Part:BBa_K3380618
No scaffold TXO ArsR regulated iSpinach construct under T7 P(BBa_K3380103) and (BBa_K3137010) T
The Edinburgh iGEM team 2020 designed a construct comprising a fluorescent RNA aptamer (iSpinach BBa_K3380150) under an ArsR transcription factor regulated promoter (BBa_K3380103). As opposed to BBa_K3380600, this does not have a scaffold. It was designed to test the ArsR regulated promoter and to assess the impact of the tRNA scaffold absence on the RNA aptamer fluorescence. Simultaneously we tested the efficiency of transcription in a cell-free extract when adding a T7 terminator (BBa_K3137010) as compared to the "run off" transcription (BBa_K3380606) from a linear DNA with no terminator. The construct is capable of exhibiting fluorescence being a transcription only construct. The T7 RNA polymerase can synthesize 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_K3380618 design. The iSpinach fluorescent RNA aptamer (shown in green) was expressed under the BBa_K3380103 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 204 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 iSpinach, 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]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI.rc site found at 25
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