M9-Minimal-Media

Prepare Stock Solutions:

• M9 salt solution (10X)

Na2HPO4 * 2 H2O 75.2 g/L

KH2PO4 30 g/L

NaCl 5 g/L

NH4Cl 5 g/L

Dissolve the salts in 800 ml ddH2O and adjust the pH to 7.2 with NaOH. Add ddH2O to a final volume of 1 L and autoclave for 15 min at 121°C.

• 20% Glucose stock solution

Glucose * H2O 220 g/L

Add ddH2O to a final volume of 1 L and autoclave for 15 min at 121°C.

• 1M MgSO4 stock solution

MgSO4 * 7 H2O 24.65 g/100 ml

Add ddH2O to a final volume of 100 ml and autoclave for 15 min at 121°C.

• 1M CaCl2 stock solution

CaCl2 * 2 H2O 14.70 g/100 ml

Add ddH2O to a final volume of 100 ml and autoclave for 15 min at 121°C.

• Biotin (1 mg/ml)

Dissolve 50 mg biotin in 45 ml ddH2O. Add small aliquots of 1M NaOH until the biotin has dissolved. Add ddH2O to a final volume of 50 ml. Sterilize the solution over a 0.22 µm filter. Prepare 1 ml aliquots and store at -20°C.

• Thiamin (1 mg/ml)

Dissolve 50 mg thiamin-HCl in 45 ml ddH2O. Add ddH2O to a final volume of 50 ml. Sterilize the solution over a 0.22 µm filter. Prepare 1 ml aliquots and store at -20°C.

• Trace elements stock solution                          

FeSO4 * 7 H2O    40.0 g/L

MnSO4 * H2O    10.0 g/L

AlCl3 * 6 H2O    10.0 g/L

CoCl2 * 6 H2O    7.3 g/L

ZnSO4 * 7 H2O    2.0 g/L

Na2MoO4 * 2 H2O    2.0 g/L

CuCl2 * 2 H2O    1.0 g/L

H3BO3    0.5 g/L

37% (12 M) HCl conc.    414 mL/L

Dissolve in HCl diluted 1:1 with ddH2O and fill up to 1 L with ddH2O; the final solution is 5 M HCl and can be stored indefinitely at RT. Sterilize the solution over a 0.22 µm filter before use.

• Yeast extract Stock (100 g/L)

Yeast extract 100 g

Add ddH2O to a final volume of 1 L. Sterilize the solution over a 0.22 µm filter.

Mix all components in a sterile bottle.

Adjusting the pH of M9- and LB-media

to adjust the pH of M9 media from 7.4 to 8.5, add 0.68ml of 2M NaOH to 100 ml M9 (be carefull, unknown precipitates appear)

to adjust the pH of M9 media from 7.4 to 6, add 4.68ml of 2M HCl to 100 ml M9

to adjust the pH of LB media from pH 7 to pH 8.5, add 0.66ml of 2M NaOH to 100ml  LB

to adjust the pH of LB media from pH 7 to 6.0, add 0.575 ml of 2M HCl to 100 ml  LB

LPM-media

50 mM Tris base

20 mM KCl

7.5 mM (NH4)2SO4

240 µM MgCl2

18.5 µM CaCl2

6.5 µM FeCl3

0.2% Glucose

0.6% Bacto peptone

• buffer with 0.1 M MOPS for pH-range 7 to 8.5, adjust pH with NaOH /

          buffer with 0.1 M MES for pH 5 to 4, adjust pH with HCl

• fill up to final volume with ddH2O

Electro-competent cells

• inoculate single colony from a fresh plate 10 mL ONC (in LB)

• 500 mL LB main culture (5 mL ONC)

• growth (at 30°C) until OD 0.35-0.4
• chill on ice 10-20 min
• centrifuge (4000 rpm, 10 min, 4°C)
• resuspend (carefully!) in 2x 400 mL ice-cold water
• centrifuge (4000 rpm, 10 min, 4°C)
• resuspend (carefully!) in 400 mL ice-cold 10% glycerol
• centrifuge (4000 rpm, 10 min, 4°C)
• resuspend in 10 mL 10% glycerol
• prepare 40 µL aliquots (on ice!)
• shock-freeze them in liquid nitrogen
• store at -80°C (up to 6 months)

Transformation (electro-competent cells)

• thaw cells on ice
• add 1-2 µl DNA (up to 2 ng) or 2-10 µl Ligationmix (on ice!)
• transfer cells into a cuvet
• electro-shock
• add 750-900 µl LB- or SOC-media
• resuspend carefully by pipetting up and down
• transfer to an new steril tube
• regenerate for 45 min at 37°C and shake at 300 rpm
• plate on LB-plate (with antibiotic)
• incubate overnight at 37°C
• check for colonies

