Team:NCKU Tainan/Measurement
Overview
To determine taurine production of the JJU-CoaBC pathway, in vitro testing was conducted by collecting supernatant and whole cell samples from the bacteria cultures. The supernatant consists of folded enzymes in the correct protein conformation and allows us to determine extracellular taurine concentrations. On the other hand, intracellular taurine concentrations can be determined from the whole cell samples, which include both folded and unfolded proteins.
After the required reactants being added to a reaction solution, extracellular and intracellular taurine concentrations were determined by high-performance liquid chromatography (HPLC).
By comparing taurine concentrations in supernatant and whole cell samples, we can gain more insight on the expression levels and taurine production abilities of the JJU and CoaBC enzymes.
Procedure
Two in vitro experiments were conducted. In the first test, plasmids containing JJU (PT7-jju) and CoaBC (PT7-coaBC) sequences were transformed into the same bacterial strain BL21(DE3). In the second experiment, PT7-jju was transformed into BD7G strain, which contains chaperone protein GroELS that aids in protein folding[1]. Sample preparations for both experiments are the same.
First, bacterial cultures were incubated at 37°C. When OD600 reached 0.6, IPTG was added to induce the trc promoter. After incubating for another 12 hours, bacterial pellets and supernatants were separated by centrifugation.
The whole cell and supernatant JJU and CoaBC samples were added in a JJU:CoaBC volume ratio of 1:1, 2:1, and 1:2. The precursor of taurine - L-cysteine, cofactor pyridoxal phosphate (PLP), and PBS buffer were added to the samples and then incubated at 37°C for one-hour reaction.
| Substance | Volume (μl) |
|---|---|
| JJU: CoaBC supernatant/whole cell (1:1, 2:1, 1:2) | 80:80, 107:53, 53:107 |
| L-cysteine (50 mM) | 20 |
| Pyridoxal phosphate (20 mM) | 5 |
| PBS buffer (pH 7.4) | 15 |
| Total | 200 |
Lastly, high-performance liquid chromatography (HPLC) was conducted to determine taurine concentration.
Results
In Vitro Test 1: JJU and CoaBC in BL21(DE3)
In the BL21(DE3) strain, JJU expression in the supernatant was not as prominent as in other samples, which means that JJU concentrations were lower outside the cells (Fig. 1).
2:1 ratio of JJU to CoaBC supernatant volume had the highest taurine concentration of around 95 mg/L (Fig. 2). Because JJU concentrations were lower in supernatant, two times as much JJU supernatant volume compared to CoaBC supernatant volume was required to produce a significant amount of taurine.
In Vitro Test 2: JJU in BD7G and CoaBC in BL21(DE3)
SDS-PAGE results confirm approximately equal expression levels of JJU and CoaBC enzymes in all samples (Fig. 3).
Lane 3: whole cell of CoaBC in BL21(DE3); Lane 4: soluble protein of CoaBC in BL21(DE3) (~45 kDa).
This time when PT7-jju was transformed into the BD7G strain, 1:1 ratio of JJU to CoaBC supernatant had the highest taurine production (Fig. 4).
This suggests that the activity level of JJU does not significantly differ from that of CoaBC, and both are equally crucial in converting L-cysteine to taurine. In addition, supernatant protein expression, in this case extracellular JJU and CoaBC levels, is one of the main determinants of considerable taurine production.
This method of comparing whole cell and supernatant protein expression and relating it to the production of taurine provides more information of the JJU and CoaBC enzymes in a low-cost and simple way. Any enzyme pathway that produces a final detectable product like taurine can be characterized with this method.
References
- Yu T-H, Yi Y-C, Shih I-Tai, Ng I-Son. Enhanced 5-Aminolevulinic Acid Production by Co-expression of Codon-Optimized hemA Gene with Chaperone in Genetic Engineered Escherichia coli. Applied Biochemistry and Biotechnology. 2019;191(1):299-312. doi:10.1007/s12010-019-03178-9
