducting electrons between hoxH and hoxF <ref>Löscher, S., Burgdorf, T., Zebger, I., Hildebrandt, P., Dau, H., Friedrich, B., & Haumann, M. (2006). Bias from H2 Cleavage to Production and Coordination Changes at the Ni−Fe Active Site in the NAD+-Reducing Hydrogenase from Ralstonia eutropha. Biochemistry, 45(38), 11658–11665.</ref>. |
===Usage and Biology=== | ===Usage and Biology=== | ||
| − | The | + | The hoxU can join in the overall function of Ni-Fe hydrogenase. |
| − | + | Get details in [https://parts.igem.org/Part:BBa_K5115063 BBa_K5115063]. | |
| + | ===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 08:58, 2 October 2024
ribozyme+RBS+hoxU+stem-loop
Introduction
This composite part is composed of hoxF 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 hoxU is a hydrogenase subunit, conducting electrons between hoxH and hoxF [4].
Usage and Biology
The hoxU can join in the overall function of Ni-Fe hydrogenase.
Get details in BBa_K5115063.
Sequence and Features
Sequence and Features
Assembly Compatibility:
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 485
Illegal BglII site found at 563
Illegal XhoI site found at 493
Illegal XhoI site found at 685 - 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 446
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI site found at 267
Illegal BsaI site found at 429
Illegal SapI.rc site found at 378
References
- ↑ 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.
- ↑ 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.
- ↑ 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.
- ↑ Löscher, S., Burgdorf, T., Zebger, I., Hildebrandt, P., Dau, H., Friedrich, B., & Haumann, M. (2006). Bias from H2 Cleavage to Production and Coordination Changes at the Ni−Fe Active Site in the NAD+-Reducing Hydrogenase from Ralstonia eutropha. Biochemistry, 45(38), 11658–11665.