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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

Maldi-TOF

Matrix-assisted Laser Desorption/Ionization – Time of flight (MALDI-TOF) is a procedure to analyze large biomolecules by their mass.

Our MALDI-TOF device

Before measurement, the analyte is co-crystallized on a metal plate within a solid matrix. 3,5-dimethoxy-4-hydroxycinnamic acid (sinapinic acid) and 2,5-dihydroxybenzoic acid (DHB) are most commonly used. Desorption of the analyte is taken by a laser beam, commonly with a wavelength of 337 nm. The matrix absorbs the laser light and the upper layer explosively vaporizes, ejecting both, matrix and analyte molecules. In addition to this vaporization, the laser beam also leads to an ionization of the analytes. The mass analysis is performed by the TOF (time of flight)-analysator. The evaporated ions are accelerated in an electric field. Typical acceleration voltages for this are 10-30 kV. The velocity of an ion depends on its mass and the charge. An ion detector converts the incoming ions into an electrical signal. The advantage of this method is the rapid analysis of a relatively large mass range.

The MALDI-TOF analysis was performed with the [http://www.bdal.com/products/maldi/ultraflextreme-series/overview.html ultrafleXtreme™] by Bruker Daltonics.

We used the MALDI-TOF Analysis to characterize the following BioBricks:

• <partinfo>K863000</partinfo>
• <partinfo>K863005</partinfo>

ÄKTAprime plus Chromatographiesystem

ÄKTAprime plus System by GE Healthcare

ÄKTA is a Swedish word that means true, genuine, or real. ÄKTAprime plus is a compact pumping system which can be used for a range of columns, chromatography media, and filters. This device is ease-of-use and present a high flexibility of liquid handling and minimizing the problem of sample loss.This system can record pH-value, pressure, conductivity, UV-absorption and the flowrate. Different programms are available like PrimeView™ software to evaluate the recorded data.

We used this system to purify our produced laccases with a Immobilized Metal Ion Affinity Chromatography strategy.

The Precellys® 24 homogenizer

The Precellys® 24 homogenizer by peqlab

[http://www.peqlab.de/wcms/en/pdf/91-PCS24.pdf The Precellys® 24] is an effective and efficient sample homogenisation to enable effective DNA, RNA or protein extraction. To disrup tumor tissues, hair, cartilages, bones, plant materials, yeasts, bacterium and fungi the samples are loaded in beads containing 2 ml tubes. The tubes can be supplied with steel, ceramic or glass beads. The homogenization effect takes place during a 3-dimensional figure of eight motion. This lead to strong acceleration of the used beads. The forces between the beads and the collisions induce the disruption of cell or the homogenisation of samples.

The first cell disruptions ot the iGEM-team Bielefeld were made by this system.

Bioreactors

For the production of the different laccases we used three different variations of bioreactors. These Bioreactors includes different kinds of sonsors:

• pO₂
• pCO₂
• pH
• airflow
• agitation
• temperatur
• antifoam/level

The process could be monitored and controlled by a bioreactor specific programm.

Infors Labfors bioreactor (3 L):

1. control panel
2. peristaltic pump for antifoam, base and acid
3. airflow sensor
4. Cooler inlet and Water inlet
5. agitator
6. correction fluid
7. CO₂-sensor

Device specifications:

• Vessel: 3.6; total volume
• Speed range: 100–1200 rpm magnetic drive
• Temperature range: ~5°C above coolant to 60°C (water jacket)
• Pump flow rate: 0.001–1.4 mL/min (small-size tubing)
• Special features: autoclavable fermenter
• Standard parameters:
• Stirrer speed, temperature, pH, pO2, antifoam/level, feed, gas

mix, gas flow

Biostat B Bioreactor (3 L) by Braun:

1. main switch
2. airflow sensor
3. thermostat
4. peristaltic pump for antifoam, base and acid
5. control panel
6. agitator
7. correction fluid
8. CO₂-sensor

Device specifications:

• Vessel: 3,5; total volume
• Speed range: 100–1200 rpm magnetic drive
• Temperature range: ~5°C above coolant to 90°C (water jacket)
• Pump flow rate: 0.001–1.4 mL/min
• Special features: autoclavable fermenter
• Standard parameters:
• Stirrer speed, temperature, pH, pO2, antifoam/level, feed, gas mix, gas flow

Bioreactor NLF (7L) by BIOENGINEERING

Device specifications:

• Vessel: 7; total volume
• Speed range: 100–3000 rpm magnetic drive
• Temperature range: up to 150°C
• Pump flow rate: 50 mL/min
• Standard parameters:
• Stirrer speed, temperature, pH, pO2, antifoam/level, feed, gas

mix, gas flow

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).

