Difference between revisions of "Part:BBa K4245003"

Revision as of 16:09, 10 October 2022

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

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
INCOMPATIBLE WITH RFC[12]
Illegal NheI site found at 366
21
COMPATIBLE WITH RFC[21]
23
COMPATIBLE WITH RFC[23]
25
COMPATIBLE WITH RFC[25]
1000
COMPATIBLE WITH RFC[1000]

@@ Line 3: / Line 3: @@
 <partinfo>BBa_K4245003 short</partinfo>
-This part produces the fluorescent RNA aptamer iSpinach-D5-G30-A32 ([https://parts.igem.org/Part:BBa_K4245000 BBa_K4245000]) under the control of IPTG. iSpinach-D5-G30-A32 is a co-crystallized, re-engineered version of iSpinach ([https://parts.igem.org/Part:BBa_K3380150 BBa_K3380150]) developed by researchers at the University of Strasbourg. They first re-engineered the original Spinach aptamer ([https://parts.igem.org/Part:BBa_K734002 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).
+This part produces the fluorescent RNA aptamer iSpinach-D5-G30-A32 (<partinfo>BBa_K4245000</partinfo>) under the control of IPTG. 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).
@@ Line 13: / Line 13: @@
-The LacI protein represses the inducible promoter ([https://parts.igem.org/Part:BBa_R0010 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.
+The LacI protein represses the inducible promoter (<partinfo>BBa_R0010</partinfo>), 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.
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