Part:BBa_K4765119
ribozyme test: constitutive expression
Introduction
This composite part is utilized to assess the cleavage efficiency of chosen ribozymes. It is regulated by the constitutive promoter and terminator. From its upstream to downstream includes stayGold, stem-loop-1, ribozyme, stem-loop-2 and mScarlet.
A ribozyme is proven to have cleavage ability when green and red fluorescence are emitted at the same time. We can assess the cleavage efficiency of ribozyme based on the ratio of red-green fluorescence intensity when the stem-loop is unchanged. We can also assess stem loop’s ability to prevent mRNA degradation based on the ratio of red-green fluorescence intensity when the ribozyme is unchanged.
Usage and Biology
This composite part is an effective tool to select the ribozyme, stem-loop, and RBS. We selected several ribozymes from Chen et al., 2022[1], and Roth et al., 2014[2]. We use this composite part to test the self-cleavage efficiency of them.
chen2022 P1 Twister: 5-AAUGCAGCCGAGGGCGGUUACAAGCCCGCAAAAAUAGCAGAGUA-3 chen2022 HHV: 5-AGACAACCAGGAGUCUAUAAAAUUUACUCUGAAGAGACUGGACGAAACCAAUAGGUCAGUAA-3 roth2014 Sm P1 reversed: 5-GGUUGGGAGGAGGAAAUGGGCCCGAACCCUGGCCGCCGCCUCAAUAACC-3 roth2014 Nvi P1 reversed: 5-GAACGAGAGACGCAAAUAGCCCGAACUCUGGCUGCCGGCGUAAUGUUC-3 roth2014 Nve P1 reversed: 5-GAAAGGGAGACGAAAUAUUCCCGAAC(C)UCUGGAAGCCGUCGUAAUUUUC-3 roth2014 Os2 P1 reversed: 5-AUAUGGGAGGAGGAAAAAGGCCCGAACCCUGGCCGCCGCCUCAAUGUAU-3 roth2014 Cb P1 reversed: 5-AAGGGUGAGACGUAACUAGUCCCGAACACUGGACGCCGACGUAAUCCUU-3 roth2014 esP3: 5-AAGCGGUUACAAGCCCGCAAAAAUAGCAGAGUAAUGUCGCGAUAGCGCGGCAUUAAUGCAGCUU-3
Characterization
Sequencing map
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| Figure1 Linker sequences between the first CDS (stayGold) and the second CDS (mScarlet)
PmeI linker was borrowed from Liu[3] to facilitate cloning, and no specific secondary RNA structure. Stem-loop 1 was used to stabilize the first RNA after ribozyme cleavage, and we tested it function in BBa_K4765129. After the ribozyme Twister, stem-loop 2 functions together with RBS to facilitate translation. T7_RBS BBa_K4162006 is shown, and a stronger RBS BBa_B0030 or a weaker RBS BBa_J61100 if needed |
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 7
Illegal NheI site found at 30
Illegal NotI site found at 1399 - 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI.rc site found at 690
Illegal BsaI.rc site found at 710
Reference
- ↑ Chen, Y., Cheng, Y., & Lin, J. (2022). A scalable system for the fast production of RNA with homogeneous terminal ends. RNA Biology, 19(1), 1077–1084. https://doi.org/10.1080/15476286.2022.2123640
- ↑ Roth, A., Weinberg, Z., Chen, A. G. Y., Kim, P. B., Ames, T. D., & Breaker, R. R. (2014). A widespread self-cleaving ribozyme class is revealed by bioinformatics. Nature Chemical Biology, 10(1), Article 1. https://doi.org/10.1038/nchembio.1386
- ↑ 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. https://doi.org/10.1021/acssynbio.2c00416
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