Materials

The following section lists the used material in the presented iGEM project:

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1. Chemicals


Chemical	Manufacturer
2 log DNA ladder	New England Biolabs
2-Mercapto Ethanol	Merck
Acetic Acid 100 %	zChL UdS
Agar Kobe I	Roth
Agarose	Peqlab, Biozym
Ampicillin	Roth
APS (Ammoniumpersulfat)	zChL UdS
BactoTM Casamino-acids	BD
BactoTM Tryptone	BD
BactoTM Yeast-Extract	BD
Boric Acid	Merck
Bovine Serum Albumin (BSA)	GE Healthcare
Bromophenol Blue	Merck
Calcium Chloride	Calbiochem
Chloramphenicol	Fagran
D(+)-Glucose	Sigma Aldrich
ddH₂O (MilliQ) (MILLIPORE 0,22 μm Membranfilter)	Biocel
DifcoTM Antibiotic Medium No. 3	BD
Dimethylsulfoxide (DMSO)	Sigma Aldrich
dNTP-Mix (10 mM)	Peqlab
EDTA	Roth
Ethanol, HPLC grade	Sigma Aldrich
Glutamate	Sigma Aldrich
Glycerol	zChL UdS
Hydrochloric Acid 37 % (HCl)	VWR
Immersion Oil	Leica
Isopropanol	Sigma Aldrich
LB-Medium (Miller)	Sigma Aldrich
Magnesium Chloride Hexahydrate	VWR
Magnesium Sulfate	zChL UdS
Maleic Acid	Roth
PEG-6000	Merck
peqGREEN	Peqlab
Potassium Chloride	Grüssing
Potassium Dihydrogen Orthophosphate	Grüssing
Potassium Hydrogen Phosphate	Grüssing
Potassium Sulfate	zChL UdS
Proline	Merck
Protein Standard	New England Biolabs
Rotiphorese Gel 30	Roth
SDS	Applichem/Roth
Sodium Chloride	VWR
Sodium Hydroxide	Grüssing
Sodium Phosphate	VWR
Succrose	Sigma Aldrich
Temed	Applichem
Tween 20	Applichem
Tetracycline	Serva
Trizma Base	Sigma Aldrich
Xylose	Sigma Aldrich

2. Enzymes


Enzyme	Manufacturer
Lysozyme	Serva
T4 Ligase	Thermo Scientific
Taq-Polymerase	New England Biolabs
Q5 High Fidelity Polymerase (Master Mix)	New England Biolabs
FastDigest MluI	Thermo Scientific
FastDigest SacI	Thermo Scientific
FastDigest SphI	Thermo Scientific
FastDigest EcoRI	Thermo Scientific
FastDigest XbaI	Thermo Scientific
FastDigest SpeI	Thermo Scientific
FastDigest PstI	Thermo Scientific
Fast AP	Thermo Scientific

3. Oligonucleotides


Abbreviation	5´-Sequence-3´
*Generation of has2-gfp* fusion gene**
SOE Has for:	TGTACACGCGTAAATCAAGGAGGTG
SOE Has rev:	CTCGCCCTTGCTCACCATTACATCTAATACCATATCGTATTGTTGACC
SOE GFP for:	ATGGTATTAGATGTAATGGTGAGCAAGGGCGAG
SOE GFP rev:	TGTACGAGCTCTTACTTGTACAGCTCGTCCATGC
Amplification of genomic DNA of B. megaterium
RBS UDP-GlcDH for	TGTACGAGCTCAAATCAAGGAGGTGAATATACAATGACTAATATCACAGTAGCGGGTACTGG
UDP-GlcDH rev:	TGTACGCATGCTTAGACATTAGCTTTAATTTTAGCTTTGTATCC
Glk for	TGTAC GAATTC ATGGATGACAAATGGTTAG
Glk rev:	TGTAC CTGCAG TTA TTATTCAAAATAAGAT
PgmB for	TGTAC GAATTC ATGAAAAAAGAAGAGCTAC
PgmB rev:	TGTAC ACTAGT TTA TTAATTTTTTGTATAC
GtaB for	TGTAC GAATTC ATGACGATAAAAAAGGCAG
GtaB rev:	TGTAC CTGCAG TTA TTAGCTGAAGTTGTTTGC
Pgi for	TGTAC TCTAGA ATGACACATGTTCGTTTTG
Pgi rev:	TGTAC CTGCAG TTA TTATTTCAAACGCTTTTC
GlmS for	TGTAC TCTAGA ATGTGCGGAATTGTAGG
GlmS rev:	TGTAC CTGCAG TTA TTATTCTACAGTAACTG
GlmM for	TGTAC GAATTC ATGGGTAAGTATTTTGG
GlmM rev:	TGTAC CTGCAG TTA TTACTCTAAGCCCATTTC
GcaD for	TGTAC GAATTC ATGTCAAAAAGATATGC
GcaD rev:	TGTAC CTGCAG TTA TTAGGATTTTTTATTAATATC
Quick Change PCR for restriction site mutagenesis
QC Has for:	GAATTTGTAGAAGATTGGTACTCTCAAGAATTTATGGGTAACCAG
QC has rev:	AAATTCTTGAGAGTACCAATCTTCTACAAATTCGTGTAATAAAGAG
QC UDP-GlcDH for:	TAATTATTAAAAACTCTGATGCTGGTAAATTATTTTTCACAACTG
QC UDP-GlcDH rev:	ACCAGCATCAGAGTTTTTAATAATTAATTCATCAAGCCCAGG
QC PgmB for:	TTGCAGCTGCTGATTGAGTTCTTCAG
QC PgmB rev:	CTGCAAGGGATCAGCCGAGTG
QC Pgi for:	AAATTCTTCTTTGTCATACTCCGTAGG
QC Pgi rev:	GAATTTGCACGCATTCAAAAAAGTGC
QC GlmS for:	AAATTCACTTGCTACGTGCACTTCTAC
QC GlmS rev:	GAATTTTCTTATAACATGCCGCTTTTATCTG
QC GlmM for:	TCGAGATCTGCAAATAAATGTGCTGC
QC GlmM for:	TCTCGATGCAGATATCTCTACAATGG
Sequencing
pSB1C3 check for:	TGCCACCTGACGTCTAAGAAACC
pSB1C3 check rev:	ATTACCGCCTTTGAGTGAGCTG

