Part:BBa_K3380509
No scaffold iSpinach aptamer under T7RNAP promoter (BBa_z0251) and T7 terminator (BBa_K3137010)
The construct is similar to the BBa_K3380500 construct, having as a reporter the iSpinach fluorescent RNA aptamer under a strong class III T7 RNA polymerase promoter (BBa_z0251) and T7 terminator (BBa_K3137010), however it lacks the F30 tRNA scaffolds (BBa_K3380101 and BBa_K3380102). Figure 1 illustrates a schematic design of the construct.
Figure 1: Construct BBa_K3380509 design. The iSpinach fluorescent RNA aptamer (shown in green) 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 to test the fluorescence intensity of the iSpinach aptamer (BBa_K3380150) when bound to fluorophores under the T7 promoter. Moreover, a terminator has been added to asses the transcription efficiency as compared to the "run off" transcription from a linear DNA with no terminator. 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 169 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
| None |