DNA

Part:BBa_K4765129

Designed by: Siliang Zhan   Group: iGEM23_Fudan   (2023-10-02)
Revision as of 23:57, 10 October 2023 by Wave (Talk | contribs) (→‎Sequencing map)


stem-loop test contributed by Fudan iGEM 2023


Introduction

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'[1] 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
bad2:    5-AAACACCCACCACAAUUUCCACCGUUU GCCGAUCGGGU UUUGU-3
bad6:    5-AAACACCCACCACAAUUUCCACCGUUU AGACGCUCGGCGUCCU UUUGU-3
bad10:   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. ↑ 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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