Part:BBa_K1758202

His-tagged lac 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 lac repressor (BBa_C0012) linked with superfolding GFP (sfGFP) with a His-Tag. Lac repressor inhibits the transcription controlled by the lac operator as it binds to the lac operator. By adding IPTG or allolactose, 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
  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 SapI.rc site found at 1146
  

Results

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

Figure 1: Proof of interaction between Cy3-labeled lacO and LacI-sfGFP

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). We characterized the his-tagged lac repressor without sfGFP with our assay. For more information, go to BBa_K1758202.

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. The two different shifts in the LacI-sfGFP EMSA result from the formation of tetramers which is typical for LacI. 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. It enhances the binding of LacI-sfGFP to its corresponding binding site.

All in all, the specific binding of LacI-sfGFP (at a protein amount of 50 pmol) to the operator DNA lacO (DNA amount: 0.05 pmol) could be proven by electrophoretic mobility shift assay (EMSA). This is the verification of the functionality of LacI as fusion protein.