Part:BBa_K1486022

Renilla Luciferase

This part is a translational unit for the Renilla luciferase. It is under the control of an Arabinose induced promoter (pBAD). Coelenterazine or benzyl-coelenterazine has to be added to obtain light output (bioluminescence).

Purpose of the biobrick

We used this biobrick in order to test different luciferase to decide which one would best suit a split luciferase complementation assay.

The split protein has also been submitted as a biobrick: BBa_K1486021 (https://parts.igem.org/Part:BBa_K1486021).

Experiment: comparision of Firefly and Renilla full and split luciferases

For any luciferase complementation assay, we should use the full protein as a positive control and the split without fusion protein as a negative control.

This experiment aimed to test which of the luciferases would best suit a complementation assay (in our case, we used the interacting CheY and CheZ proteins, see this page (https://parts.igem.org/Part:BBa_K1486054)). We compared this part to the part BBa_K325108 from Cambridge 2010 team (https://parts.igem.org/Part:BBa_K325108), EPIC Firefly.

As shown in the graph below, we could determine that Firefly luciferase was more suitable. For the same concentration of substrate, it has a more stable and higher signal. Moreover, the difference between the background noise (negative control, non fused split luciferase) and the full luciferase is bigger for Firefly luciferase, which is also preferable.

Sequence and Features

Assembly Compatibility:

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

  Illegal NheI site found at 1205
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 1144
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 979
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal SapI site found at 961