Part:BBa_K5241007

PelB-PkhppD-6xH-20b（+）

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

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal EcoRI site found at 43
  Illegal EcoRI site found at 52
  Illegal EcoRI site found at 967
  Illegal PstI site found at 925
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal EcoRI site found at 43
  Illegal EcoRI site found at 52
  Illegal EcoRI site found at 967
  Illegal PstI site found at 925
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal EcoRI site found at 43
  Illegal EcoRI site found at 52
  Illegal EcoRI site found at 967
  Illegal XhoI site found at 1075
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal EcoRI site found at 43
  Illegal EcoRI site found at 52
  Illegal EcoRI site found at 967
  Illegal PstI site found at 925
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal EcoRI site found at 43
  Illegal EcoRI site found at 52
  Illegal EcoRI site found at 967
  Illegal PstI site found at 925
  Illegal NgoMIV site found at 342
  Illegal NgoMIV site found at 1140
  Illegal AgeI site found at 319
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 316