Part:BBa_K2876002

RNAPolIII alpha subunit

This is the alpha subunit of a RNAPolIII modified so that it can be fused to a protein at its C terminal. This gene was sourced from an Addgene plasmid (https://www.addgene.org/53734), and was deposited by Anna Hochschild from her paper Activation of Prokaryotic Transcription Through Arbitrary Protein-Protein Contacts (Dove, Joung, Hochschild, Nature 1997).

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 8
  
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 480
  
• 1000
  COMPATIBLE WITH RFC[1000]