Basic 6His purification with pET expression

A highly stable laccase from Bacillus subtilis strain R5: gene cloning and characterization

Same protocol as "Basic 6His purification with pET expression" but Expression was induced by 4 mM IPTG in the presence of 10 mM copper sulfate. After 4 h of post-induction at 37 °C and 120 rpm, shaking was stopped in order to provide microaerobic conditions.

DH5alpha/BL21 competent cells

Acrylamide Gel

LB Agar

If you need an antibiotic, see to the table below :
Antibiotic color code Volume to add in LB Final [x]
Ampicilline 2 [x] :
- 25 mg/mL
- 100 mg/mL
4 blues 4 µL/mL 100 µg/mL
Kanamycine 2 [x] :
- 10 mg/mL
- 100 mg/mL
1 red 5 µL/mL 50 µg/mL
15 mg/ mL
1 black 1 µL/mL 15 µg/mL
30 mg/mL
2 green 1.6 µL/mL 50 µg/mL

Note : if you’re using different antibiotics you should divide your volume according to what you need in separate erlenmeyer.


Mini prep

PCR on colonies




Laccase activities

Laccase activity was assayed using 2,2'-azino-bis (3-ethylbenzthiazoline-6-sulphonic acid) (ABTS) as substrate. Oxidation of ABTS was detected by following the absorbance at 420 nm (e420 nm =36000 M−1·cm−1) during 2 min with a spectrophotometer (Cary 60, Agilent Technologies, USA). The reaction mixture (1 mL) contained 10 µL of appropriately diluted enzyme sample, 890 µL of acetate buffer (100 mM pH 5.7) and 100 µL of 50mM ABTS in Milli Q® Water at 30°C. The ABTS was added to initiate the reaction. One unit (U) of laccase was defined as one micromole of substrate oxidized per minute in these described conditions.

In addition, a 96-well plate assay was used to test the activity of the laccases. The experiments were done in two different pHs, pH 5 and pH 7, of phosphate-citrate buffer (100 mM). Absorbances were measured at A420 in one minute intervals with the Tecan M200 plate reader.

Phage Display

ELISA screening

Killer digestion

Resin reuse