Difference between revisions of "Part:BBa K4245000"

Latest revision as of 16:13, 10 October 2022

iSpinach-D5-G30-A32 Aptamer

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.

Sequence and Features

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
COMPATIBLE WITH RFC[12]
21
COMPATIBLE WITH RFC[21]
23
COMPATIBLE WITH RFC[23]
25
COMPATIBLE WITH RFC[25]
1000
COMPATIBLE WITH RFC[1000]

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 <partinfo>BBa_K4245000 short</partinfo>
-BBa_K4245000 is a composite part producing a fluorescent RNA aptamer called Spinach. As shown in Figure 3, BBa_K4245000 consists of an inducible promoter (BBa_R0010) that is repressed by the LacI gene, a Spinach Aptamer (BBa_K734002) with stabilizing tRNA scaffolds designed by Austin Texas iGEM 2012, and a T7 terminator (BBa_K731721).
+iSpinach-D5-G30-A32 is a co-crystallized, re-engineered version of iSpinach (<partinfo>BBa_K3380150</partinfo>) developed by researchers at the University of Strasbourg. They first re-engineered the original Spinach aptamer (<partinfo>BBa_K734002</partinfo>) 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).
+[[File:Characterization-figure-3.png|center|350px]]
+<center>
+  <p>''Figure 1. DFHBI and iSpinach-D5-G30-A32 aptamer binding to form RNA-fluorophore complex.''</p>
+</center>
-The LacI protein represses the inducible promoter (BBa_R0010), thus stopping downstream transcription of the Spinach aptamer (BBa_K734002). When IPTG is present, it inhibits LacI from repressing the promoter, allowing transcription of the aptamer. Once DFHBI (3,5-difluoro-4-hydroxybenzylidene imidazolinone) binds to the aptamer, quantifiable green fluorescence is produced.
 <!-- Add more about the biology of this part here