Part:BBa_K2013008

TPA 1,2-dioxygenase

This part contains one of sequences(ISF6_0227) that relates to encode (terephthalic acid )TPA 1,2-dioxygenase.This is the first. TPA is incorporated through the TPA transporter (TPATP) and catabolized by TPA 1,2-dioxygenase (TPADO).The result is that TPA is catabolized into 1,2-dihydroxy-3,5-cyclohexadiene-1,4-dicarboxylate.In addition&#65292we did codon optimization before synthesizing it to encode the target product successfully in E. coli.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 43
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal SapI.rc site found at 307
  

Experimental Validation

This part is validated through Four ways: amplification, PCR, Enzyme cutting and Sequence.

Amplification

Enzyme:KOD

Primer-F:5′-GAATTCGCGGCCGCTTCTAGAGTACTAGAGTCACACATGAGGGTACTAGATG-3′

Primer-R:5′- CAGCACATGGGCGTTTAATAATACTAGTAGCG-3′

Results

PCR

Enzyme:Taq

Primer-F:5′-CCACCTGACGTCTAAGAAAC-3′

Primer-R:5′-GTATTACCGCCTTTGAGTGA-3′

Results

Double digestion

After the assembly ,the plasmid was transferred into the Competent E. coli top10. After culturing overnight in LB,we minipreped the plasmid for double digestion .The first cutting procedure was performed with EcoRI and EcoRV restriction endonuclease. The second cutting procedure was performed with PstI and NcoI restriction endonuclease.The plasmid was cutted in a 25μL system at 37 ℃ for 1 hours. The Electrophoresis was performed on a 1％ Agarose glu

Results