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Contents 1 Recipes 1.1 Media 1.1.1 LB-broth 1.1.2 M9 Swimming Agar 1.1.3 Tryptone swimming agar 1.2 Antibiotics 1.3 Supplements 1.4 Buffer and Solution 1.4.1 5x M9 salts stock solution 1.4.2 CaCl2 Buffer for competent cells 1.4.3 50x TAE for agarose gels 2 E. coli 2.1 Non-mutated strains 2.1.1 DH10B 2.1.1.1 Genotype 2.1.1.2 Genetical Characteristics 2.1.1.3 Reference 2.1.2 DH5α 2.1.2.1 Genotype 2.1.2.2 Genetical Characteristics 2.1.2.3 Reference 2.1.3 BL21DE3 2.1.3.1 Genotype 2.1.3.2 Genetical Characteristics 2.1.3.3 Reference 2.1.4 XL1 blue 2.1.4.1 Genotype 2.1.4.2 Genetical Characteristics 2.1.4.3 Reference 2.1.5 MG1655 2.1.5.1 Genotype 2.1.5.2 Genetical Characteristics 2.1.5.3 Reference 2.1.6 RP437 2.1.6.1 Genotype 2.1.6.2 Genetical Characteristics 2.1.6.3 Reference 2.2 Mutated strains 2.2.1 ∆tar 2.2.1.1 Genotype 2.2.1.2 Genetical Characteristics 2.2.1.3 Reference 2.3 Storage 2.3.1 Glycerol Stock 2.3.2 LB-agar

Recipes

Media

Recipes for liquid and solid media for bacterial cultivation. Stir until dissolved; sterilize by autoclaving 121 °C. Do not forget a magnetic stirrer.

LB-broth

• 1 % (w/v) bacto tryptone
• 0.5 % (w/v) bacto yeast extract
• 1 % (w/v) NaCl
• if required: ad 1 % (w/v) agar

M9 Swimming Agar

• 1.25 % (v/v) glycerol
• 20 % (v/v) 5x M9 salt stock solution
• 0.1 % (v/v) of CaCl₂ x 2 H₂O stock solution (20 mg/mL)
• 0.1 % (v/v) MgSO₄ stock solution (0.12 g/mL)
• 0.3 % (w/v) of agar

Tryptone swimming agar

• 1 % (w/v) bacto tryptone
• 0.5 % (w/v) NaCl
• 0.3 % (w/v) agar

Antibiotics

• Stock solutions and concentrations of antibiotics.

Reagent	Abbreviation	Stock Conc. [mg/mL]	Working Conc. [µg/mL]	Dilution	Solvent	Notes
Ampicillin	Amp	100	100	1,000x	ddH2O	Culture plates with Amp can be stored at 4 °C for about 2 weeks. Stock solutions can be stored at 4 °C for 2 weeks but can last as long as 4-6 months when stored at -20 °C.
Chloramphenicol	Cm	20	20	1,000x	EtOH
Kanamycin	Kan	50	50	1,000x	ddH2O	Plates may only last a few weeks at 4 °C.

Supplements

• Aminoacid mix (containing all aminoacids except tryptophane): stocksolution 0.2 % (w/v), varying amounts used in swimming assays, view methods and notebook of group 1
• Caffeine: stocksolution 10 mM, 100 µL applied to whatmanpaper in swimming assays, view methods
• D-Aspartate: stocksolution 10 mM, 100 µL applied to whatmanpaper in swimming assays, view methods
• 2-Ethyl-1-Hexanole: stocksolution 5 mM, 100 µL applied to whatmanpaper in swimming assays, view methods
• Geraniol: stocksolution 5 mM, 100 µL applied to whatmanpaper in swimming assays, view methods
• L-Aspartate-4-Benzylester: stocksolution 10 mM, 100 µL applied to whatmanpaper in swimming assays, view methods
• L-aspartate: stocksolution 10 mM, varying amounts used in swimming assays, view methods and notebook of group 1
• Leucin: stocksolution 30.49 mM, add to M9-agar: 4 mL (final concentration 0.305 µM)
• Methionin: stocksolution 26.8 mM, add to M9-agar: 4 mL (final concentration 0.268 µM)
• Sodium Cyclamate: stocksolution 10 mM, 100 µL applied to whatmanpaper in swimming assays, view methods
• Tryptone: stocksolution 0.5 % (w/v), varying amounts used in swimming assays, view methods and notebook of group 1
• Vanillin: stocksolution 10 mM, 100 µL applied to whatmanpaper in swimming assays, view methods

Buffer and Solution

Recipes for stocks of buffers and solutions.

