Part:BBa_K3584008

p14 pro-rbs-tyrR-rbs-tyrP-PPO

Assembly Compatibility:

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

  Illegal BglII site found at 798
  Illegal BglII site found at 1140
  Illegal BglII site found at 3083
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 604
  Illegal AgeI site found at 2035
  
• 1000
  COMPATIBLE WITH RFC[1000]

In Escherichia coli, the TyrR protein can both repress and activate the transcription operons required for the biosynthesis and uptake of aromatic amino acids (tyrosine, phenylalanine, and tryptophan). In BBa_K3584008, the promoter of the tyrP gene (encodes a tyrosine-specific transporter) is activated by the TyrR dimer in the presence of tyrosine. Then, the polyphenol oxidase(PPO) gene is expressing.

Contribution

BBa_K3584008 is composite part containing the coding sequences of TyrR gene and polyphenol oxidase (PPO) proteins which are controlled by constitutive promoter p14 and tyrP promoter, respectively.

Figure 1

Engineering Success

Degradation activity of P-Cresol in engineering bacteria induced by tyrosine We added 0, 50, 100, 500 four concentration gradients of tyrosine to induce PPO expression in the medium of pSU2718-p15A-PPO/E.coli Nissle 1917 Nissle 1917 strain, and compared the degradation efficiency of catechol after 12 hours of inoculation. It was found that the yellow getting darker gradually with the increase of tyrosine concentration, indicating that more catechol was degraded (Fig.2).

Figure 2

                           Fig. 2 The situation of catechol degraded by pSU2718-p15A-PPO/E.coli Nissle 1917 strain

Figure 3

                                   Fig. 3 The enzymatic activity of the crude enzyme solution on catechol

We further obtained the PPO crude enzyme solution by ultrasonic decomposition from sample with different b tyrosine concentration, and then detected the PPO enzyme activity with catechol as the substrate. It was found that the enzymatic activity of the crude enzyme solution on catechol increased significantly with the increase of the concentration of tyrosine (Fig.3), and tyrosine could indeed induce the expression of active p-cresol degrading enzyme.