Reporter

Part:BBa_K3147000:Design

Designed by: Thomas Bessede   Group: iGEM19_Montpellier   (2019-10-12)

sfGFP fuse to TEV-cleavable ssrA tag


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 41
    Illegal NheI site found at 53
    Illegal NheI site found at 76
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 82
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


Design Note

The TEV cutting site used is the classic recognition site, composed of 6 amino acids: ENLYFQS.

We placed the ssrA in C-ter because it is well known to be working on this side of the protein. [5]

The construction has a RiboJ insulator Part:BBa_ K1679038 and a BCD2 Part:BBa_ J364100.

RiboJ : After transcription, RiboJ becomes a section of functional RNA which comprises the sTRSV- ribozyme and an additional 23-nt hairpin immediately downstream to help expose the RBS. STRSV can cleaves the mRNA autocatalytically at a defined residue, which had the effect of cleaving extraneous RNA leaders that arose from transcription from promoters with different start sites.[6] Attributed to the biochemical function mentioned above, RiboJ has insulating capability. We use it to buffer synthetic circuits from genetic context.

BCD2 : This is one of the bicistronic design elements designed by Mutalik et al. in the 2013 Nature publication [7] (citation below). Specifically, this is bicistronic design element number two (or BCD2, for short).

This sequence was synthesized by IDT DNA for iGEM Headquarters.

This share is strongly expressed.

Reference

[5] Sunohara, T., Abo, T., Inada, T., & Aiba, H. (2002). The C-terminal amino acid sequence of nascent peptide is a major determinant of SsrA tagging at all three stop codons. RNA (New York, N.Y.), 8(11), 1416–1427. doi:10.1017/s1355838202020198

[6] Lou C, Stanton B, Chen Y J, et al. Ribozyme-based insulator parts buffer synthetic circuits from genetic context[J]. Nature biotechnology, 2012, 30(11): 1137-1142.

[7]Mutalik, V. K. et al. "Precise and reliable gene expression via standard transcription and translation initiation elements." Nature Methods 10, 354–360 (2013).