Part:BBa_K4011013
ptac-RiboJ-TnaA-RL-FMO-B0015
Long Description: pTac-RiboJ-TnaA-RL-FMO-B0015 is an expression cassette in E. coli expressing TnaA-RL-FMO BBa_K4011005 used to convert 6-Halogen-Trpytophan (6-X-Trp) into di-halogenated indigoid dyes such as tyrian purple. TnaA-RL-FMO is composed of two seperate domains: TnaA and FMO, fused with a flexible linker. pTac-RiboJ BBa_K3552015 is an IPTG-inducible promoter suited to all strains of E. coli. B0015 BBa_B0015 is a strong terminator. 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. TnaA and FMO are fused together with the common rigid linker EAAAKEAAAK (Lee et al, 2021).
The pTac promoter system is suitable for all strains of E. coli, contrary to the T7 system, which is only suited for E. coli BL21. RiboJ is an riboenzyme insulator which cleaves itself after transcription, insulating the ribosomal binding site from the RNA sequence of the pTac promoter.
Source
TnaA-FL-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 flexible linker is EAAAKEAAAK.
3. Transformed and expressed in E. coli DH5α ΔTnaA to negate influence of endogenous TnaA in measurements.
Characterization
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 4158
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 2565
- 1000COMPATIBLE WITH RFC[1000]
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