Part:BBa_K3380506
iSpinach aptamer under T7RNA polymerase promoter (BBa_z0251) and T7 terminator (BBa_K3137010)
The Edinburgh iGEM team 2020 designed a construct comprising a fluorescent RNA aptamer (iSpinach BBa_K3380150) flanked by a tRNA scaffold (F30 BBa_K3380101 and BBa_K3380102) under a strong class III T7 RNA polymerase promoter (BBa_z0251) and T7 terminator (BBa_K3137010). The Figure 1 illustrates a schematic design of the construct.
Figure 1: Construct BBa_K3380506 design. The iSpinach fluorescent RNA aptamer (shown in green) was flanked by the F30 upstream and downstream scaffolds (shown in blue) to protect it from the RNAse degradation (from the cell free extract), it was expressed under the Class III strong T7 RNA polymerase promoter BBa_z0251 (shown in black) and BBa_K3137010 terminator (shown in black) to test the transcription efficiency with a terminator.
Usage and Biology
It can be used in cell-free extracts or buffers containing T7 RNA polymerase, chemical energy (ATP), NTPs, fluorophore and adequate cofactors and pH. In contrast to the similar BBa_K3380500 iSpinach construct, it has a T7 RNA polymerase terminator (BBa_K3137010). It is designed to test the fluorescence intensity of the iSpinach aptamer flanked by F30 tRNA scaffold when binding to the DFHBI fluorophore. Moreover, the terminator was added to test if it increases the transcription efficiency of the construct and subsequently the fluorescence. The construct is capable of exhibiting fluorescence being a transcription only construct. The iSpinach aptamer is used in tandem with DFHBI fluorophore to exhibit green fluorescence. It might exhibit lower fluorescence, due to degradation by the RNAses, depending on the cell extract it is used in. However, the absence of the scaffold can also improve the fluorescence, as in the example of Broccoli fluorescent RNA aptamer.
The construct functioning solely on transcription and having a small length of only 235 nucleotides has multiple advantages over the conventional fluorescent proteins. The time required for the fluorescence formation is much shorter than compared to the time needed for the synthesis of the fluorescent proteins. Moreover, there is less burden exhibited on the cell (if expressed in cells) or it requires less resources (NTPs, energy if expressed in cell free extract) when expressing the transcription only construct. Also, individual parts can be "de novo" synthesized and ligated rather than expressing them in cells using plasmids, which is more expensive and requires more time.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI.rc site found at 25
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