Part:BBa_K3380508
DIR2s-Apt 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 (DIR2s-Apt BBa_K3380151) 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). Figure 1 illustrates a schematic design of the construct.
Figure 1: Construct BBa_K3380508 design. The DIR2s-Apt fluorescent RNA aptamer (shown in red) 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 was designed to test the fluorescence intensity of the DIR2s-Apt aptamer flanked by F30 tRNA scaffold when binding to the OTB or DIR fluorophores. Moreover, the terminator was added to test if it increases the transcription efficiency of the construct and subsequently the fluorescence. The DIR2s-Apt fluorescent aptamer is used with DIR fluorophore to exhibit red fluorescence and OTB to exhibit blue 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 214 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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