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Latest revision as of 03:15, 27 October 2012

Materials

This is where we are going to list all our materials, devices and equipment that we have used.

Devices

Tecan Infinite Microplate Reader

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 fermenter

Infors Labfors bioreactor (3 L):

control panel
peristaltic pump for antifoam, base and acid
airflow sensor
Cooler inlet and Water inlet
agitator
correction fluid
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

Biostat B Bioreactor (3 L) by Braun:

main switch
airflow sensor
thermostat
peristaltic pump for antifoam, base and acid
control panel
agitator
correction fluid
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

HSG medium

14.9 g L^-1 glycerine
13.5 g L^-1 soy peptone
7 g L^-1 yeast extract
2.5 g L^-1 NaCl
2.3 g L^-1 K₂HPO₄
1.5 g L^-1 KH₂PO₄
0.249 g L^-1 MgSO₄ * 7 H₂O

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

HSG Autoinduction medium

The medium is based on HSG-medium. Add the following components after heat sterilization of 900 mL HSG 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	Sodium phosphate [mM]	Imidazole [mM]
Binding buffer	50	20
Elution buffer	50	500

Buffer for Talon-HisTag affinity chromatography

Buffer	Sodium phosphate [mM]	NaCl [mM]	Imidazole [mM]
Binding buffer	50	300	0
Elution buffer	50	300	150

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.

