Difference between revisions of "Part:BBa K3771006"

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JJU-6xHis

Description

JJU (cysteine lyase) is an enzyme weighing approximately 43.1 kDa. JJU catalyzes the beta replacement reaction of L-cysteine and sulfite to form L-cysteate and hydrogen sulfide[1].

Usage

JJU10 enzyme was used in vitro testing of taurine production. The sequence for JJU10 enzyme and trc promoter were ligated and transformed into E. coli to calculate taurine production using high-performance liquid chromatography (HPLC). A 6xHis-tag is added to the C-terminal of the JJU10 protein, allowing for confirmation of JJU10 expression by western blot using the anti-6xHis antibody.

Figure.1 Taurine pathways in E. coli [1,2].

JJU is an enzyme that is part of the JJU-CoaBC taurine production pathway, one of three possible taurine synthesis pathways. Its main function is to convert L-cysteine to L-cystate, which then becomes taurine by CoaBC[1].

Characterization

The jju sequence synthesized by IDT was amplified by PCR and ligated to trc promoter (BBa_K864400) in the pET28a cloning vector.

Figure 3. Agarose gel electrophoresis results showing amplified jju gene fragment

Taurine Production of JJU in E. coli BL21(DE3) strain

In our in vitro testing of taurine production by JJU, JJU was transformed into E. coli BL21(DE3) strain. Supernatant (S) and whole cell (WC) samples were collected to confirm extracellular and intracellular protein expression by SDS-PAGE.

Figure 4. Confirmation of JJU expression by SDS-PAGE

Whole cell and supernatant JJU and CoaBC samples were collected and added in a JJU:CoaBC volume ratio of 1:1, 2:1, and 1:2. High-performance liquid chromatography (HPLC) was conducted to determine taurine concentration. Because JJU concentrations were lower, two times as much JJU supernatant volume compared to CoaBC supernatant volume was required to produce a significant amount of taurine. As shown in figure 2, 2:1 ratio of JJU to CoaBC supernatant volume had the highest taurine concentration of around 95 mg/L.

Figure 5. (in vitro 1 taurine)

Taurine Production of JJU in E. coli BD7G strain

Since JJU expression in BL21(DE3) strain was not prominent in the supernatant, we performed another in vitro test in which P_T7-jju was transformed into BD7G strain instead of the BL21(DE3) strain. The BD7G strain contains chaperone protein GroELS that aids in protein folding [1]. SDS-PAGE results confirm JJU expression in both supernatant and whole cell samples.

Figure 6. (PAGE in vitro 2)

The whole cell and supernatant JJU and CoaBC samples are collected and added in a JJU:CoaBC volume ratio of 1:1, 2:1, and 1:2. When P_T7-jju was transformed into the BD7G strain, 1:1 ratio of JJU to CoaBC supernatant had the highest taurine production, as shown in figure 20. This suggests the activity level of JJU does not significantly differ from that of CoaBC, and both are equally crucial and effective in converting L-cysteine to taurine.

Figure 7. (in vitro 2 taurine)

References

BRENDA - Information on EC 4.4.1.10 - cysteine lyase. Brenda-enzymes.org. Published 2021. Accessed October 6, 2021. https://www.brenda-enzymes.org/enzyme.php?ecno=4.4.1.10

Sequence and Features