Part:BBa_K4765119

ribozyme test: constitutive expression

contributed by Fudan iGEM 2023

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

Figure 1. 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

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
INCOMPATIBLE WITH RFC[12]
Illegal NheI site found at 7
Illegal NheI site found at 30
Illegal NotI site found at 1399
21
COMPATIBLE WITH RFC[21]
23
COMPATIBLE WITH RFC[23]
25
COMPATIBLE WITH RFC[25]
1000
INCOMPATIBLE 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

[1] 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

[2] 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

[3] 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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