primer name	length	sequence
F	28	GTTTCTTCGAATTCGCGGCCGCTTCTAG
R	29	GTTTCTTCCTGCAGCGGCCGCTACTAGTA
pSB1C3-5aox1-f	60	CGCTAAGGATGATTTCTGGAATTCGCGGCCGCTTCTAGAGAGATCTAACATCCAAAGACG
pSB1C3-5aox1-r	30	GGTGGCGGCGGGCGTTTCGAATAATTAGTT
5aox1-mfalpha1-f	68	AGAAGATCAAAAAACAACTAATTATTCGAAACGCCCGCCGCCACCATGAGATTTCCTTCAATTTTTAC
5aox1-mfalpha1-r	20	AGCTTCAGCCTCTCTTTTCT
mfalpha1-aarI-taox1-f	80	GTATCTCTCGAGAAAAGAGAGGCTGAAGCTACACGCAGGTGGTATGTATCACCTGCGTGTCT TGCTAGATTCTAATCAAG
mfalpha1-aarI-taox1-r	20	TAAGCTTGCACAAACGAACT
taox1-phis4-f	60	GTACAGAAGATTAAGTGAGAAGTTCGTTTGTGCAAGCTTATCATGCCATGGACAAGATTC
taox1-phis4-r	20	GGCCGCTCGAGTATTCAGAA
phis4-kozak-his4-f	72	AATAGTTTACAAAATTTTTTTTCTGAATACTCGAGCGGCCCCCGCCGCCACCATGACATTTCCCTTGCTACC
phis4-kozak-his4-r	30	TTATTATTTCTCCATACGAACCTTAACAGC
his4-3aox1-f	60	TCACCGCAATGCTGTTAAGGTTCGTATGGAGAAATAATAACGAGTATCTATGATTGGAAG
his4-3aox1-r	20	AAAACAAGATAGTGCCCCTC
3aox1-pSB1C3-f	60	AGTCTGATCCTCATCAACTTGAGGGGCACTATCTTGTTTTTACTAGTAGCGGCCGCTGCA
3aox1-pSB1C3-r	20	CTCTAGAAGCGGCCGCGAAT
taox-his4-f	61	GTACAGAAGATTAAGTGAGAAGTTCGTTTGTGCAAGCTTAAGATCTCCTGATGACTGACTC
taox-his4-r	27	CTCGGATCTATCGAATCTAAATGTAAG
his4-3aox1-f02	60	TTATTTAGAGATTTTAACTTACATTTAGATTCGATAGATCCGAGTATCTATGATTGGAAG
his4_gi537483_f	46	ACGTgaattcgcggccgcttctagagAGATCTCCTGATGACTGACT
his4_gi537483_r	41	ctgcagcggccgctactagtaGATCTATCGAATCTAAATGT
5'AOX_FW	45	ACGTgaattcgcggccgcttctagagAGATCTAACATCCAAAGACG
5'AOX_RV	41	CTGCAGCGGCCGCTACTAGTACGTTTCGAATAATTAGTTGT
3'AOX_FW	45	ACGTgaattcgcggccgcttctagagCGAGTATCTATGATTGGAA
3'AOX_RV	41	CTGCAGCGGCCGCTACTAGTAAAAACAAGATAGTGCCCCTC
pAOX1_FW	44	ACGTgaattcgcggccgcttctagagcatccgacatccacaggtc
pAOX1_RV	45	CTGCAGCGGCCGCTACTAGTATTCTCAAGTTGTCGTTAAAAGTCG
tAOX1_FW	45	ACGTGAATTCGCGGCCGCTTCTAGAGCTTGCTAGATTCTAATCAA
tAOX1_RV	41	CTGCAGCGGCCGCTACTAGTATAAGCTTGCACAAACGAACT
Ko_alpha_FW	58	ACGTgaattcgcggccgcttctagagcccgccgccaccATGAGATTTCCTTCAATTTT
Ko_alpha_RV	41	CTGCAGCGGCCGCTACTAGTAAGCTTCAGCCTCTCTTTTCT
B.pumi_LAC_FW	44	ACGTGAATTCGCGGCCGCTTCTAGATGAACCTAGAAAAATTTGT
B.pumi_LAC_RV	41	CTGCAGCGGCCGCTACTAGTATTACTGGATGATATCCATCG
B.halo_FW	44	ACGTGAATTCGCGGCCGCTTCTAGATGAAAAAATCATATGGAGT
B.halo_RV	41	CTGCAGCGGCCGCTACTAGTATTACTCAGGCATATTTGGAA
T.thermo_LAC_FW	44	ACGTGAATTCGCGGCCGCTTCTAGATGCTGGCGCGCAGGAGCTT
T.thermo_LAC_RV	41	CTGCAGCGGCCGCTACTAGTACTAACCCACCTCGAGGACTC
E.coli_LAC_FW_T7	79	ACGTGAATTCGCGGCCGCTTCTAGAGtaatacgactcactatagggagagaggagaaaaATGCAACGTCGTGATTTCTT
E.coli_LAC_RV_HIS	62	CTGCAGCGGCCGCTACTAGTATTATTAGTGATGGTGATGGTGATGTACCGTAAACCCTAACA
Xcc_LAC_FW_T7	79	ACGTGAATTCGCGGCCGCTTCTAGAGtaatacgactcactatagggagagaggagaaaaATGTCATTCGATCCCTTGTC
Xcc_LAC_RV_HIS	62	CTGCAGCGGCCGCTACTAGTATTATTAGTGATGGTGATGGTGATGTGCCTCCACCCGCACTT
B.pumi_LAC_FW_T7	79	ACGTGAATTCGCGGCCGCTTCTAGAGtaatacgactcactatagggagagaggagaaaaATGAACCTAGAAAAATTTGT
B. pumi_LAC_RV_HIS	62	CTGCAGCGGCCGCTACTAGTATTATTAGTGATGGTGATGGTGATGCTGGATGATATCCATCG
E.coli_LAC_FW_T7	79	ACGTGAATTCGCGGCCGCTTCTAGAGtaatacgactcactatagggagagaggagaaaaATGCAACGTCGTGATTTCTT
E.coli_LAC_RV_HIS	62	CTGCAGCGGCCGCTACTAGTATTATTAGTGATGGTGATGGTGATGTACCGTAAACCCTAACA