Britton-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

SOC medium

• Add the following components to 900 ml of distilled H₂O
  • 20 g Bacto Tryptone
  • 5 g Bacto Yeast Extract
  • 2 ml of 5 M NaCl
  • 2.5 ml of 1 M KCl
  • 10 ml of 1 M MgCl₂
  • 10 ml of 1 M MgSO₄
  • 20 ml of 1 M glucose
• Adjust to 1L with distilled H2O
• Sterilize by autoclaving

YPD medium

For 1 L of YPD medium:

• 20 g Peptone
• 10 g Yeast extract
• 20 g Dextrose (add 50 mL sterile stock solution (40% dextrose))
• Adjust pH to 6.5
• For plates: add 17 g/L agar.

Yeast Nitrogen Base (YNB) 10x stock solution

• Dissolve 69 g Yeast Nitrogen Base (without aminoacids; with ammonium sulfat) in 500 mL bidest water and filter sterilize.
• Store at 4 °C.
• Durable for one year.

Biotin 500x stock solution

• Dissolve 20 mg biotin in 100 mL of 0.05 M NaOH solution and filter sterilize.
• Store at 4 °C.
• Durable for one year.

Minimal dextrose medium (MD)

Medium for selection of recombinant His⁺ clones For 1 L MD agar plates you need:

• Autoclave 2 g dextrose and 15 g agar
• add 100 mL sterile 10x YNB stock solution and
• 2 mL sterile 500x biotin stock solution.

Minimal methanol media (MM)

Medium for protein induction. For 1 L MM medium you need:

• 5 mL 100% methanol
• 100 mL sterile 10x YNB stock solution and
• 2 mL sterile 500x biotin stock solution.
• 893 mL sterile bidest. water

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

special buffers for resuspending the cell pellet

for KRX and KRX with [http://partsregistry.org/wiki/index.php?title=Part:BBa_K863015 BBa_K863015] and [http://partsregistry.org/wiki/index.php?title=Part:BBa_K863005 BBa_K863005]:

• sodium acetate 100 mM
• adjust to pH 5

for KRX with [http://partsregistry.org/wiki/index.php?title=Part:BBa_K863010 BBa_K863010]:

• sodium acetate 20 mM
• adjust to pH 5

for KRX with [http://partsregistry.org/wiki/index.php?title=Part:BBa_K863000 BBa_K863000]: [http://www.aeisner.de/rezepte/puffer1.html Mc Ilvaine buffer]

• solution A: 21,01 g/L citric acid
• solution B: 35,60 g/L disodium phosphate dihydrate
• mix 38,6 mL of solution B and 61,4 mL of solution A
• adjust to pH 4

for KRX with [http://partsregistry.org/wiki/index.php?title=Part:BBa_K863020 BBa_K863020]:

• 1 mM CuSO₄
• 15 mM PBS mit K₂HPO₄
• 100 mM NaCl
• adjust to pH 7 and shake before use

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

Colloidal Coomassie Brilliant Blue G-250 staining solution

for 1 L staining solution

• dissolve 50 g L^-1 (NH4)₂SO₄ in ddH₂O
• add 10 % (v/v) ethanol
• dissolve 0.2 g L^-1 Coomassie Brilliant Blue G-250
• add 2 % (v/v) phosphoric acid
• fill up to 1 L with ddH₂O

Fairbanks Coomassie staining solutions

Solution A:

• 25 % (v/v) ispropanol
• 10 % (v/v) acetic acid
• 0,5 g L^-1 Coomassie Brilliant Blue R-250

Solution B:

• 10 % (v/v) ispropanol
• 10 % (v/v) acetic acid
• 0,05 g L^-1 Coomassie Brilliant Blue R-250

Solution C:

• 10 % (v/v) acetic acid
• 0,025 g L^-1 Coomassie Brilliant Blue R-250

Solution D:

• 10 % (v/v) acetic acid

Solutions for Gibson assembly

5x isothermal reaction buffer for Gibson assembly

• 3 mL of 1 M Tris-HCl (pH 7.5)
• 150 µL of 2 M MgCl2
• 60 µL of 100 mM dGTP
• 60 µL of 100 mM dATP
• 60 µL of 100 mM dTTP
• 60 µL of 100 mM dCTP
• 300 µL of 1 M DTT
• 1.5 g PEG-8000
• 300 µL of 100 mM NAD

storage -20˚C

Hanks Buffered Saline Solution (HBSS)

• 0.137 M NaCl
• 5.4 mM KCl
• 0.25 mM Na2HPO4
• 0.44 mM KH2PO4
• 1.3 mM CaCl2
• 1.0 mM MgSO4
• 4.2 mM NaHCO3

Primers

This is a list of primers we have used.

Used enzymes

Used Kits

Substrates

Hormones

• Estradiol
• Estrone
• [http://www.tcichemicals.com/eshop/en/eu/commodity/E0037/ Ethinyl estradiol]

Analgesics

• Diclofenac
• Ibuprofen
• Naproxen

Polycyclic aromatic hydrocarbons

• Naphthalin
• Acenaphthen
• Phenanthren
• Anthracen

Used chemicals

• Acetonitril
• Methanol