Part:BBa_K2876010

lambda+linker

lambda backbone + tri-alanine linker

In 1997, Dove, Joung, and Hochschild showed that they could initiate prokaryotic transcription through the interactions of two proteins fused to the alpha subunit of RNA polymerase and a lambda repressor, respectively. The system fused the lambda repressor to a bait protein and the alpha subunit of RNA polymerase to a target protein (Figure 1). The interaction of the bait and target proteins stabilizes the binding of the RNA polymerase allowing the initiation of transcription [1]. This P2H (Prokaryotic two-hybrid system) developed by Dove et al was used primarily as a way to screen protein-protein interactions, and was spun into the BacterioMatch kit produced by Agilent [2]. Its utility as a detection mechanism, however, remained unexplored.

Inspired by the yeast two-hybrid system designed iGEM Tsinghua 2017 [3], we fused antibodies and dCas9 proteins to the subunits of the P2H system, creating a versatile platform capable of detecting specific DNA sequences, small molecules, and proteins. Our DNA detection system works with dCas9-protein fusions, which can be targeted to specific DNA sequences (FIGURE 2: DNA detection figure), and is called casP2H (cas9 prokaryotic two hybrid system). Our protein and small molecule system works by fusing single-chain-antibodies specific to different epitopes of a target protein or small molecule, and is called SCAP2H (single chain antibody prokaryotic two hybrid system).

This detection system is unique because it utilizes transcription. Detection-induced transcription allows genes to be activated by the presence of a user-selected molecule, therefore creating a genetic output to a chosen input. For example, a therapeutic protein could be transcribed in response to a disease biomarker.

Sequence and Features

Assembly Compatibility:

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

  Illegal NotI site found at 723
  
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]