Part:BBa_K4011005

TnaA-RL-FMO

TnaA-RL-FMO is a fused protein used to convert 6-Halogen-Trpytophan (6-X-Trp) into di-halogenated indigoid dyes such as tyrian purple. It is composed of two seperate domains: TnaA and FMO, fused with a rigid linker. This is a part in a part collection where we enable the production of indigo, tyrian purple, and related dyes from tryptophan in E. coli.

The part collection includes: Parts expressing Fre-SttH to convert Trp to 6-X-Trp. BBa_K4011003 and BBa_K4011012. Parts expressing fusion protein TnaA-FMO to convert 6-X-Trp into indigoid dyes. BBa_K4011004, BBa_K4011005, BBa_K4011013, BBa_K4011014, BBa_K4011015, and BBa_K4011019.

Our part collection can be used to help and inspire future teams to design and perfect different indigoid dye production pathways in E. coli, adding to the collection.

Usage and Biology

TnaA is an tryptophanase found in E. coli. It converts tryptophan (trp) and other related molecules, such as 6-Halogen-trp (6-X-trp) into indole or 6-X-indole. Its specific reaction formula is L-tryptophan + H₂O ⇌ indole + pyruvate + NH₃. Indole, play important roles in signaling in bacterial cells. FMO is a monooxygenase found in Methylophaga aminisulfidivorans. It adds a hydroxyl group onto numerous molecules, in our case adding a hydroxyl onto the third carbon on indole or 6-X-indole, allowing for spontaneous dimerization of 3-hydroxyl-indole or 3-hydroxyl-6-X-indole into indigo or tyrian purple dyes. They are fused together with the common rigid linker EAAAKEAAAK (Lee et al, 2021).

Source

TnaA-RL-FMO is composed of two main domains: tryptophanase (TnaA) from E. coli and flavin-containing monooxygenase (FMO) from Methylophaga aminisulfidivorans.

Design Considerations

1. All codons were optimized for E. coli<.i> based on <i>E. coli codon bias.

2. The sequences for the rigid linker is EAAAKEAAAK.

3. Transformed and expressed in E. coli DH5α ΔTnaA to negate influence of endogenous TnaA in measurements.

Characterization

Assembly Compatibility:

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

  Illegal BamHI site found at 2812
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 1219
  
• 1000
  COMPATIBLE WITH RFC[1000]