Composite

Part:BBa_K4245001

Designed by: Manasvi Gupta, Natalie Tan, Vandana Vijay Kumar, Christina Cho, Nick Lockhart, Hari Mudigonda, Janet Standeven   Group: iGEM22_Lambert_GA   (2022-08-17)
Revision as of 13:33, 6 October 2022 by NickL (Talk | contribs)


Spinach aptamer with LacI repression

This part produces the fluorescent RNA aptamer Spinach (BBa_K734002) under the control of IPTG. Originally generated by the Jaffrey Lab at Cornell University, the Spinach aptamer is a fluorescent light-up aptamers (FLAP) that binds to 3’5-difluoro-4-hydroxybenzylidene imidazolinone (DFHBI), a small dye derived from the GFP fluorophore, to produce fluorescence (469 nm/501 nm) (Paige et al., 2011). As shown in Figure 1, the aptamer and DFHBI bind together to produce green fluorescence, which has roughly 50% of the fluorescence intensity of enhanced GFP (Neubacher & Hennig, 2018). However, FLAPs can be more effective than GFP in biosensing as they bind to a fluorophore after transcription (RNA), while GFP requires additional translation for expression. Similar to other FLAPs, Spinach is expressed within a transfer RNA (tRNA) scaffold, which shields the RNA from misfolding and degradation (Paige et al., 2011).


Figure 1. DFHBI and Spinach aptamer binding to form RNA-fluorophore complex.

The LacI protein represses the inducible promoter (BBa_R0010), which stops downstream transcription of the Spinach aptamer. When IPTG is present, LacI is inhibited, allowing for the transcription of the aptamer. Once DFHBI binds to the aptamer, the RNA-fluorophore complex produces a quantifiable green fluorescence.

This part works identically to BBa_K4245002, a composite part producing iSpinach, and BBa_K4245003, a composite part producing iSpinach-D5-G30-A32; the only difference being the aptamer produced. This disparity allows accurate comparison of the basic aptamer parts. hello hello


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 361
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


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Categories
Parameters
None