Difference between revisions of "Part:BBa K4765120"

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===Introduction===
 
===Introduction===
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This composite part is utilized to assess the cleavage efficiency of Twister P1， stem-loop’s ability of preventing mRNA degradation and the strength of different ribosome binding site (RBS). It is regulated by T7 promoter ,lac operator and T7 terminator. From its upstream to understream includes StayGold, stem-loop, Twister P1 and mScarlet.
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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.
  
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A ribozyme is proved 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.  
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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.  
  
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We can also assess stem-loop’s ability of preventing mRNA degradation based on the ratio of red-green fluorescence intensity when the ribozyme is unchanged.
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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.
  
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Furthermore, the orginal T7_RBS can be replaced by other RBSs. We can test the strength of one RBS based on ratio of red-green fluorescence intensity.
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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===
 
===Usage and Biology===
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This composite part is an easy and effective tool to select the fit ribozyme, stem-loop and RBS.
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This composite part is an effective tool to select the ribozyme, stem-loop, and RBS.
 +
We selected several ribozymes Chen et al., 2022<ref>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</ref>, and Roth et al., 2014<ref>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</ref>. We use this composite part to test the self-cleavage efficiency of them:
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<pre>chen2022 P1 Twister:      5-AAUGCAGCCGAGGGCGGUUACAAGCCCGCAAAAAUAGCAGAGUA-3
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chen2022 HHV:              5-AGACAACCAGGAGUCUAUAAAAUUUACUCUGAAGAGACUGGACGAAACCAAUAGGUCAGUAA-3
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roth2014 Sm P1 reversed:  5-GGUUGGGAGGAGGAAAUGGGCCCGAACCCUGGCCGCCGCCUCAAUAACC-3
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roth2014 Nvi P1 reversed:  5-GAACGAGAGACGCAAAUAGCCCGAACUCUGGCUGCCGGCGUAAUGUUC-3
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roth2014 Nve P1 reversed:  5-GAAAGGGAGACGAAAUAUUCCCGAAC(C)UCUGGAAGCCGUCGUAAUUUUC-3
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roth2014 Os2 P1 reversed:  5-AUAUGGGAGGAGGAAAAAGGCCCGAACCCUGGCCGCCGCCUCAAUGUAU-3
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roth2014 Cb P1 reversed:  5-AAGGGUGAGACGUAACUAGUCCCGAACACUGGACGCCGACGUAAUCCUU-3
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roth2014 esP3:            5-AAGCGGUUACAAGCCCGCAAAAAUAGCAGAGUAAUGUCGCGAUAGCGCGGCAUUAAUGCAGCUU-3</pre>
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Different from [https://parts.igem.org/Part:BBa_K4765119 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===
 
===Characterization===
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| '''Figure1 Linker sequences between the first CDS (stayGold) and the second CDS (mScarlet)'''  
 
| '''Figure1 Linker sequences between the first CDS (stayGold) and the second CDS (mScarlet)'''  
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PmeI linker was borrowed from Liu<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. https://doi.org/10.1021/acssynbio.2c00416</ref> to facilitate cloning, and no specific secondary RNA structure. Stem-loop 1 was used to stabilize the first RNA after ribozyme cleavage, and we test it function in [https://parts.igem.org/Part:BBa_K4765129 BBa_K4765129]. After the ribozyme Twister<ref>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</ref>, stem-loop 2 function together with RBS to facilitate translation. T7_RBS [https://parts.igem.org/Part:BBa_K4162006 BBa_K4162006] is shown, and a stronger RBS [https://parts.igem.org/Part:BBa_B0030 BBa_B0030] or a weaker RBS [https://parts.igem.org/Part:BBa_J61100 BBa_J61100] if needed
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PmeI linker was borrowed from Liu<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. https://doi.org/10.1021/acssynbio.2c00416</ref> 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 [https://parts.igem.org/Part:BBa_K4765129 BBa_K4765129]. After the ribozyme Twister, stem-loop 2 functions together with RBS to facilitate translation. T7_RBS [https://parts.igem.org/Part:BBa_K4162006 BBa_K4162006] is shown, and a stronger RBS [https://parts.igem.org/Part:BBa_B0030 BBa_B0030] or a weaker RBS [https://parts.igem.org/Part:BBa_J61100 BBa_J61100] if needed
 
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|}
  
 
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<span class='h3bb'>Sequence and Features</span>
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===Sequence and Features===
 
<partinfo>BBa_K4765120 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K4765120 SequenceAndFeatures</partinfo>
  
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<partinfo>BBa_K4765120 parameters</partinfo>
 
<partinfo>BBa_K4765120 parameters</partinfo>
 
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===Reference===

Latest revision as of 15:36, 12 October 2023


ribozyme test: T7 leaky expression

contributed by Fudan iGEM 2023

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

contributed by Fudan iGEM 2023
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


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NotI site found at 1409
  • 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 700
    Illegal BsaI.rc site found at 720


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