Part:BBa_K5115019
ribozyme+RBS+hypF+stem-loop
Introduction
This composite part is composed of hypF 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 hypF is a hydrogenase subunit which assists in the assembly of the hydrogenase complex by stabilizing its structure during maturation[4].
Usage and Biology
The hypF can help with the overall function of Ni-Fe hydrogenase.
Get details in BBa_K5115063.
Sequence and Features
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.
- ↑ Anne K. Jones, Oliver Lenz, Angelika Strack, Thorsten Buhrke, and, & Friedrich*, B. (2004, October 2). NiFe Hydrogenase Active Site Biosynthesis: Identification of Hyp Protein Complexes in Ralstonia eutropha† (world) [Research-article]. ACS Publications; American Chemical Society.
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