Protocols

Genetic Engineering

Whole Genome Isolation

• For the isolation following kit has been used: Fermentas GeneJET™ Plasmid Miniprep Kit
• Standard Protocol:
  • Centrifuge 10 mL of over-night liquid culture
  • Resuspend pellet in 800 µL of resuspension solution
  • Ribolyse three times for 60 s with 6500 rpm (30 s break between every run, 400 mg beads)
  • Centrifuge 3 min at 10,000 rpm
  • Transfer 500 µL of supernatant into new 2 mL tube
  • Add 500 µL of lysis buffer, invert 4 - 6 times
  • Add 700 µL of neutralisation buffer, invert 4 - 6 times
  • Centrifuge 10 min at 12,000 rpm
  • Transfer 2 x 600 µL of supernatant into spin column (centrifuge down each time for 30 s at 12,000 rpm)
  • Two wash steps with 750 µL of washing buffer
  • Dry column
  • Elute in 75 µL of elution buffer

Generating electrocompetent cells

• Material:
  • 550 mL LB-Medium
  • 1 L cooled bidest. H2O
  • 150 mL cooled 10 % glycerine
  • 10 pre-cooled 50 mL Falcons

• Protocol:
  • Inoculate 2x3 mL LB with bacterial stock; incubate over night at 37 °C and 200 rpm
  • Inoculate 2x250 mL LB with the over night cultures in 1-litre-flask at 37 °C and 140 rpm
  • Incubate until OD600 0,4-0,6
  • Cool the culture 15-30 minutes on ice
  • Onwards all steps at 4°C
  • Divide the cultures into cooled 50 mL Falcons and centrifugate at 4000 rpm, 4 °C for 15 minutes, make sure to slowly accelerate and deccelerate
  • Discard supernatant
  • Resuspend pellet in 5 mL cooled bidest H2O (and don't get frustrated while doing it, keep shaking gently)
  • Pool two suspensions each, add bidest H2O up to 50 mL and centrifugate again (see centrifugation above)
  • Discard supernatant
  • Resuspend pellet in 5 mL cooled bidest H2O
  • Add bidest H2O up to 50 mL and centrifugate again (see centrifugation above)
  • Discard supernatant
  • Resuspend pellet in 5 mL cooled 10 % glycerine
  • Transfer suspensions in two 50 mL Falcons and centrifugate again (see centrifugation above)
  • Discard supernatant
  • Add volume of 10 % glycerine that is approximately equal to the volume of the pellet and resuspend
  • Divide cells in 50 μL aliquots and freeze in liquid N2 immediately
  • Store at -80 °C

Transformation via electroporation

• Thaw 50 µL competent E.coli cells on ice, dilute with icecold 50 µL glycerine (10 %) if necessary
• Add 0.5-5 µL plasmid to 50 µl electrocompetent cells
• Store cells on ice for 1 minute
• Electroporate at U = 2.5 kV, C = 25 µF, R = 400 Ώ
• Transfer transformation reaction to 450 µL SOC-Medium and shake 1 h at 37 °C
• Centrifuge 2 min at 2000 rpm and plate on selective LB-Medium
• Incubate over night at 37 °C

Analytics

SDS-PAGE

• Pouring the polyacrylamide gel
  • Make a master mix for the stacking and separating gel without adding ammonium persulfate and TEMED
  • Aliquote 6,5 mL for each separating and 2,5 mL for each stacking gel
  • Add ammonium persulfate and TEMED to each separating gel aliquote and pour the solution quickly into your gel casting form. Leave about 2 centimeters below the bottom of the comb for the stacking gel
  • Layer isopropanol on top of the gel
  • Leave the separating gel at room temperature for >60 minutes to polymerize
  • Remove isopropanol and wait until the surface is dry
  • Add ammonium persulfate and TEMED to each separating gel aliquote and pour the solution quickly into your gel casting form
  • Insert comb without getting bubbles stuck underneath
  • Leave the gel at room temperature for >60 minutes to polymerize

• For storage:
  • Remove sealing and store the gel wrapped in moistened paper towel at 4°C

• Preparing the sample
  • Mix your protein mixture 4:1 with Laemmli-buffer (30 mL protein solution + 10 mL Laemmli-buffer)
  • Heat for 5 minutes at 95 °C

• Running the gel
  • Remove sealing, put the polymerized gel into gel box and pour SDS running buffer into the negative and positive electrode chamber
  • Remove comb without destroying the gel pocket
  • Pipet the sample into the gel pockets, adjusting the volume according to the amount of protein in your sample. Make sure to include a lane with molecular weight standards (PageRuler Prestained Protein Ladder™ (Fa. Fermentas)) to determinate the molecular weight of your sample
  • Connect the power lead and run the stacking gel with 10 mA until the blue dye front enters the separating gel
  • Raise amperage up to 20 mA for running the separating gel
  • When the distance of the lowest molecular weight standard lane to the gel end is down to 0.5 cm stop the electrophoresis by turning off the power supply

NADH-Assay

• This method has been used for measurement of intracellular NADH concentration.

