Difference between revisions of "Part:BBa K3739044"
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====1. Identification==== | ====1. Identification==== | ||
| − | After transforming into the ''Vibrio natriegens'' by electroporation, colony PCR was done to certify that the plasmid was correct, and the experimental results were shown in | + | After transforming into the ''Vibrio natriegens'' by electroporation, colony PCR was done to certify that the plasmid was correct, and the experimental results were shown in '''fig. 1'''.<br/> |
| − | + | [[File:T--XMU-China--14-F1.png|300px]]<br/> | |
Fig. 1 Colony PCR of pET-28a(+)_J23100-RBS-tnaA-his in ''Vibrio natriegens'' | Fig. 1 Colony PCR of pET-28a(+)_J23100-RBS-tnaA-his in ''Vibrio natriegens'' | ||
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The purified tnaA enzyme activity was measure by the amount of indole produced from tryptophan within a specific time. The concentration of indole was calculated through the standard curve, which draws as the method as fellow: | The purified tnaA enzyme activity was measure by the amount of indole produced from tryptophan within a specific time. The concentration of indole was calculated through the standard curve, which draws as the method as fellow: | ||
After dissolving 5 mg of indole in a small amount of toluene, transfer the solution 50 mL volumetric flask, dilute with toluene to tick marks, and mix. Transfer 10, 20, 30, 40, and 50 μl solution above into five clean tubes respectively and dilute with toluene to 1.0 ml, then added 0.4 mL distilled water, 3ml PDAB (5%, v/v) and n-butanol (5%, v/v) sulfate mixed chromogenic solution, and mix. Stand for 30 min and measure the absorbance at 570 nm. Taking the absorbance as the X-axis, the concentration as the Y-axis, draw the standard curve and obtain the equation. | After dissolving 5 mg of indole in a small amount of toluene, transfer the solution 50 mL volumetric flask, dilute with toluene to tick marks, and mix. Transfer 10, 20, 30, 40, and 50 μl solution above into five clean tubes respectively and dilute with toluene to 1.0 ml, then added 0.4 mL distilled water, 3ml PDAB (5%, v/v) and n-butanol (5%, v/v) sulfate mixed chromogenic solution, and mix. Stand for 30 min and measure the absorbance at 570 nm. Taking the absorbance as the X-axis, the concentration as the Y-axis, draw the standard curve and obtain the equation. | ||
| − | 20 µl PLP solution (0.20 mg/ml), 10 µl GSH solution (0.005 mol/l), and 270 µl tnaA enzyme solution were added in several 15 ml tubes respectively and then covered with 1.0 ml toluene. After keeping the temperature at 37 °C for 5 min, 100 µl tryptophan solution with different concentrations was added into the 15 tubes respectively, which were put in a shaker for 10min at 37 °C. Then 3 ml of PDAB (5%, v/v) and n-butanol sulfate mixed chromogenic solution (5%, v/v) was added to stop the reaction. After 30 min, absorbance was measured at 570 nm, which was used to calculate the Km and kcat. (Fig. 2) | + | 20 µl PLP solution (0.20 mg/ml), 10 µl GSH solution (0.005 mol/l), and 270 µl tnaA enzyme solution were added in several 15 ml tubes respectively and then covered with 1.0 ml toluene. After keeping the temperature at 37 °C for 5 min, 100 µl tryptophan solution with different concentrations was added into the 15 tubes respectively, which were put in a shaker for 10min at 37 °C. Then 3 ml of PDAB (5%, v/v) and n-butanol sulfate mixed chromogenic solution (5%, v/v) was added to stop the reaction. After 30 min, absorbance was measured at 570 nm, which was used to calculate the Km and kcat. ('''Fig. 2''')<br/> |
| − | + | [[File:T--XMU-China--14-F2.png|300px]]<br/> | |
Fig. 2 The relationship of 1/enzyme activity and 1/concentration of Indole | Fig. 2 The relationship of 1/enzyme activity and 1/concentration of Indole | ||
Latest revision as of 00:05, 22 October 2021
J23100-B0030-tnaA-his-B0010
TnaA can induce cells to produce l-tryptophan and then synthesize indoles
Biology
tnaA encodes a tryptophan enzyme that converts tryptophan into indole. In 2021, XMU-China used tnaA to produce indole to inhibit the formation of mussel byssus.
Usage
In order to obtain purified tnaA, we add a his-tag (6∗His) at the N-terminal of tnaA. We used BBa_K081005 to construct the expression system and obtained the composite BBa_K3739044, which is assembled on the expression vector pET-28a(+) by standard assembly. The constructed plasmid was transformed into Vibrio natriegens, then the positive transformants were selected by kanamycin and confirmed by colony PCR and sequencing.
Characterization
1. Identification
After transforming into the Vibrio natriegens by electroporation, colony PCR was done to certify that the plasmid was correct, and the experimental results were shown in fig. 1.
Fig. 1 Colony PCR of pET-28a(+)_J23100-RBS-tnaA-his in Vibrio natriegens
2. Enzyme activity of TnaA
The purified tnaA enzyme activity was measure by the amount of indole produced from tryptophan within a specific time. The concentration of indole was calculated through the standard curve, which draws as the method as fellow:
After dissolving 5 mg of indole in a small amount of toluene, transfer the solution 50 mL volumetric flask, dilute with toluene to tick marks, and mix. Transfer 10, 20, 30, 40, and 50 μl solution above into five clean tubes respectively and dilute with toluene to 1.0 ml, then added 0.4 mL distilled water, 3ml PDAB (5%, v/v) and n-butanol (5%, v/v) sulfate mixed chromogenic solution, and mix. Stand for 30 min and measure the absorbance at 570 nm. Taking the absorbance as the X-axis, the concentration as the Y-axis, draw the standard curve and obtain the equation.
20 µl PLP solution (0.20 mg/ml), 10 µl GSH solution (0.005 mol/l), and 270 µl tnaA enzyme solution were added in several 15 ml tubes respectively and then covered with 1.0 ml toluene. After keeping the temperature at 37 °C for 5 min, 100 µl tryptophan solution with different concentrations was added into the 15 tubes respectively, which were put in a shaker for 10min at 37 °C. Then 3 ml of PDAB (5%, v/v) and n-butanol sulfate mixed chromogenic solution (5%, v/v) was added to stop the reaction. After 30 min, absorbance was measured at 570 nm, which was used to calculate the Km and kcat. (Fig. 2)
Fig. 2 The relationship of 1/enzyme activity and 1/concentration of Indole
References
1. Hu, M.; Zhang, C.; Mu, Y.; Shen, Q.; Feng, Y., Indole affects biofilm formation in bacteria. Indian J Microbiol 2010, 50 (4), 362-8.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 7
Illegal NheI site found at 30 - 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 545
- 1000COMPATIBLE WITH RFC[1000]