5x M9 salts stock solution

Amount	Substance
64 g	Na2HPO4 × 7 H2O
15 g	KH2PO4
2.5 g	NaCl
5.0 g	NH4Cl
ad 1000 ml	ddH2O

• Stir until dissolved

CaCl₂ Buffer for competent cells

Amount	Substance	Notes
7.351 g	CaCl2 × 7 H2O	Alternative 0.1 M CaCl2
15%	Glycerol
ad 500 ml	ddH2O

• Stir until dissolved; sterilize by sterile filtration.

50x TAE for agarose gels

Amount	Substance
242 g	Tris base
57.1 mL	Glacial acetic acid
18.6 g	EDTA
ad 1000 mL	ddH2O

• Stir until dissolved. Working concentration is 1x (40 mL of 50x stock ad 2 L ddH₂O).

E. coli

• E. coli strain list - List of the working E. coli strains including genotypes, genetic markers, and alleles.

Non-mutated strains

These strains may not contain any plasmids or resistances, because they should be used for transformation or other experiments.

DH10B

Genotype

• F- endA1 recA1 galE15 galK16 nupG rpsL ΔlacX74 Φ80lacZΔM15 araD139 Δ(ara,leu)7697 mcrA Δ(mrr-hsdRMS-mcrBC) λ-

Genetical Characteristics

• Antibiotic resistance: none

Reference

• Invitrogen

DH5α

Genotype

• F- endA1 glnV44 thi-1 recA1 relA1 gyrA96 deoR nupG Φ80dlacZΔM15 Δ(lacZYA-argF)U169, hsdR17(rK- mK+), λ–

Genetical Characteristics

• Antibiotic resistance: none

Reference

• Invitrogen

BL21DE3

Genotype

• E. coli B F- dcm ompT hsdS(rB- mB-) gal [malB+]K-12(λS)

Genetical Characteristics

• Antibiotic resistance: none!

Reference

• Merck Millipore

XL1 blue

Genotype

• endA1 gyrA96(nalR) thi-1 recA1 relA1 lac glnV44 F'[ ::Tn10 proAB+ lacIq Δ(lacZ)M15] hsdR17(rK- mK+)

Genetical Characteristics

• Antibiotic resistance: none!

Reference

MG1655

Genotype

• F-λ-, rph-1

Genetical Characteristics

• Antibiotic resistance: none!

Reference

• Coli Genetic Stock Center

RP437

Genotype

• F-, thr-1, araC14, leuB6(Am), fhuA31, lacY1, tsx-78, λ-, eda-50, hisG4(Oc), rfbC1?, rpsL136(strR), xylA5, mtl-1, metF159(Am), thiE1

Genetical Characteristics

• Antibiotic resistance: none!

Reference

• Coli Genetic Stock Center

Mutated strains

∆tar

Genotype

• F-, Δ(araD-araB)567, ΔlacZ4787(::rrnB-3), λ-, Δtar-739::kan, rph-1, Δ(rhaD-rhaB)568, hsdR514

Genetical Characteristics

• Antibiotic resistance: Kanamycin

Reference

• Coli Genetic Stock Center

Storage

Storage of E. coli strains - How to keep E. coli strains.

Glycerol Stock

The glycerol stock is prepared as follow:

• 900 µL over night culture in LB-liquid medium
• 900 µL 50 % sterile glycerol

Glycerol is extremely viscous. Hence, use cut off tips to pipette. Glycerol stocks were vortexed to ensure uniformly mixture and shock frozen in N₂ or dry ice. Then, cultures were stored at -80 °C. Do NOT forget to fill in the according GMO S1 construction sheet!

LB-agar

LB-agar plates were prepared as follow:

• 1% agar (w/v) was added to 1 L LB-broth
• LB-agar was autoclaved
• LB-agar was poured in plates

The E. coli strains were plated out and stored at 4°C. The plates were renewed every two weeks.

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