• Protocol:
  • Inoculate an overnight culture (30 mL with 1 mL of pre-culture)
  • Centrifugate (5 min at 5000 g) of 6-10 mL overnight culture (OD 4-6), adjust volume between different samples for the approximation of the number of cells
  • Discard supernatant and wash pellet 3 times with 1 mL of PBS buffer
  • Resuspend pellet in 1 mL PBS buffer
  • Cell disruption by Ribolysation (3 x 30 sec at 6500 rpm)
  • Centrifugation for 10 min at maximum speed
  • Store supernatant at - 20 ° C or direct measurement with Tecan Infinite® M200 platereader

• Tecan Infinite® M200 platereader parameters:
  • Sample volume = 100 μL clear supernatant
  • Excitation = 340 nm
  • Emission = 460 nm
  • Concentration calculation by NADH calibration curve

Hexadecan Assay

• This assay has been used to measure cell membrane hydrophobicity.

• Protocol
  • Inoculate an overnight culture (30 mL with 1 mL of pre-culture)
  • Centrifugate (5 min at 4000 g) of 2 mL overnight culture (OD 4-6)
  • Discard supernatant and wash pellet 3 times with 1 mL of PBS buffer
  • Resuspend pellet in 1 mL 0,9% NaCl and measurement of OD600
  • Add x μL of washed cells to 0,9% NaCl for a final volume of 3 mL . Final OD600 should be approximately 0,3 (denoted as A0, calculate exact value)
  • Add 3 mL Hexadecan and vortex for 60 sec
  • Incubation for 15 min
  • Discard the upper organic phase and measure OD600 of the aqueous phase (denoted as A)
  • Hydrophobicity can be calculated using the equation: affinity [%] = 100 x [1 – (A/A0)]

Cold osmotic shock

• Release of periplasmic protein fraction from E. coli by cold osmotic shock

• Modified protocol from Neu & Heppel (1965)
  • Centrifuge E. coli cell suspension for 5 min at 14,000 g (4 °C) to collect the cells
  • Discard the entire supernatant
  • Resuspend the cells in ice-cold cell fractionating buffer 1. The resulting volume should be 1/4 of the former suspension volume
  • Incubate for 20 min on ice. Invert the suspension at regular intervals to counteract sedimentation
  • Centrifuge the cell suspension for 15 min at 14,000 g (4 °C)
  • Discard the entire supernatant
  • Resuspend the cells in ice-cold cell fractionating buffer 2. The resulting volume should be 1/4 of the former suspension volume
  • Incubate for 10 up to 20 min on ice under regular invertion
  • Centrifuge the cell suspension for 15 min at 14,000 g (4 °C)
  • Save the supernatant, which contains the periplasmatic proteins and membrane proteins when using Cell fractionating buffer 2.2 and 2.3
  • If the periplasmatic protein fraction is turbid, re-centrifuge and filter it through a 0.2 µm filter

Cultivation

Cultivation in liquid LB-Medium

Media

Chloramphenicol stock solution

• Solubilize 20 mg mL-1 Chloramphenicol in 100 % Ethanol
• Store at -20 °C

DNA loading buffer

• 50 % (v/v) glycerol
• 1 mM EDTA
• 0.1 % (w/v) bromphenol blue
• Solve in ddH2O

LB medium

• 10 g Trypton
• 5 g yeast extract
• 10 g NaCl
• 12 g Agar-Agar (for plates)
• Adjust pH to 7.4

M9 minimal medium

• For 1 L M9 mineral medium 867 mL sterile water is needed(for plates add 16 g Agar-Agar as well)
• Then add in the following order:
  • 00 µL 1 M CaCl2
  • 1 M CaCl2
  • 1 M CaCl2-H20
  • 14.70 g/100 mL

• 100ml 5x M9 salt solution
  • 10x M9 salt solution
    • Na2HPO4-2H2O 75.2 g/L
    • KH2PO4 30 g/L
    • NaCl 5 g/L
    • NH4Cl 5 g/L