T.thermo_LAC_FW_T7	79	ACGTGAATTCGCGGCCGCTTCTAGAGtaatacgactcactatagggagagaggagaaaaATGCTGGCGCGCAGGAGCTT
T.thermo_LAC_RV_HIS	62	CTGCAGCGGCCGCTACTAGTATTATTAGTGATGGTGATGGTGATGACCCACCTCGAGGACTC
Tv_lac5_FW_oS	38	acgtcacctgcgtgtagctggtatcggtcctgtcgccg
Tv_lac5_RV	39	acgtcacctgcgtgtcaagTTACTGGTCGCTCGGGTCGC
Tv_lac5.P.FW	43	gaattcgcggccgcttctagATGTCGAGGTTTCACTCTCTTCT
Tv_lac5.S.RV	41	CTGCAGCGGCCGCTACTAGTATTACTGGTCGCTCGGGTCGC
Pc_lac35.P.FW	43	gaattcgcggccgcttctagATGTCGAGGTTCCAGTCCCTCTT
Pc_lac35.S.RV	41	CTGCAGCGGCCGCTACTAGTATCAGAGGTCGCTGGGGTCAA
Pc_lac35_FW_oS	38	acgtcacctgcgtgtagctgccatagggcctgtggcgg
Pc_lac35_RV	39	acgtcacctgcgtgtcaagTCAGAGGTCGCTGGGGTCAA
GFP_FW_SV	39	acgtcacctgcgtgtagctCGTAAAGGAGAAGAACTTTT
GFP_RV_SV	41	acgtcacctgcgtgtcaagTTATTTGTATAGTTCATCCATG
J23117_RBS_FW	70	aattcgcggccgcttctagagttgacagctagctcagtcctagggattgtgctagcaaagaggagaaata
J23117_RBS_RV	70	ctagtatttctcctctttgctagcacaatccctaggactgagctagctgtcaactctagaagcggccgcg
J23103_RBS_FW	70	aattcgcggccgcttctagagctgatagctagctcagtcctagggattatgctagcaaagaggagaaata
J23103_RBS_RV	70	ctagtatttctcctctttGCTAGCATAATCCCTAGGACTGAGCTAGCTATCAGctctagaagcggccgcg
J23110_RBS_FW	70	aattcgcggccgcttctagagtttacggctagctcagtcctaggtacaatgctagcaaagaggagaaata
J23110_RBS_RV	70	ctagtatttctcctctttgctagcattgtacctaggactgagctagccgtaaactctagaagcggccgcg
J23103_K_FW	25	CTGACAGCTAGCTCAGTCCTAGGTA
J23110/117_K_FW	25	TTTACGGCTAGCTCAGTCCTAGGTA
T7_K_FW	26	TAATACGACTCACTATAGGGAAAGAG
CBDcex_2AS-Link_Frei	56	CTGCAGCGGCCGCTACTAGTATTAACCGGTGCTGCCGCCGACCGTGCAGGGCGTGC
CBDcex_Freiburg-Prefix	54	GCTAGAATTCGCGGCCGCTTCTAGATGGCCGGCGGTCCGGCCGGGTGCCAGGTG
CBDcex_T7RBS	80	TGAATTCGCGGCCGCTTCTAGAGTAATACGACTCACTATAGGGAAAGAGGAGAAATAATGGGT CCGGCCGGGTGCCAGGT
CBDclos_2ASlink_compl	63	CTGCAGCGGCCGCTACTAGTATTAACCGGTGCTGCCTGCAAATCCAAATTCAACATATGTATC
CBDclos_Freiburg-Prefix	57	GCTAGAATTCGCGGCCGCTTCTAGATGGCCGGCTCATCAATGTCAGTTGAATTTTAC
CBDclos_T7RBS	73	CCGCTTCTAGAGTAATACGACTCACTATAGGGAAAGAGGAGAAATAATGTCAGTTGAATTTTACAACTCTAAC
Cex_Freiburg_compl	54	ACGTCTGCAGCGGCCGCTACTAGTATTAACCGGTGCCGACCGTGCAGGGCGTGC
Clos_Freiburg_compl	56	ACGTCTGCAGCGGCCGCTACTAGTATTAACCGGTTGCAAATCCAAATTCAACATAT
Eco_Freiburg	53	ACGTGAATTCGCGGCCGCTTCTAGATGGCCGGCCAACGTCGTGATTTCTTAAA
Eco_Freiburg_compl	53	ACGTCTGCAGCGGCCGCTACTAGTATTAACCGGTTACCGTAAACCCTAACATC
GFP_Frei	54	TACGGAATTCGCGGCCGCTTCTAGATGGCCGGCATGCGTAAAGGAGAAGAACTT
GFP_Freiburg	54	ACGTGAATTCGCGGCCGCTTCTAGATGGCCGGCCGTAAAGGAGAAGAACTTTTC
GFP_Freiburg_compl	61	ACGTCTGCAGCGGCCGCTACTAGTATTAACCGGTTTTGTATAGTTCATCCATGCCATGTGT
GFP_His6_compl	74	CTGCAGCGGCCGCTACTAGTATTAACCGGTGTGATGGTGATGGTGATGTTTGTATAGTTCATCCATGCCATGTG
GFP_FW	48	ATGCGAATTCGCGGCCGCTTCTAGAGTCCCTATCAGTGATAGAGATTG
S3N10_Cex_compl	40	TTGTTGTTGTTCGAGCTCGAGCCGACCGTGCAGGGCGTGC
S3N10_Clos_compl	40	TTGTTGTTGTTCGAGCTCGAGCTGCCGCCGACCGTGCAGG
S3N10_GFP	40	CAATAACAATAACAACAACCGTAAAGGAGAAGAACTTTTC
5AOX-Phenotype-FW	21	GACTGGTTCCAATTGACAAGC
TT-Phenotype-RV	21	GCAAATGGCATTCTGACATCC
A.th_cDNA_FW_T7	79	ACGTgaattcgcggccgcttctagagtaatacgactcactatagggaaagaggagaaaaATGTCACATTCCTTCTTCAA
A.th_cDNA_HIS_RV	62	CTGCAGCGGCCGCTACTAGTATTATTAGTGATGGTGATGGTGATGTTCATAGCAAGGCGGCA

