Part:BBa_K3594004

Guaiacol Degrading Enzyme System

This composite part is used as the device to degrade guaiacol, one of the aggregation pheromones. It consists basic parts BBa_K3594006 and BBa_K3594014, which is respectively Cytochrome P450 (CYP) and Ferredoxin Reductase (FR), and Plux, which functions as a promotor.

The process we do in order to determine the expressing of protein

The analysis of SDS-PAGE of CYP

Lane 3 and 4 are plamsmids after overnight culture, and then diluted to M9 and cultured to an OD approximately equal to 0.6,which means that there is no induction. Lane 5 and 6 are plasmids adding IPTG for 3h under 37 degrees centigrade.

Pre-inoculum of PSB4K5_pTac_CYP_FR strains and control PSB4K5 strains were cultured overnight in M9 medium with 10 g/L glucose at 30 °C with orbital shaking at 220 rpm. Guaiacol assimilation experiments were carried out in 250mL shake flasks with 150mL of M9 medium and 1.5 ml inoculum in the same conditions described for the pre-inoculum with guaiacol 5mM. Aliquots were withdrawn regularly for measurement of optical density at 600nm (OD600). Then we used TPR_China’ NAHR sensor analysis of the concentration of guaiacol.

Sequence and Features

Assembly Compatibility:

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

  Illegal BglII site found at 1332
  Illegal XhoI site found at 394
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 34
  Illegal NgoMIV site found at 355
  Illegal NgoMIV site found at 406
  Illegal NgoMIV site found at 841
  Illegal NgoMIV site found at 1000
  Illegal NgoMIV site found at 1180
  Illegal NgoMIV site found at 1305
  Illegal AgeI site found at 1416
  
• 1000
  COMPATIBLE WITH RFC[1000]