4. Media and Solutions


Solution	Ingredients	Quantity
10 x KPP Buffer	KH₂PO₄ K₂HPO₄ to 100 ml ddH₂O autoclaved	2,71 g 12.5 g
Stacking Gel Buffer	Tris SDS Adjust pH value to 6.7 to 100 ml ddH₂O	6.05 g 0.4 g
Stacking Gel (4 %)	ddH₂O Stacking Gel Buffer Rotiphorese Gel 30 Temed 10 % APS	1193 µl 500 µl 267 µl 5 µl 10 µl
Separating Gel Buffer	Tris SDS Adjust pH value to 8.8 To 100 ml ddH₂O	18.17 g 0.4 g
Separating Gel (12 %)	ddH₂O Separating Gel Buffer Rotiphorese Gel 30 Temed 10 % APS	1350 µl 1000 µl 1600 µl 10 µl 20 µl
5 x SDS Sample Buffer	Tris 2-ME SDS Glycerol Bromophenol Blue Adjust pH value to 6.5 with HCl to 50 ml ddH₂O	1.21 g 3.487 g 5 g 10 ml 25 mg
Na₃PO₄ Solution (100 mM)	Na₃PO₄ Solve in 80 ml ddH₂O Adjust pH value to 6.5 with H₃PO₄ to 100 ml ddH₂O	1.64 g
MgSO₄ Solution (1 M)	MgSO₄ to 100 ml ddH₂O	12.04 g
Lysis Buffer	Na₃PO₄ Solution MgSO₄ Solution add 5 mg Lysozyme per ml Lysis Buffer before use	1 ml 1 ml
PBS-T	1 x PBS Tween 20	300 µl 300 µl
Blocking Buffer	BSA 1 x PBS Tween 20	0.2 g 40 ml 40 µl
10 x TBE	Trizma-Baze Boric Acid EDTA to 1000 ml ddH₂O	162 g 27.5 g 9.3 g
12 % Proline	Proline to 25 ml ddH₂O sterile filtered	3 g
2 x AB3	Antibiotic Medium NO. 3 (Difco) to 200 ml ddH₂O autoclaved	7 g
2 x SMM	Maleic Acid NaOH MgCl₂ x 6H2O Succrose Solve in 150 ml ddH₂O Adjust pH value to 6.5 with HCl to 250 ml ddH₂O. sterile filtered	1.16 g 0,8 g 2.03 g 85.58 g
20 % Glucose	Glucose to100 ml ddH₂O steril filtered	20 g
3.5 ml CR5-Topagar-Mixture	Solution A Solution B 8 x CR5-Salts 12 % Proline 20 % Glucose	1.75 ml 1 ml 0.346 ml 0.175 ml 0.175 ml
8 x CR5-Salts	K₂SO₄ KH₂PO₄ MgCl₂ x 6H₂O CaCl₂ to 625 ml ddH₂O autoclaved	1.25 g 0.25 g 50 g 11 g
Loading Dye for Agarose Gels	25 mM Tris-HCl Adjust pH value to 7.8 with HCl Bromophenol Blue Xylene Cyanol Glycerol EDTA	4 ml 3 mg 3 mg 6 ml
Ampicillin Stock Solution (100 mg/ml)	Ampicillin Ethanol ddH₂O	1000 mg 5ml 5ml
Chloramphenicol Stock Solution (10 mg/ml)	Chloramphenicol to 10 ml Ethanol	100mg
CR5-Topagar	SolutionA: Succrose MOPS NaOH to 250 ml ddH₂O Adjust pH value to 7.3 with NaOH Steril filtered Solution B: Agar Casamino Acids (Difco) Yeast Extract to 142.5 ml ddH₂O autoclaved	51.5 g 3.25 g 0.3 g 2 g 0.1 g 5 g
LB-Agar	LB-Medium (Sigma Aldrich) Agar to 400 ml ddH₂O autoclaved	10 g 6 g
LB-Medium	LB-Medium (Sigma Aldrich) to 400 ml ddH₂O autoclaved	12 g
PEG-P	PEG-6000 1 x SMM autoclaved	20 g 50 ml
SMMP	2 x AB3 2 x SMM	25ml 25 ml
SOC-Medium	Solution 1: Bacto Tryptone Yeast Extrakt 1 M KCl 1 M MgCl₂ 1 M MgSO₄ 5 M NaCl to 990 ml ddH₂O autoclaved Solution 2: 40 % Glucose (w/v) sterile filtered Add Solution 1 to Solution 2	20 g 5 g 2,5 ml 10 ml 10 ml 2 ml 10 ml
TB-Medium	Yeast Extrakt Tryptone Glycerol to 900 ml ddH₂O autoclaved Add 100 ml 10 x KPP Buffer	24 g 12 g 4 ml
Tetracyclin Stammlösung (10 mg/ml)	Tetracycline to 10 ml Ethanol	100 mg
Xylose Solution (250 mg/ml)	Xylose to 10 ml ddH₂O	2.5 g

