Coding

Part:BBa_K863010

Designed by: Isabel Huber   Group: iGEM12_Bielefeld-Germany   (2012-09-18)

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.


293px-Lakaze.jpeg 160px-Lakaza_po_3%2C6_urah.jpeg


We found that while BBa_K863005 shows excellent activity, BBa_K863010 shows no activity under the same conditions. 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
    COMPATIBLE WITH RFC[1000]


[edit]
Categories
//cds/enzyme
//function/degradation
//proteindomain/degradation
Parameters
None