Screen of our setup of the Infinite Reader

For measuring the Laccase activity we detected the level of oxidized ABTS via optical density at 420nm. The device we were able to use was a [http://www.tecan.com/platform/apps/product/index.asp?MenuID=1812&ID=1916&Menu=1&Item=21.2.10.1 Tecan Infinite Reader M200]. The program setup was in some parts adapted to the needs of our probes (like duration of the measurement) and in some parts standardized.
Used setup for Laccase activity measurements: Temperature: 25°C; Orbital shaking before each measuring cycle (time depends on duration of each cycle); Number of flashes: 30

Media, buffer and other solutions

Ampicillin stock solution

• Solubilize 100 mg mL^-1 Ampicillin
• Store at -20 °C

Chloramphenicol stock solution

• Solubilize 20 mg mL^-1 Chloramphenicol in 100 % Ethanol
• Store at -20 °C

TAE buffer

For 1 L of 50 x TAE buffer you need:

• 242.48 g Tris
• 41.02 g Sodiumacetate
• 18.612 g EDTA
• Adjust pH to 7.8 with acetic acid
• Solve in dH₂O

10 mL of the stock is diluted in 1 L dH₂O for the gel electrophoresis (0.5 x TAE buffer).

Briton Robinson Buffer

• 0,1 mM acetic acid
• 0,1 mM boric acid
• 0,1 mM phosphoric acid
• adjust to pH 5 with sodium hydroxide

DNA loading buffer

• 50 % (v/v) glycerol
• 1 mM EDTA
• 0.1 % (w/v) bromphenol blue
• Solve in ddH₂O

LB medium

For 1 L of LB medum:

• 10 g Trypton
• 5 g Yeast extract
• 10 g NaCl
• 12 g Agar-Agar (for plates)
• Adjust pH to 7.4

Autoinduction medium

The Autoinduction medium is based on LB-medium. Add the following components after heat sterilization of 900 mL LB medium.

• 5 mL of a 200 g L-1 steril L-rhamnose stock solution -> final concentration 2 g L-1
• 2.5 mL of a 200 g L-1 steril glucose stock solution -> final concentration 1 g L-1
• if necessary add antibiotics:

Cm: 1 mL or 3 mL of a 20 μg mL-1 Cm stock solution -> final concentration 20 or 60 mg L-1

Amp: 1 mL or 3 mL of a 100 μg mL-1 Amp stock solution -> final concentration 100 or 300 mg L-1

• fill up to 1 L with steril ddH2O

YPD media

For 1 L of YPD media:

• 20 g Peptone
• 10 g Yeast extract
• 20 g Dextrose (add 50 mL sterile stock solution (40% dextrose))
• Adjust pH to 6.5

BEDS buffer

For 1 L of BEDS buffer:

• 1,85 g bicine-sodium salt,
• 30 mL ethylene glycol (final concentration 3%(v/v))
• 50 mL (v/v) dimethyl sulfoxide (DMSO)(final concentration 5%(v/v))
• 182.17 g sorbitol
• Adjust pH at 8,3

Dithiothreitol-Solution (DTT)

For 50mL of DTT-Solution:

• 7.71 g Dithiothreitol

Buffers for His-Tag affinity chromatography

• Adjust pH to 7.4 - 7.6

Buffer for Ni-NTA-HisTag affinity chromatography

Buffer for Talon-HisTag affinity chromatography

SDS-PAGE gel

The following amouts are for one gel. Stacking gel 5 %:

• 775 μL H₂O
• 1.25 mL 0,25 M Tris (pH 6,8)
• 425 μL Bis/Acrylamide (0,8 %, 30 %)
• 50 μL 5 % SDS
• 25 μL 10 % Ammonium persulfate
• 3 μL TEMED

Separating gel 12 %:

• 1.5 mL H₂O
• 2.8 mL 1 M Tris (pH 8,8)
• 3.0 mL Bis/ Acrylamide (0,8%, 30%)
• 150 μL 5% SDS
• 37.5 μL 10% Ammonium persulfate
• 5 μL TEMED

SDS running buffer

• 25 mM Tris [pH 8,3]
• 192 mM Glycerol
• 0.1 % SDS

4x Laemmli-buffer

• 250 mM Tris-HCl
• 40 % [v/v] Glycerol
• 20 % [v/v] 2-Mercapthoethanol
• 80 g L^-1 SDS
• 0.04 g L^-1 BPB

Primers

This is a list of primers we have used.

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