Difference between revisions of "Part:BBa K4011012"

Latest revision as of 20:22, 17 December 2021

pTac-RiboJ-Fre-SttH-B0015

The DNA circuit of ptac-RiboJ-Fre-SttH-B0015 is constructed from an IPTG inducible promoter of E.coli: ptac-RiboJ, a fusion protein which can express halogenase: Fre-SttH, and a strong terminator: B0015. This complete DNA circuit can achieve the halogenation of 6th carbon of 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

Fre-SttH is a fusion protein used to halogenate the sixth carbon of the tryptophan in the dye production. Fre-SttH is composed of two separate domains: Fre is from E.coli and SttH is from Streptomyces toxytricini. They are fused by a rigid linker with the protein sequence EAAAKEAAAK. SttH is a trp-6-haloganese that requires FADH2 as a cofactor to convert trp and halogen ions into 6-X-trp, and is highly insoluble in E.coli. Therefore, Fre, a highly-soluble flavin reductase which reduces FAD to FADH2 from E.coli, is fused with SttH as a N-terminal soluble tag, enabling the protein to become soluble and eliminating the need for costly FADH2 cofactors to be added. We express Fre-SttH in ptac system and measured its enzymatic activity using HPLC (Lee et al, 2021).

Characterization

SDS-PAGE

We expressed Fre-SttH under T7 promoter in E. coli BL21(DE3) (Fig. 1A). SDS-PAGE of the sample showed decent Fre-SttH expression but suboptimal water solubility, since the supernatant sample has less intense target band compared to the whole cell sample (Fig. 1B).

For producing tyrian purple, Fre-SttH and TnaA has to be in two different strains, so we used a ΔTnaA E. coli strain, courtesy of Sha Zhou. However, our ΔTnaA E. coli was supplied as E. coli DH5α, which was incompatible with the T7 promoter.

Because of the need for a ΔTnaA E. coli strain, we decided to switch from the T7 system to the E. coli DH5α compatible ptac system. We constructed two ptac plasmids, ptac-Fre-SttH and ptac-histag-Fre-SttH, and transformed them into E. coli DH5α ΔTnaA (Fig. 1C).

We then induced expression of both proteins and performed SDS-PAGE. Results show that histag-Fre-SttH expression and solubility were poor, but Fre-SttH had extremely high expression and solubility (Fig. 1D). Thus, ptac-Fre-SttH in E. coli DH5α ΔTnaA was used for all further experiments.

Figure 1: Construction and expression of Fre-SttH proteins. A) Schematic representing E. coli BL21 (DE3) transformed with histag-Fre-SttH plasmid. B) SDS-PAGE analysis of histag-Fre-SttH expressed by E. coli BL21 (DE3) represented by A. C) Schematic representing E. coli DH5a ΔTnaA transformed with histag-Fre-SttH and Fre-SttH plasmids. D) SDS-PAGE analysis of Fre-SttH and histag-Fre-SttH expressed by E. coli DH5a ΔTnaA represented by C.

HPLC

In order to model the enzymatic dynamics of Fre-SttH, we induced Fre-SttH expression and added the substrates. We then took samples of the culture in 6-hour-intervals for 24 hours for HPLC, and calculated the relative concentrations of trp and 6-X-trp (Fig. 2C & D). To learn about the detailed experimental setup, visit our experiment and measurement pages.

For sample added with trp and NaBr, the concentration of trp decreased from approximately 1.5 mM to 0.5mM, while the concentration of 6-Br-Trp increased from approximately 0mM to 1.0mM (Fig. 2A). We obtained similar result from sample with trp and NaCl, which the trp concentration went from approximately 1.7mM to 0.4mM and 6-Cl-Trp concentration increased from 0 to 1.3mM (Fig. 2B). For both samples, the yield of trp to 6-X-trp conversion by Fre-SttH was near 100%. As trp still remains after 24h in both cultures, a fermentation time of 36 or 48h might yield a higher concentration of 6-X-Trp, should it be desired. From this, we acquired several enzymatic constants for Michaelis-Menten equation, which was vital to our modeling team.

Figure 2: Quantifying the production of 6-X-Trp in Fre-SttH. A) Concentration-time graph of Trp and 6-Br-Trp in ptac-Fre-SttH in E. coli DH5a ΔTnaA with 1.5mM Trp and NaBr. B) Concentration-time graph of Trp and 6-Cl-Trp in a culture of ptac-Fre-SttH in E. coli DH5a ΔTnaA with 1.7mM Trp and NaCl. C) HPLC results, from top to bottom, of Trp, 6-Br-Trp, 040 + NaBr culture at 0, 6, 12, 18, and 24h. D) HPLC results, from top to bottom, of Trp, 6-Cl-Trp, 040 + NaCl culture at 0, 6, 12, 18, and 24h.

Production of dyes using Fre-SttH supernatant

We attempted to produce dyes using Fre-SttH and TnaA-FMO 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 TnaA-FMO cultures 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. The successful production of tyrian red and tyrian purple in all cultures confirms the viability and stability of Fre-SttH expression and activity.

Figure 3: 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.

Modelling: Determining Vmax and Km of Fre-SttH

Part of our measurement is to show how trp is converted into 6-Br-trp (by TnaA-FL-FMO) over time. We added about 1.7mM trp in a Fre-SttH saturated solution, then measured the concentrations of trp and 6-Br-trp every 6 hours for 4 times (24h in total). We obtained the raw data and fitted a natural exponential curve (equation) to the data in order to describe the relationship between time and trp concentration (note: we chose the natural exponential equation because it showed the highest R^2 among other equations we tried fitting). After that, we determined the reaction velocity at x-values (times) 6(h), 12(h), 18(h), and 24(h) (i.e. different trp concentrations) by taking the gradients of the natural exponential equation at the above points, thus obtaining 4 reaction velocities with respect to their substrate concentrations. Then, we fitted the Michealis-Meten equation to the calculated data and determined Vmax and Km.

Sequence and Features

References

Lee, J., Kim, J., Song, J.E. et al. Production of Tyrian purple indigoid dye from tryptophan in Escherichia coli. Nat Chem Biol 17, 104–112 (2021). https://doi.org/10.1038/s41589-020-00684-4