Difference between revisions of "Part:BBa K4011013"

Revision as of 16:45, 21 October 2021

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 based on 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

TnaA and FMO are two vital but separate enzymes for converting trp/6-Br-trp to our indigo and tyrian purple dye. To increase the overall reaction speed, we fused these two proteins together into TnaA-linker-FMO.

We designed and engineered three strains of E. coli DH5α ΔTnaA: ptac-TnaA-rbs-FMO (RBS; in this strain TnaA and FMO are expressed as separate proteins), ptac-TnaA-rigid linker-FMO (RL), and ptac-TnaA-Flexible linker-FMO (FL) (Fig. 1A & 1C). As TnaA is expressed as a tetramer and FMO a dimer, we put the TnaA tetramer at the center of the fused protein, with FMO forming two dimers to each side of the TnaA tetramer (Fig. 1B).

After culturing and inducing the expression, the three strains, the SDS-PAGE showed separate expression of TnaA (60kDa) and FMO (54kDa) for RBS, and expression of one fused protein at 114 kDa for RL and FL (Fig. 1D). This indicated expected expression of our fused proteins.

Figure 1: Measuring dyes production of different TnaA-FMO strains from Trp, 6-Cl-Trp, and 6-Br-Trp. A) Pictures of TnaA-RL-FMO, TnaA-FL-FMO, and TnaA-RBS-FMO with Trp, 6-Cl-Trp, or 6-Br-Trp added. B) Comparison of production titers of TnaA-RL-FMO, TnaA-FL-FMO, and TnaA-RBS-FMO with Trp, 6-Cl-Trp, or 6-Br-Trp added, pictured in A. Error bars denote two standard deviations from the mean.

We then cultured and induced RBS, RL, and FL with IPTG. After 20 hours, 1mM of either trp, 6-Cl-trp, or 6-Br-trp was added as substrate, and the relative dye concentration produced by each strain was calculated by using a standard calibration curve (Fig. 2B). The comparison between RBS, RL, and FL shows that there is similar production of tyrian red and tyrian purple, and a significant difference between indigo production of RL and FL. Titers of indigo is approximately 0.30mM (60% yield) for FL and RBS, and 0.20mM (40% yield) for RL. Titers of tyrian red is approximately 0.30mM (60% yield) for RL, FL, and RBS. Titers of tyrian purple is approx. 0.25mM (50% yield) for FL and 0.20mM (40% yield) for RL and RBS.

Figure 2: Measuring dye production of different TnaA-FMO strains from Fre-SttH + Trp + NaCl/NaBr. A) Pictures of different TnaA-FMO strains with supernatant of Fre-SttH + Trp + NaCl/NaBr added. B) Comparison of dye production titers of TALEsp2-TnaA-RBS-FMO and TALEsp2-TnaA-FL-FMO. C) Comparison of dye production titers of ptac-TnaA-RL-FMO, ptac-TnaA-FL-FMO, and ptac-TnaA-RBS-FMO.

After confirming the efficacy of Fre-SttH and TnaA-FMO, we wanted to compare TnaA-rbs-FMO with our TnaA-FL-FMO. Therefore, drawing inspiration from GreatBaySZ 2019’s TnaA-rbs-FMO expression system using a constitutive promoter system(TALEsp2), we designed and constructed TALEsp2-TnaA-FL-FMO.

Instead of using 1mM standard samples of trp or 6-X-trp, we attempted to produce dyes from trp and NaX salts. We induced Fre-SttH expression, took the sample supernatant and added it to the ptac-TnaA-FMO and TALEsp2-TnaA-FMO cultures, and compared the titers of the all using supernatant from Fre-SttH cultures as substrate (Fig. 3). There is no significant difference between any of the samples. Both TALEsp2 cultures produced titers of approx. 0.09mM for tyrian purple and 0.23mM for 6, 6’di-chloro-indigo. RL, FL, and RBS both achieved titers of 0.12 and 0.28mM for tyrian purple and tyrian red respectively.

Figure 3: Construction and expression of TnaA-FMO proteins. A) Schematic representing TnaA-RBS-FMO, TnaA-Flexible linker-FMO, and TnaA-Rigid linker-FMO transformed into E. coli DH5a ΔTnaA. B. Schematic representing the structure of TnaA-linker-FMO. C) The sequences between TnaA and FMO, with the rbs and amino acids labelled. FL and RL stands for flexible linker and rigid linker respectively. D) SDS-PAGE analysis of TnaA-RBS-FMO, TnaA-RL-FMO, and TnaA-FL-FMO.

Sequence and Features