Part:BBa_K3380617
TXO ArsR regulated construct under T7 P(BBa_K190015) and (BBa_K3137010) T expressing DIR2s-Apt
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 an ArsR transcription factor regulated promoter (BBa_K190015) and T7 terminator (BBa_K3137010). It was designed to test the ArsR regulated promoter. The construct is capable of exhibiting fluorescence being a transcription only construct. Simultaneously we tested the efficiency of transcription in a cell-free extract when adding a terminator as compared to the "run off" transcription (BBa_K3380605) from a linear DNA with no terminator. The T7 RNA polymerase is capable of synthesizing transcripts via run-off transcription in the absence of a terminator (because the DNA template is linear). Figure 1 illustrates a schematic design of the construct.
Figure 1: Construct BBa_K3380617 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 BBa_K190015 promoter (shown in black) and BBa_K3137010 T7 terminator (shown in black). The promoter has an ArsR binding site (shown in yellow) that represses the transcription in the absence of Arsenic.
Usage and Biology
The construct could be used in a As biosensor in cell-free extracts or buffers containing T7 RNA polymerase, chemical energy (ATP), NTPs, fluorophore and adequate cofactors and pH. The construct functioning solely on transcription and having a small length of only 251 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.
The efficiency of the ArsR transcription factor repression can be tested using a cell-free extract under a class III T7 strong promoter and a T7 terminator. The ArsR proteins from the cell extracts can be used to induce the repression of the DNA template transcription (containing the fluorescent RNA aptamer). In the presence of arsenic, the ArsR homodimer would unbind the ArsR binding site allowing the T7 RNA polymerase to bind and initiate the transcription of the DIR2s-Apt, resulting in the fluorescence. In the absence of arsenic, the fluorescence would not be observed. A more detailed explanation of the ArsR transcription factor can be found in BBa_K190015.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
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