Part:BBa_K3380600

TXO ArsR regulated construct under T7 promoter (BBa_K3380103) expressing iSpinach

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 an ArsR transcription factor regulated promoter (BBa_K3380103). 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 the absence of a terminator in a cell-free extract. 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_K3380600 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 BBa_K3380103 promoter (shown in black). The promoter has an ArsR binding site (shown in yellow) that repressed the transcription in the absence of Arsenic.

Usage and Biology

The construct could be used in a As biosensor.

The efficiency of the ArsR transcription factor repression was tested using the Cuprividus Metallidurans CH34 cell extract under a class III strong promoter. The ArsR proteins were used from the cell extract to induce the repression of our DNA template transcription. In the presence of arsenic, the ArsR homodimer would unbind the ArsR binding site allowing the t& 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. Figure 2 shows the behaviour of the BBa_K3380600 construct in the C. Metallidurans CH34 cell extract using T7 RNA polymerase and 7 μM As(III).

Figure 1. C. Metallidurans cell-free extract containing the construct under the ArsR transcription factor repression expressing iSpinach Normalised Fluorescence over time in minutes.The T7 RNA polymerase and 7 μM As(III) were added at time=0min.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 25