Difference between revisions of "Part:BBa K4765120"

Revision as of 15:32, 12 October 2023

ribozyme test: T7 leaky expression

Introduction

This composite part is utilized to assess the cleavage efficiency of chosen ribozymes. From its upstream to downstream includes stayGold, stem-loop-1, ribozyme, stem-loop-2, and mScarlet. It is regulated by T7 promoter,lac operator, and T7 terminator.

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 the stem-loop’s ability to prevent mRNA degradation based on the ratio of red-green fluorescence intensity when the ribozyme is unchanged.

Furthermore, the original T7_RBS can be replaced by other RBSs. We can test the strength of one RBS based on the ratio of red-green fluorescence intensity.

Usage and Biology

This composite part is an effective tool to select the ribozyme, stem-loop, and RBS. We selected several ribozymes 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

Different from BBa_K4765119, leaky expression of T7 promoter was used. We found the RFP expression level is higher then BBa_K4765119, which is better for our functional characterization of various Parts.

Characterization

Sequencing map

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 its 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

Reference

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