Difference between revisions of "Part:BBa K4765129"

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This year we further improved the ribozyme-assisted polycistronic co-expression system (pRAP).
 
This year we further improved the ribozyme-assisted polycistronic co-expression system (pRAP).
  
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The major problem of polycistronic vectors, which contain two or more target genes under one promoter, is the much lower expression of the downstream genes compared with that of the first gene next to the promoter<ref>Kim, K.-J., Kim, H.-E., Lee, K.-H., Han, W., Yi, M.-J., Jeong, J., & Oh, B.-H. (2004). Two-promoter vector is highly efficient for overproduction of protein complexes. Protein Science: A Publication of the Protein Society, 13(6), 1698–1703. https://doi.org/10.1110/ps.04644504</ref> Compared with multiple promoter system, in pRAP, self-interaction of the polycistron can be avoid and each cistron can initiate translation with comparable efficiency. In pRAP system , the RNA sequences of ribozyme conduct self-cleaving, and the polycistronic mRNA transcript is thus co-transcriptionally converted into individual mono-cistrons in vivo.
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The major problem of polycistronic vectors, which contain two or more target genes under one promoter, is the much lower expression of the downstream genes compared with that of the first gene next to the promoter<ref>Kim, K.-J., Kim, H.-E., Lee, K.-H., Han, W., Yi, M.-J., Jeong, J., & Oh, B.-H. (2004). Two-promoter vector is highly efficient for overproduction of protein complexes. Protein Science: A Publication of the Protein Society, 13(6), 1698–1703. https://doi.org/10.1110/ps.04644504</ref>. Compared with multiple promoter system, in pRAP, self-interaction of the polycistron can be avoid and each cistron can initiate translation with comparable efficiency. In pRAP system , the RNA sequences of ribozyme conduct self-cleaving, and the polycistronic mRNA transcript is thus co-transcriptionally converted into individual mono-cistrons ''in vivo''.
  
 
Stem-loop is a key regulatory element in pRAP, it affects protein concentration by regulating the rate of mRNA degradation. By regulating the intensity of stem-loop, we can control the expression of proteins.  
 
Stem-loop is a key regulatory element in pRAP, it affects protein concentration by regulating the rate of mRNA degradation. By regulating the intensity of stem-loop, we can control the expression of proteins.  

Revision as of 08:26, 12 October 2023


stem-loop test contributed by Fudan iGEM 2023


Introduction

This year we further improved the ribozyme-assisted polycistronic co-expression system (pRAP).

The major problem of polycistronic vectors, which contain two or more target genes under one promoter, is the much lower expression of the downstream genes compared with that of the first gene next to the promoter[1]. Compared with multiple promoter system, in pRAP, self-interaction of the polycistron can be avoid and each cistron can initiate translation with comparable efficiency. In pRAP system , the RNA sequences of ribozyme conduct self-cleaving, and the polycistronic mRNA transcript is thus co-transcriptionally converted into individual mono-cistrons in vivo.

Stem-loop is a key regulatory element in pRAP, it affects protein concentration by regulating the rate of mRNA degradation. By regulating the intensity of stem-loop, we can control the expression of proteins.

This compoiste part is a stem-loop-deleted version of BBa_K4765120, which includes stayGold and mScarlet. Its red-green fluorescence intensity ratio can be compared with BBa_K4765120 to assess the stem-loop's ability to prevent mRNA degradation.

Usage and Biology

We use this composite part to test the following stem-loops'[2] ability to prevent mRNA degradation. 

nsl:     5-AAACACCCACCACAAUUUCCACCGUUU UUUGU-3
liu2023: 5-AAACACCCACCACAAUUUCCACCGUUU CCCGACGCUUCGGCGUCGGG UUUGU-3
new2:    5-AAACACCCACCACAAUUUCCACCGUUU CCCCGUCGGCUGCU UUUGU-3
new6:    5-AAACACCCACCACAAUUUCCACCGUUU AGACGCUCGGCGUCCU UUUGU-3
new10:   5-AAACACCCACCACAAUUUCCACCGUUU ACUGGGGGGAUCGAGGUCUUU UUUGU-3
old2:    5-AAACACCCACCACAAUUUCCACCGUUU GCCGAUCGGGU UUUGU-3
old6:    5-AAACACCCACCACAAUUUCCACCGUUU AGACGCUCGGCGUCCU UUUGU-3
old10:   5-AAACACCCACCACAAUUUCCACCGUUU GGCGGCGCUACAGCGUCGU UUUGU-3

Characterization

Sequencing map

contributed by Fudan iGEM 2023
Figure 1. Sequencing result of nsl (no stem-loop before Twister ribozyme cleavage site).
Sanger sequencing verified that we have removed the stem-loop before ribozyme sequence, from BBa_K4765120. We also construct plasmids with stem-loop new2, new6, new10, bad2, bad6, bad10, all of which were designed by our Software RAP, and fully characterized using functional assays.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NotI site found at 1389
  • 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. ↑ Kim, K.-J., Kim, H.-E., Lee, K.-H., Han, W., Yi, M.-J., Jeong, J., & Oh, B.-H. (2004). Two-promoter vector is highly efficient for overproduction of protein complexes. Protein Science: A Publication of the Protein Society, 13(6), 1698–1703. https://doi.org/10.1110/ps.04644504
  2. ↑ 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