Difference between revisions of "Part:BBa K5241007"

Revision as of 00:56, 30 September 2024

PelB-PkhppD-6xH-20b（+）

Short Description:Based on Pseudomonas_koreensis_D26 [gene=hppD][protein=4-hydroxyphenylpyruvate dioxygenase], a gene with a PelB signal peptide and 6His tag is synthesized, aiming for the tyrosinase to be localized to the bacterial periplasmic space.

Gene Circuit Diagram:

Description:

1. On the laminar flow hood, open the glycerol tube and streak on an LB plate with ampicillin. Make markings.

2. On the laminar flow hood, open the plasmid tube and add sterile water according to the label.

3. Transform the plasmid into E. coli BL21(DE3) or BL21(DE3)pLysS. Take 100 microliters to spread on an LB plate with ampicillin, and inoculate the rest into LB liquid medium with ampicillin.

4. E. coli BL21(DE3) or BL21(DE3)pLysS (containing Pahdd-6xH in pET-21b(+)) is grown in LB medium with ampicillin at 37°C with overnight shaking. Then, inoculate 1/50 of the culture into 200 mL of the same medium and grow at 30°C with a shaking speed of 180 rpm. When the OD600 reaches 0.6, add 100 µg/mL IPTG, and further incubate the bacterial culture under the same conditions to observe melanin production.

Result:

When attempting to induce the expression of PeLB-PkhppD-6XH for melanin production in Escherichia coli BL21 (DE3) / BL21 (DE3) pLys using L-arabinose, the expression outcome was not as effective as that of the hppD gene , with only a small amount of melanin being expressed.

Reference documentation

[1] Melati, R., Indriyani, A., Gaffar, S., Sriwidodo, & Maksum, I. P. (2019). Comparison of extracellular secretion of recombinant human epidermal growth factor using TorA and PelB signal peptides in Escherichia coli BL21 (DE3). Asian Journal of Pharmaceutical and Clinical Research, 12(11), 35316. https://doi.org/10.22159/ajpcr.2019.v12i11.35316

Sequence and Features