Difference between revisions of "Part:BBa K3380600"

Revision as of 02:46, 12 October 2022

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 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 218 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 was tested using the Cuprividus Metallidurans CH34 cell extract under a class III T7 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 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. 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). Note similar construct with the binding site preceding the promoter was tested in construct BBa_K3380603.

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= 0 min. 100ng of DNA template was used. The measurements were done in triplicates. The black triangle exhibits fluorescence of the construct BBa_K3380500 with 7 μM As(III) added. The green triangle exhibits fluorescence of the BBa_K3380600 construct with 7 μM As(III) and the blue dot exhibits fluorescence with the BBA_K3380600 construct with no metal addition. The normalised fluorescence = (RFU of the sample- RFU blank)/ femtomole of dsDNA. The RFU blank is measured without the DNA template and the femtomole dsDNA can be calculated by knowing the transcript size and DNA concentration. The details of the construct assembly and the exact concentrations of parts used can be found under the Experience page of the similar construct BBa_K3380500 [1]

Improvement by Edinburgh-UHAS_Ghana 2022

We improved this part in three different ways.
1. We designed a similar part (Part:BBa_K4390103) using a different T7 promoter (Part:BBa_I712074), which does not have a BsaI site. This means the part is now compatible with JUMP assembly, as well as other type IIS assembly methods that use BsaI
2. We designed a TXO ArsR-regulated T7 promoter biosensor expressing Broccoli instead of iSpinach. Broccoli binds DFHBI more tightly than iSpinach, hence producing brighter fluorescence (See Figure 1).
3. We expanded the use of the iSpinach biosensor, by producing analogues of this biosensor regulated by MerR and PbrR respectively.

Figure 1: The brightness of various aptamers in combination with various fluorophores

Sequence and Features