Team:Bielefeld-Germany/Protocols/Materials

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

Contents

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 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 ultrafleXtreme™ by Bruker Daltonics.


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



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

Yeast Nitrogen Base (YNB)

for 500 mL Yeast Nitrogen Base Medium:

  • 67 g Yeast Nitrogen Base (without aminoacids;with ammonium sulfat)
  • for Agar-plates: add 5 g Agar-Agar

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 BBa_K863015 and BBa_K863005:

  • sodium acetate 100 mM
  • adjust to pH 5

for KRX with BBa_K863010:

  • sodium acetate 20 mM
  • adjust to pH 5

for KRX with BBa_K863000: 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 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

Fixer solution

  • 10 % [v/v] acetic acid
  • 25 % [v/v] 2-propanol
  • 65 % [v/v] H2O

Primers

This is a list of primers we have used.

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 || GTATCTCTCGAGAAAAGAGAGGCTGAAGCTACACGCAGGTGGTATGTATCACCTGCGTGTCTTGCTAGAT
TCTAATCAAG 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 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 Cex_Freiburg_compl || 54 || ACGTCTGCAGCGGCCGCTACTAGTATTAACCGGTGCCGACCGTGCAGGGCGTGC Clos_Freiburg_compl || 56 || ACGTCTGCAGCGGCCGCTACTAGTATTAACCGGTTGCAAATCCAAATTCAACATAT Eco_Freiburg || 53 || ACGTGAATTCGCGGCCGCTTCTAGATGGCCGGCCAACGTCGTGATTTCTTAAA Eco_Freiburg_compl || 53 || ACGTCTGCAGCGGCCGCTACTAGTATTAACCGGTTACCGTAAACCCTAACATC 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
primer name length sequence
5'AOX_RV 41 CTGCAGCGGCCGCTACTAGTACGTTTCGAATAATTAGTTGT

Used enzymes

Enzyme Producer
AarI Fermentas
AgeI Fermentas
DpnI Fermentas
EcoRI Fermentas
GoTaq DNA-polymerase Promega
NgoMIV NEB
Pfu DNA-polymerase Promega
PstI Fermentas
Phusion HF DNA-polymerase Finnzymes
Shrimp alkaline phosphatase Fermentas
SpeI Fermentas
T4-DNA-Ligase Fermentas
taq DNA-polymerase Bioline
XbaI Fermentas

Used Kits

Function Name
Plasmid purification Fermentas GeneJET™ Plasmid Miniprep Kit
Plasmid purification Promega PureYield™ Plasmid Preps
PCR Cleanup Promega Wizard® SV Gel and PCR Clean-Up
PCR core system Promega GoTaq® PCR Core System I

Substrates

Hormones

Analgesics

Polycyclic aromatic hydrocarbons

  • Naphthalin
  • Acenaphthen
  • Phenanthren
  • Anthracen

Used chemicals


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