Part:BBa_K1758203

His-tagged arsenic repressor fused to superfolder GFP under the control of T7 promoter

Usage and Biology

Uses T7 promotor, a strong RBS (BBa_K525998) and the arsenic repressor linked with superfolding GFP (sfGFP) with a His-Tag. Arsenic repressor inhibits the transcription controlled by the arsenic operator as it binds to the arsenic operator. By adding arsenic, the repressor changes its conformation and is released from the operator. Thus, the transcription can started. By linking sfGFP, we want to detect bound protein via fluorescence. We also characterized the arsenic repressor and arsenic operator. For more information, go to BBa_K1758301 and BBa_K1758302.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 186
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal SapI.rc site found at 421
  

Results

After protein expression we could see fluorescence in the cell pellet.

Figure 1: Proof of interaction between Cy3-labeled arsO and ArsR-sfGFP. + refers to the presence of dithiothreitol in the reaction.

The iGEM Team Bielefeld created an in vitro cell-free system based on the binding of a purified repressor protein to purified DNA. Our aim was to establish an alternative assay type that works in vitro on paper. In our developed assay, a repressor protein forms a complex with a plasmid containing the corresponding operator sequence. The repressor changes its conformation upon binding of the targeted substrate (further referred to as "analyte") and the bond to the DNA is broken. This disruption will be detected via a loss of a fluorescence signal caused by elution of labeled protein or DNA. We call this system "Plasmid Repressor Interaction Assay" (PRIA) (see figure 2).

Figure 2: Illustration of PRIA with immobilized DNA and a sfGFP-tagged repressor protein bound to its operator site. Due to the addition of the analyte, the repressor is released The signal measured is generated by the release of the tagged protein.

For the approach of a paper-based test strip with immobilized DNA, super folding Green Fluorescent Protein (sfGFP)-tagged repressor proteins are required for the generation of a signal upon release. Here, we tagged the repressor for the detection of arsenic with sfGFP C-terminally. Their specific binding to the operator DNA could be proven by electrophoretic mobility shift assay (EMSA). Upon increasing amounts of protein binding to the DNA the electrophoretic mobility of the DNA fragments decreases. This results in a shift between protein-DNA complexes and free DNA. It is visible that the labeled operator sites without protein added to them run faster in the gel compared to the operator sites occupied by the corresponding binding proteins (see figure 1). So all purified fusion proteins retained their DNA-binding function. Dithiothreitol was added to the reaction (marked with a +). It simulates reducing conditions, which are normally present in the cell. This can influence the performance of the repressors.

All in all, the specific binding of ArsR-sfGFP (at a protein amount of 50 pmol) to the operator DNA arsO (DNA amount: 0.5 pmol) could be proven by electrophoretic mobility shift assay (EMSA). This is the verification of the functionality of ArsR as fusion protein.