Difference between revisions of "Part:BBa K4245002"
 
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<partinfo>BBa_K4245002 short</partinfo>
 
<partinfo>BBa_K4245002 short</partinfo>
  
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This part produces the fluorescent RNA aptamer iSpinach (<partinfo>BBa_K3380150]</partinfo>) under the control of IPTG. iSpinach is a mutant of Spinach (<partinfo>BBa_K734002</partinfo>) developed by researchers at the University of Strasbourg. Compared to Spinach, iSpinach is less salt-sensitive, has higher thermal stability, and produces more fluorescence (Autour et. al, 2016). iSpinach 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 is expressed within a transfer RNA (tRNA) scaffold, which shields the RNA from misfolding and degradation (Paige et al., 2011).  
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This part produces the fluorescent RNA aptamer iSpinach (<partinfo>BBa_K3380150</partinfo>) under the control of IPTG. iSpinach is a mutant of Spinach (<partinfo>BBa_K734002</partinfo>) developed by researchers at the University of Strasbourg. Compared to Spinach, iSpinach is less salt-sensitive, has higher thermal stability, and produces more fluorescence (Autour et. al, 2016). iSpinach 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 is expressed within a transfer RNA (tRNA) scaffold, which shields the RNA from misfolding and degradation (Paige et al., 2011).  
  
  

Latest revision as of 16:07, 10 October 2022


iSpinach aptamer with LacI repression

This part produces the fluorescent RNA aptamer iSpinach (BBa_K3380150) under the control of IPTG. iSpinach is a mutant of Spinach (BBa_K734002) developed by researchers at the University of Strasbourg. Compared to Spinach, iSpinach is less salt-sensitive, has higher thermal stability, and produces more fluorescence (Autour et. al, 2016). iSpinach 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 is expressed within a transfer RNA (tRNA) scaffold, which shields the RNA from misfolding and degradation (Paige et al., 2011).


Characterization-figure-3.png

Figure 1. DFHBI and iSpinach 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


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 375
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]