Team:Goettingen/Project/Materials

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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-Medium

• 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.

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

• Stir until dissolved

CaCl₂ Buffer for competent cells

• Stir until dissolved; sterilize by sterile filtration.

50x TAE for agarose gels

• 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. 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

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

BL21

Genotype

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

Genetical Characteristics

• Antibiotic resistance: none!

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!

MG1655

Genotype

F-λ-, rph-1

Genetical Characteristics

• Antibiotic resistance: none!

Reference

http://cgsc.biology.yale.edu/Strain.php?ID=4837

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

http://cgsc.biology.yale.edu/Strain.php?ID=111960

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!

Storage on 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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