Part:BBa_K4245003
iSpinach-D5-G30-A32 aptamer with LacI repression
This part produces the fluorescent RNA aptamer iSpinach-D5-G30-A32 (BBa_K4245000) under the control of IPTG. iSpinach-D5-G30-A32 is a co-crystallized, re-engineered version of iSpinach (BBa_K3380150) developed by researchers at the University of Strasbourg. They first re-engineered the original Spinach aptamer (BBa_K734002) to enhance fluorescence production and promote intermolecular interactions during crystallization. However, further research identified that with few mutations in the basal stem and UNCG loop, iSpinach-D5-G30-A32 optimizes iSpinach’s production and crystallization, improving its folding capacity (Millan et. al, 2017). iSpinach-D5-G30-A32 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 (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 since they bind to a fluorophore after transcription (RNA), while GFP requires additional translation for expression. Similar to other FLAPs, iSpinach-D5-G30-A32 is expressed within a transfer RNA (tRNA) scaffold, which shields the RNA from misfolding and degradation (Paige et al., 2011).
Figure 1. DFHBI and iSpinach-D5-G30-A32 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.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 366
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
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