Used enzymes

Enzyme	Producer
AarI	[http://www.fermentas.de/product_info.php?info=p166&language=de Fermentas]
AgeI	[http://www.fermentas.de/product_info.php?info=p247 Fermentas]
DpnI	[http://www.fermentas.de/product_info.php?info=p296 Fermentas]
EcoRI	[http://www.fermentas.de/product_info.php?info=p336 Fermentas]
GoTaq DNA-polymerase	[http://www.promega.com/products/pcr/routine-pcr/gotaq-pcr-core-systems/ Promega]
NgoMIV	[http://www.neb.com/nebecomm/products/productR0564.asp NEB]
lysozym	[http://www.carlroth.com/catalogue/catalogue.do;jsessionid=488EF42170F5810F6A64C9ED26264DD7?favOid=000000010000a36800020023&act=showBookmark&lang=de-de&market=AT Carl Roth]
Pfu DNA-polymerase	[http://www.promega.com/products/pcr/routine-pcr/pfu-dna-polymerase/ Promega]
PstI	[http://www.fermentas.de/product_info.php?info=p458 Fermentas]
Phusion HF DNA-polymerase	[http://www.finnzymes.com/pcr/phusion_high_fidelity_dna_polymerase.html Finnzymes]
Shrimp alkaline phosphatase	[http://www.fermentas.de/product_info.php?info=p592 Fermentas]
SpeI	[http://www.fermentas.de/product_info.php?info=p202 Fermentas]
T4-DNA-Ligase	[http://www.fermentas.de/product_info.php?info=p580 Fermentas]
BIOTAQ DNA-polymerase	[http://www.bioline.com/h_prod_detail.asp?itemid=219 Bioline]
XbaI	[http://www.fermentas.de/product_info.php?info=p247 Fermentas]
T5 exonuclease	[http://www.neb.com/nebecomm/products/productM0363.asp NEB]
taq DNA Ligase	[http://www.neb.com/nebecomm/products/productM0208.asp NEB]
PvuII	[http://www.neb.com/nebecomm/products/productr0151.asp NEB]
HindIII	[http://www.neb.com/nebecomm/products/productr0104.asp NEB]
KOD Hot Start DNA Polymerase	[http://www.merckmillipore.com/germany/life-science-research/kod-hot-start-dna-polymerase/EMD_BIO-71086/p_iFCb.s1O874AAAEj2Bl9.zLX Merck Millipore]
NotI	[http://www.neb.com/nebecomm/products/productr0189.asp NEB]

Used Kits

Function	Name
Plasmid purification	[http://www.fermentas.de/product_info.php?info=p874 Fermentas GeneJET™ Plasmid Miniprep Kit]
Plasmid purification	[http://www.promega.com/b/de/minis/default.aspx?utm_source=Promega&utm_medium=Online&utm_campaign=Online_DEPureYield100Preps_Promega_HP_Banner Promega PureYield™ Plasmid Preps]
PCR Cleanup	[http://www.promega.com/products/dna-and-rna-purification/dna-fragment-purification/wizard-sv-gel-and-pcr-clean_up-system/ Promega Wizard® SV Gel and PCR Clean-Up]
PCR core system	[http://www.promega.com/products/pcr/routine-pcr/gotaq-pcr-core-systems/ Promega GoTaq® PCR Core System I]
Protein quantitation	[http://www.carlroth.com/media/_nl-nl/usage/K880.pdf Roti®-Nanoquant]
gDNA Isolation	[http://www.promega.com/resources/protocols/technical-manuals/0/wizard-genomic-dna-purification-kit-protocol/ Promega Wizard genomic DNA purification system kit]

