Difference between revisions of "Part:BBa K4245002"
Manasvigupta (Talk | contribs) |
|||
| Line 4: | Line 4: | ||
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). | 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 as 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). | 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 as 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). | ||
| + | |||
This part works identically to BBa_K4245001, a composite part producing Spinach, 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. | This part works identically to BBa_K4245001, a composite part producing Spinach, 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. | ||
[[File:Characterization-figure-3.png|350px|link=]] | [[File:Characterization-figure-3.png|350px|link=]] | ||
| + | |||
Figure 1. DFHBI and iSpinach aptamer binding to form RNA-fluorophore complex. | Figure 1. DFHBI and iSpinach aptamer binding to form RNA-fluorophore complex. | ||
Revision as of 20:23, 6 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 as 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).
This part works identically to BBa_K4245001, a composite part producing Spinach, 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.
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
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 375
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]