Part:BBa_K5241005
Pahdd-6xH in pET-21b(+)
Short Description：Based on Pseudomonas aeruginosa PAO1 [gene=hppD] [protein=4-hydroxyphenylpyruvate dioxygenase], a Pahdd-6xH gene with a 6xH tag is synthesized in pET-21b(+), with the anticipated protein localization within the bacterial cytoplasm.
Gene Circuit Diagram:
Description：
1.Bacterial strains and cultural conditions
P. aeruginosa PAO1 [25] and E. coli JM109 [26] were grown at 37°C under 170 rpm agitation in rich LB medium or in M9 minimal medium supplemented with suitable carbon sources. Glucose, malic acid and arabinose were used at 10 mM concentration for supporting bacterial growth. Liquid cultures were started by inoculating fresh media with 1/100 volume of overnight cultures; culture volumes never exceeded 1/5 of the total volume of the Enlermeyer flasks to allow proper aeration. When needed, ampicillin was added as a selection antibiotic at a final concentration of 200 μg/mL.
2.Purification of HPD 6XHis-Tagged Protein
E. coli JM109 (pVO hpd 6XHis) was grown O/N at 37°C with agitation in LB medium supplemented with 200 µg/mL ampicillin. 1/50 of the culture was then inoculated into 200 mL of the same medium and grown at 37°C with agitation 180 rpm, up to OD600 equal to 0.6. Arabinose was added at 0.1% W/V and the bacterial culture was further incubated in the same conditions for two hours. Before starting the whole purification procedure, the 1 mL culture pellet was analyzed by SDSPAGE to verify recombinant protein expression.
3.GFP, Induced Expression
Escherichia coli BL21(DE3) or BL21(DE3)pLysS (containing pET-21b(+)-eGFP-pc) is grown in LB medium supplemented with ampicillin at 37°C with overnight shaking. Then, 1/50 of the culture is inoculated into 200 mL of the same medium and grown at 30°C with a shaking speed of 180 rpm. When the OD600 reaches 0.6, 100 µg/mL IPTG is added, and the bacterial culture is further incubated under the same conditions to observe the production of melanin.
Result：
We successfully expressed the hppD gene using IPTG induction, and the 2×BL21(DE3) strain performed better than the 2×BL21(DE3) pLysS; the expression of melanin was more effective than that of the PkhppD gene.
Reference documentation
[1]ChiaraScanferla EnricoCaruso Viviana TeresaOrlandi FabrizioBolognese. Bacterial melanin production by heterologous expression of 4‑hydroxyphenylpyruvate dioxygenase from Pseudomonas aeruginosa[J]. International Journal of Biological Macromolecules, 2019, 133: 1072-1080.
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal EcoRI site found at 2950
Illegal EcoRI site found at 3121
Illegal EcoRI site found at 3595
Illegal PstI site found at 3826 - 12INCOMPATIBLE WITH RFC[12]Illegal EcoRI site found at 2950
Illegal EcoRI site found at 3121
Illegal EcoRI site found at 3595
Illegal PstI site found at 3826 - 21INCOMPATIBLE WITH RFC[21]Illegal EcoRI site found at 2950
Illegal EcoRI site found at 3121
Illegal EcoRI site found at 3595
Illegal BglII site found at 587
Illegal BglII site found at 2316
Illegal BglII site found at 3830
Illegal BamHI site found at 562
Illegal BamHI site found at 1630
Illegal XhoI site found at 676
Illegal XhoI site found at 1624
Illegal XhoI site found at 3763 - 23INCOMPATIBLE WITH RFC[23]Illegal EcoRI site found at 2950
Illegal EcoRI site found at 3121
Illegal EcoRI site found at 3595
Illegal PstI site found at 3826 - 25INCOMPATIBLE WITH RFC[25]Illegal EcoRI site found at 2950
Illegal EcoRI site found at 3121
Illegal EcoRI site found at 3595
Illegal PstI site found at 3826
Illegal AgeI site found at 524 - 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI site found at 75
Illegal BsaI site found at 1388
Illegal BsaI.rc site found at 2149
Illegal BsaI.rc site found at 2617
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