Part:BBa_K259007

AraC Promoter fused with RBS

This part has the arabinose regulated promoter (AraC/pBAD) upstream of a ribosome binding site. This is ideal for inserting a protein to be expressed downstream of this part as it's expression can be regulated by the presence of the concentration of arabinose in the growing environment/media of the cells.

What this part actually does

This composite part includes the AraC/pBAD promoter and a strong ribosome binding site designed from the Anderson family of RBS.

The AraC/pBAD promoter can be used with prokaryotic chassis to regulate expresion of proteins.

When the AraC/pBAD promoter is placed upstream of your canditate translational unit (protein to be expressed) it allows control of the trancription of the said protein. This is done via controlling the concentration of arabinose sugar in the environment/solution of bacterial growth.

Controlling the transcription will not ensure translation. The ribosome binding site is ideally placed a small distance away from the promoter site to ensure that the ribosome will bind and start translating the protein coding sequence found downstream.

Part assessment

The AraC-RBS was tested by promoter induction at various concentrations of L-Arabinose sugar. Concentrations used were 0.05% 0.1% 0.2% 0.5% and 1.0% . Two constructs were tested

1.AraC-RBS-FhuA-GFP
2.AraC-RBS-GFP(E0040)-Terminator(B0014)

Construct 1. This causes reduction in optical density after promoter induction with L-Arabinose. Construct 2. This does not cause reduction in optical density after promoter induction with L-Arabinose.

Optical density at A600nm is indicative of cell density. L-Arabinose concentration is in weight per volume.

The graph below illustrates that the part can induce promoter with as little as 0.05%(w/v) [L-Arabinose].

The graph shows results of cell density post promoter induction illustrating the promoter-RBS efficiency at different L-Arabinose concentration. The x-axis is [L-Arabinose] concentration and the y-axis is optical density at A600. ARFG is the AraC-RBS-FhuA-GFP construct and ARG is the AraC-RBS-GFP-Terminator construct. Clearly the AraC-RBS construct is effective at initiating the promoter with minimal L-Arabinose sugar, from as little as 0.05%.

Sequence and Features

Assembly Compatibility:

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

  Illegal BamHI site found at 294
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 125
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal SapI site found at 107