Part:BBa_K4245001
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
Figure 3. Diagram of the BBa_K4245001 construct.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 361
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
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