• 20 % glucose
  • 20 % (w/v)glucose 200 g/L
  • For 500 mL stock solution add 100 g glucose to 440 mL water. Autoclave for 15 at 121°

• 1 M MgSO4 </p>
  • 1 M MgSO4-7H20 24.65 g/100 mL

• 1 mL Biotin (1 mg/mL)
  • Biotin (1 mg/mL) 50 mg/50 mL
  • For 50 mL stock solution dissolve 50 mg biotin in 45 mL water. Add water to a final volume of 50 mL. Sterilize the solution over a 0.22-µm filter. Prepare 1 mL aliquots and store at -20°.

• 1 mL Thiamin (1 mg/mL)
  • Thiamin-HCl (1 mg/mL) 50 mg/50 mL
  • For 50 mL stock solution dissolve 50 mg thiamin-HCl in 45 mL water. Add water to a final volume of 50 mL. Sterilize the solution over a 0.22-µm filter. Prepare 1 mL aliquots and store at -20°.

• 10 mL 100x trace elements solution
  • 100x trace elements solution
    • EDTA 5 g/L 13.4 mM
    • FeCl3-6H2O 0.83 g/L 3.1 mM
    • ZnCl2 84 mg/L 0.62 mM
    • CuCl2-2H2013 mg/L 76 µM
    • CoCl2-H2O 10 mg/L 42 µM
    • H3BO3 10 mg/L 162 µM
    • MnCl2-4H20 1.6 mg/L 8.1 µM
    • Dissolve 5g EDTA in 800 mL water and adjust the pH to 7.5 with NaOH. Then add the other components in the quantities mentioned below and add water to a final volume of 1L. Sterilize the solution over a 0.22µm filter</p>

• Unknown
  • 498 mg FeCl3(anhydrous)
  • 84 mg ZnCl2
  • 765 µL 0.1 M CuCl2-2H20 1.70 g/100 mL
  • 210 µL 0.2 M CoCl2-H2O 4.76 g/100 mL
  • 1.6 mL 0.1 M H3BO3 0.62 g/100 mL
  • 8.1 µL 1M MnCl2-4H20 19.8 g/100 mL

SOC medium

• Add the following components for 900 ml of distilled H2O:
  • 20 g Trypton
  • 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.

Buffers & Solutions

Biotin 500x Stock

• Dissolve 20 mg biotin in 100 mL of 0.05 M NaOH solution and filter sterilize
• Store at 4 °C
• Durable for one year

TAE-Buffer

• 1 L of 50x TAE buffer
  • 242.48 g Tris
  • 41.02 g Sodiumacetate
  • 18.612 g EDTA
  • Adjust pH to 7.8 with acetic acid
  • Solve in dH2O

• • Dilute 20 mL 50x stock in 1L dH2O for 1x Buffer for PAGE

Cell Fractioning Buffers

CFB 1 (pH 8)

• 0.2 M Tris
• 200 g L -1 sucrose
• 0.1 M EDTA

CFB 2.1 (pH 8)

• 0.01 M Tris
• 0.005 M MgSO4

CFB 2.2 (pH 8)

• 0.01 M Tris
• 0.005 M MgSO4
• 1% Trition
• 2% SDS

CFB 2.3 (pH 8)

• 0.01 M Tris
• 0.005 M MgSO4
• 1% Trition
• 0.2% SDS

CFB 2.4 (pH 8)

• 0.01 M Tris
• 0.005 M MgSO4
• 1% Trition
• 1% SDS

Contents 1 Genetic Engineering 1.1 Whole Genome Isolation 1.2 Generating electrocompetent cells 1.3 Transformation via electroporation 2 Analytics 2.1 SDS-PAGE 2.2 NADH-Assay 2.3 Hexadecan Assay 2.4 Cold osmotic shock 3 Cultivation 3.1 Cultivation in liquid LB-Medium 4 Media 4.1 Chloramphenicol stock solution 4.2 DNA loading buffer 4.3 LB medium 4.4 M9 minimal medium 4.5 SOC medium 5 Buffers & Solutions 5.1 Biotin 500x Stock 5.2 TAE-Buffer 5.3 Cell Fractioning Buffers 5.3.1 CFB 1 (pH 8) 5.3.2 CFB 2.1 (pH 8) 5.3.3 CFB 2.2 (pH 8) 5.3.4 CFB 2.3 (pH 8) 5.3.5 CFB 2.4 (pH 8)