Part:BBa_K2017006

RSIAT-TEV linker + Luciferase

Firefly luciferase protein. It oxidates its substrate luciferin, emitting light during the process. It can act as a reporter protein when used with a promoter. This part is made to be fused downstream to other coding sequence, so it includes the RSIAT-TEV linker in order to let the luciferase acquire the correct tertiary structure. The TEV region is the recognition site for the NiaTEV protease. The protease cuts on its site, letting the luciferase without the upstream coding sequence. The luciferase does not include ATG (Met) to initiate translation, as it needs to be fused to other coding sequence on the 5'. The translation must begin in the coding sequence fused upstream. The linker includes a random nucleotide in 5', to change the reading frame of the luciferase. That way, it will not be translated unless an indel is produced in the coding region to which the part is fused.

Sequence and Features

Assembly Compatibility:

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

  Illegal BglII site found at 3
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal SapI.rc site found at 855
  

Usage and Biology

Luciferase protein is a useful reporter. It emits light when it oxidates luciferin. In our gRNA testing system, we need to have a truncated luciferase. We have inserted an additional nucleotide before the coding sequence, and we have removed the ATG (Met) that initiates translation. This avoids the luciferase to be expressed unless it is put back to its reading frame. This occurs when it is inserted in our [http://2016.igem.org/Team:Valencia_UPV/Hardware/Reagents#Ourdevice_id gRNA testing system device] and the CRISPR/Cas9 cuts the target. Upstream the luciferase, it is put a linker. In the gRNA testing system, when the device is transcribed and translated, the gene target is attached to the luciferase. When the protein folds, the gene target could interact with the luciferase and affect its tertiary structure. The upstream linker avoids this by creating a distance between the gene target and the luciferase. The linker, theoretically, does not interact with the luciferase.

This part is one of the standard parts that can be used to create our gRNA testing system device, together with a promoter, terminator and a gene target of your choice.