Difference between revisions of "Part:BBa K5115019"
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===Introduction=== | ===Introduction=== | ||
| − | This composite part is composed of | + | 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. |
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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 hoxF is a hydrogenase subunit responsible for electron transport. Under anaerobic conditions, NADH is oxidized to NAD+ on the surface of hoxF subunit<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 heterologously expressed codon-optimized MTA can endowing E.coli with detoxifying capability. |
| + | |||
| + | Get details in [https://parts.igem.org/Part:BBa_K5115050 BBa_K5115050] | ||
Revision as of 07:08, 1 October 2024
ribozyme+RBS+hypF+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 hoxF is a hydrogenase subunit responsible for electron transport. Under anaerobic conditions, NADH is oxidized to NAD+ on the surface of hoxF subunit[4].
Usage and Biology
The heterologously expressed codon-optimized MTA can endowing E.coli with detoxifying capability.
Get details in BBa_K5115050
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal XhoI site found at 499
Illegal XhoI site found at 2338 - 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 104
Illegal NgoMIV site found at 325
Illegal NgoMIV site found at 349
Illegal NgoMIV site found at 710
Illegal NgoMIV site found at 2110
Illegal NgoMIV site found at 2510 - 1000COMPATIBLE WITH RFC[1000]
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.