Part:BBa_K5394228

Lux B:Venus - BRET

Lux B: Venus is a chimeric protein made up of Lux B (Part:BBa_K216000) from Photobacterium phosphoreum ATCC11060 and a mutated cp157 Venus fluorescent protein.

Since cp157Venus is similarly bright to the regular Venus, a maximum of six times enhancement in brightness through BRET was expected. However, when the researchers fused luciferase to cp157Venus, they observed a ten times enhancement, which was more than anticipated. This unexpected increase might be due to changes in luciferase's properties from the fusion with cp157Venus.

Circularly permuted fluorescent proteins (cpFPs) have been used to develop biosensors that can monitor various intracellular events. When the N-terminus and C-terminus, are connected via a peptide linker new ends are created in a different part of the protein, close to the chromophore, the part that makes it glow. This enhanced the mobility and spectral properties of cpFPs.

This protein engineering was conducted by using a reference from the Addgene LuxB: Venus bacterial luciferase expression plasmid from "Enhanced brightness of bacterial luciferase by bioluminescence resonance energy transfer" by Kaku et. Al . The sequence map was downloaded and cp157 was identified, The circularly permuted protein was broken down into its domain and placed in its original order so it lined up the venus sequence. The mutations were then changed on an amino acid level. The protein domains were once again circularly permuted at AA157 and the order of the protein domains was changed to the advantageous configuration. This was then turned into a nucleotide sequence and plugged into the circuit on benchling.

mVenus already had the the following mutations:Q69M, A206K, F46L and S175G. Added mutations include F64L, M153T andV163A

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 991
  Illegal BamHI site found at 1027
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]