Part:BBa_K1758204

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

Usage and Biology

Uses T7 promotor and a strong RBS (BBa_K525998), the blc repressor linked with superfolding GFP (sfGFP) with a His-Tag. The blc repressor reacts to gamma-Hydroxybutyric acid (GHB) and gamma-Butyrolactone (GBL). gamma-Hydroxybutyric acid is a component of date rape drugs. GBL is the precursor molecul of GHB which is in chemical balance with GHB. As we wanted to construct a new biosensor for date rape drugs, we used the blc repressor and the blc operator. The Blc repressor inhibits the transcription controlled by the blc operator as it binds to the blc operator. By adding GHB and GBL, 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.

Sequence and Features

Assembly Compatibility:

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

  Illegal NheI site found at 800
  
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 508
  Illegal NgoMIV site found at 749
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal SapI.rc site found at 893
  

Results

After protein expression, purification and concentrating the proteins we could see fluorescence in the protein aliquot.

Figure 1: Proof of the interaction between Cy3-labeled P_blc and BlcR-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, sfGFP-tagged repressor proteins are required for the generation of a signal upon release. We tagged the repressors for the detection of components of the date rape drugs 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. In some cases dithiothreitol (DTT) was added to the reaction (marked with +/-). It simulates reducing conditions, which are normally present in the cell. This can influence the performance of the repressors. In the case of BlcR it had no effect. The specific binding of BlcR-sfGFP (at a protein amount of 5 pmol) to the operator DNA P_blc (DNA amount: 0.05 pmol) could be proven by electrophoretic mobility shift assay (EMSA). This is the verification of the functionality of BlcR as fusion protein.