Substrates

Hormones

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

Analgesics

Polycyclic aromatic hydrocarbons

[http://www.merckmillipore.com/chemicals/naphthalene/MDA_CHEM-820846/p_Kreb.s1LaPgAAAEWeOEfVhTl Naphthalin]
[http://www.sigmaaldrich.com/catalog/product/aldrich/215376?lang=de&region=DE Acenaphthen]
[http://www.chemicalbook.com/ChemicalProductProperty_DE_CB8854465.htm Phenanthren]
[http://www.chemcas.com/msds112/cas/1600/120-12-7.asp Anthracen]

Used chemicals

55px

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@@ Line 206: / Line 206: @@
 The Autoinduction medium is based on [https://2012.igem.org/Team:Bielefeld-Germany/Protocols/Materials#LB_medium 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 [https://2012.igem.org/Team:Bielefeld-Germany/Protocols/Materials#Chloramphenicol_stock_solution Cm stock solution] -> final concentration 20 or 60 mg L-1
+::Amp: 1 mL or 3 mL of a 100 μg mL-1 [https://2012.igem.org/Team:Bielefeld-Germany/Protocols/Materials#Ampicillin_stock_solution Amp stock solution] -> final concentration 100 or 300 mg L-1
+* fill up to 1 L with steril ddH2O
+==HSG Autoinduction medium==
+The medium is based on [https://2012.igem.org/Team:Bielefeld-Germany/Protocols/Materials#HSG_medium HSG-medium].
+Add the following components after heat sterilization of 900 mL HSG medium.
 * 5 mL of a 200 g L-1 steril L-rhamnose stock solution -> final concentration 2 g L-1
@@ Line 690: / Line 701: @@
 == Polycyclic aromatic hydrocarbons ==
-*Naphthalin
+*[http://www.merckmillipore.com/chemicals/naphthalene/MDA_CHEM-820846/p_Kreb.s1LaPgAAAEWeOEfVhTl Naphthalin]
-*Acenaphthen
+*[http://www.sigmaaldrich.com/catalog/product/aldrich/215376?lang=de&region=DE Acenaphthen]
-*Phenanthren
+*[http://www.chemicalbook.com/ChemicalProductProperty_DE_CB8854465.htm Phenanthren]
-*Anthracen
+*[http://www.chemcas.com/msds112/cas/1600/120-12-7.asp Anthracen]
 = Used chemicals =

Team:Bielefeld-Germany/Protocols/Materials

From 2012.igem.org

Latest revision as of 03:15, 27 October 2012

Contents

Devices

Tecan Infinite Microplate Reader

Maldi-TOF

ÄKTAprime plus Chromatographiesystem

The Precellys® 24 homogenizer

Bioreactors

Infors labfors fermenter

Biostat B Bioreactor (3 L) by Braun

Bioreactor NLF (7L) by BIOENGINEERING

Media, buffer and other solutions

Ampicillin stock solution

Chloramphenicol stock solution

TAE buffer

Britton-Robinson Buffer

DNA loading buffer

LB medium

HSG medium

Autoinduction medium

HSG Autoinduction medium

SOC medium

YPD medium

Yeast Nitrogen Base (YNB) 10x stock solution

Biotin 500x stock solution

Minimal dextrose medium (MD)

Minimal methanol media (MM)

BEDS buffer

Dithiothreitol-Solution (DTT)

special buffers for resuspending the cell pellet

Buffers for His-Tag affinity chromatography

Buffer for Ni-NTA-HisTag affinity chromatography

Buffer for Talon-HisTag affinity chromatography

SDS-PAGE gel

SDS running buffer

4x Laemmli-buffer

Colloidal Coomassie Brilliant Blue G-250 staining solution

Fairbanks Coomassie staining solutions

Solutions for Gibson assembly

5x isothermal reaction buffer for Gibson assembly

Hanks Buffered Saline Solution (HBSS)

Primers

Used enzymes

Used Kits

Substrates

Hormones

Analgesics

Polycyclic aromatic hydrocarbons

Used chemicals