Part:BBa_K863010

tthl laccase from Thermus thermophilus with T7 promoter, RBS and His-tag

tthl (Laccase from Thermus thermophilus) with T7, RBS and HIS tag

Usage and Biology

Slovenia HS characterized this part in 2015.

Escherichia coli BL21 (DE3) bacteria were transformed with the expression plasmids (BBa_K863005 and BBa_K863010) and grown in 10 ml at 37 °C in LBC medium overnight. To express both recombinant proteins, 10 ml of overnight cultures shaker cultures were grown at 37 °C in LB broth supplemented with 30 µg/ml chloramphenicol and shaking with 225 rpm. Expression of the recombinant protein was induced by addition of IPTG to a final concentration of 1 mM, when the cell density reached an OD600 of 0.8. After induction, cells were grown for 5 h and then collected by centrifugation at 6000g for 10 min. The cell pellet collected from 400 ml of bacterial culture was resuspended in 20 ml of resuspension buffer (50 mM HEPES pH 7.5, 500 mM NaCl, 20 mM imidazole) and sonified 3 × 6 min on ice. Following centrifugation at 30 000 × g for 10 min to remove insoluble debris, the supernatant was applied to a Ni-NTA Superflow Cartridge (Qiagen) connected to ÄKTA FPLC system, washed with the resuspension buffer and eluted in the same buffer, but containing 250 mM imidazole. The peak fractions were collected and 15 µl of each fraction was collected and resolved on 12 % SDS-PAGE.

We found that while BBa_K863005 shows excellent activity, BBa_K863010 shows no activity under the same conditions.

IPTG Induction of K863010 (2019 PuiChing_Macau)

Figure 1. Western Blot and SDS-PAGE (coomassie blue) of K863000 and K863010 laccase. Both laccase should be with His-tag.

Consistent with characterization by previous iGEM teams, we found no expression of laccase with K863010 plasmid (contains a fungal laccase, expected to be IPTG regulated), as shown in Figure 1. To the best of our knowledge, there is no functional IPTG-regulated fungal laccase in the iGEM part registry. We here successfully add one (K3021002), as an improvement of the part K863010, which cannot be expressed. For detail information of K3021002, refer to 2019 PuiChing_Macau K3021002

Sequence and Features

Assembly Compatibility:

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

  Illegal XhoI site found at 1408
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 475
  Illegal NgoMIV site found at 962
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal SapI.rc site found at 790