5. Equipment


Equipment	Manufacturer
300 ml Erlenmeyer Flask	Schott Duran
300 ml Baffled Flask	Thermo Scientific
2000 ml Erlenmeyer Flask	Schott Duran
Ultra Low Temoerature Freezer	Heraeus
Freezer	Sigma Aldrich
Refrigerator	Liebherr
Autoclave: Systec VX-150	Systec
Cover Slips	Roth
Ice Machine	Ziegra
Electroporation Cuvettes (gap width 0.1 cm)	Sigma-Aldrich
Elektroporator: Easyject Prima	EquiBio
Falcon Tubes: 15 ml 50 ml	Sarstedt
Electrophoresis systems: PowerPACTM High-Current Power Supply Mini PROTEANTM 3 Cell PerfectBlue Gel System Mini S	Biorad Biorad Peqlab
Western Blot System: Blotting Device: OwlTM Separation System Power Supply: Voltcraft DPS-4005PFC	Thermo Fisher Scientific Conrad Electronic
Fluorescence microscopy: Inverse Fluorescence microscope Axiovert 200M CCD Digital camera Coolsnap HQ2	Zeiss Photometrics
Scale (MXX2001)	Denver Instruments
Micro scale (BP 210 S)	Sartorius
Gloves: Nitrile Latex	VWR
Imaging System Chemi DOC XRS+	Biorad
Incubator: Innova 4230	Innova
Cuvettes: Polystyrol/Polystyrene 10 x 4x 45 mm Quarz	Sarstedt
Magnetic Mixer IKAMAG® RCT	IKA® Labortechnik
Microliter Pipettes:	Brand
Microliter Pipette Tips:	Sarstedt
Microwave: Severin 700	Severin
Microscope slides	Roth
PCR-Cycler: Primus advanced 96	Peqlab
pH-Meter: 766 Calimatic	Knick
Photometer: UV2100 UV/VIS Recording Spectrophotometer	Shimadzu
Petri Dishes	Sarstedt
Reusable Glass Media Bottles with Cap	Fisher Scientific
Reaction Tubes: 1,5 ml 2 ml PCR-Softtubes	Sarstedt
Speedvac	UNIVAPO 100H
Thermomixer 5436	Eppendorf
Compartment Dryer	Memmert
Mini UV-Table (60-ECX-20M)	Peqlab
Water Bath SUB6 Grant	Biolab
Vortex Genie 2	Bender & Hobein
Centrifuges: Biofuge pico Multifuge 1L	Heraeus

6. Kits


Kit	Manufacturer
QIAprep Spin Miniprep Kit	Qiagen
QIAquick Gel Extraction Kit	Qiagen
Gibson Assembly TM Cloning Kit	NEB
DNA Distribution Kit 2014	iGEM
Nexttec TM 1-Step DNA Isolation Kit for Bacteria	NexttecTM

7. Strains


Strain	Genotype	Reference
E. coli TOP10 F`	F'{lacIqTn10(TetR)} mcrA ∆(mrr-hsdRMS-mcrBC) Φ80lacZ∆M15 ∆lacX74 recA1 araD139 ∆(ara-leu) 7697 galU galK rpsL endA1 nupG	Invitrogen
B. megaterium MS941	ΔnprM	Wittchen et al., 1995

8. Antibodies


Antibody	Manufacturer
Anti-GFP, N-terminal antibody produced in rabbit	Sigma Aldrich
Anti-Rabbit IgG peroxidase produced in goat	Sigma Aldrich

Team:Saarland/Materials

From 2014.igem.org

Materials

1. Chemicals

2. Enzymes

3. Oligonucleotides

4. Media and Solutions

5. Equipment

6. Kits

7. Strains

8. Antibodies

Impressum/Copyright