Transformation (chemically competent cells for Gibson Cloning)

• thaw cells on ice
• add 2 µl DNA (on ice!)
• place mixture on ice for 30 min
• heat-shock at 42°C for 1 min (thermomixer)
• transfer on inc for 2 min
• add 750-900 µl LB- or SOC-media
• regenerate for 45 - 60 min at 37°C and shake at 300 rpm
• plate on LB-plate (with antibiotic)
• incubate overnight at 37°C
• check for colonies

PCR with Q5-polymerase with proofreading activity from NEB

X µl template (up to 1 ng)

2.5 µl fw_primer (10 µM)

2.5 µl rev_primer (10 µM)

0.5 µl Q5-Polymerase

10 µl Q5-Buffer 5x

10 µl GC-Enhancer (additional)

1 µl dNTP´s (10 mM)

26.6 - X µl ddH20

50 µl in total    

PCR with GoTaq-polymerase without proofreading activity from Fermentas

X µl template (< 0.05 µg/10 µl)

0.5 µl fw_primer  (10 µM)

0.5 µl rev_primer  (10 µM)

0.4 µl MgCl2

0.2 µl dNTP´s (10 mM)

0.05 µl GoTaq-Polymerase

2 µl Polymerasepuffer 5xGreen

6.35 - X µl ddH20

10 µl in total    

Gibson-Cloning (NEBuilder HiFi DNA Assembly Cloning Kit)

• Set up the following reaction on ice:

2 – 3 fragments: vector:insert = 1:2 (use 50 – 100 ng of vector)

X µl total amount of fragments (0.03 – 0.2 pmol)

10 µl NEBuilder HiFi DNA Assembly Master Mix

10 - X µl ddH20

20 µl in total   

4 – 6 fragments: vector:insert = 1:1 (use 50 – 100 ng of vector)

X µl total amount of fragments (0.2 – 0.5 pmols)

10 µl  NEBuilder HiFi DNA Assembly Master Mix

10 - X µl ddH20

20 µl in total     

• Incubate sample at 50°C for 15 min for 2 – 3 fragments or 60 min for 4 – 6 fragments
• Transform 2 µl of the assembly reaction into competent cells

Restriction Digest (Fast Digest Enzymes from ThermoScientific)

• set up following reaction:

1 µl enzyme (XbaI, EcoRI, PstI, BcuI)

2 µl FastDigest Green Buffer 10x

X µl DNA (plasmid DNA up to 1 µg or ~0.2 µg PCR product)

17 - X µl ddH0

20 µl in total    

• incubate for at least 15 min at 37°C
• inactivate for 5 min at 80°C or purify with PCR-purification kit

Ligation

1 µl T4-Liagse

2 µl T4 Ligase Buffer 10x

X µl insert DNA (1:1 molar ratio over vector)

Y µl vector DNA (20 – 100 ng)

17 – (X+Y) µl ddH20

20 µl in total    

• incubate for at least 15 min at 17 - 22°C
• inactivate for 10 min at 65°C or purify with PCR-purification kit
• ready for transformation

Miniprep (Wizard® Plus SV Miniprep DNA Purification System from Promega)

• pellet 1 – 10 ml ONC
• resuspend pellet with 250 µl Cell Resuspension Solution
• add 250 µl Cell Lysis Solution and invert to mix
• add 10 µl Alkaline Protease Solution and invert to mix
• incubate for 5 min at RT
• add 350 µl Neutralizations Solution and invert to mix
• centrifuge at top speed for 10 min
• insert Spin Column into Collection Tube
• decant cleaned lysate into Spin Column
• centrifuge at top speed for 1 min
• discard flow through
• add 750 µl Wash Solution
• centrifuge at top speed for 1 min
• discard flow through
• add 250 µl Wash Solution
• centrifuge at top speed for 5 min
• discard flow through
• centrifuge for another 5 min to get rid of ethanol
• transfer Spin Column to a new reaction tube
• add 20 – 50 µl nuclease free water
• centrifuge at top speed for 1 min

Miniprep (Wizard® SV Gel and PCR Clean-Up System from Promega)

A: Dissolving a gel slice

• transfer gel into a reaction tube
• add 10 µl Membrane Binding Solution per 10 mg gel slice
• Incubate at 50 – 65°C until the gel is completely dissolved
• Vortex

B: Processing PCR products

• Add an equal volume of Membrane Binding Solution

• Insert Spin Column into Collection Tube
• Transfer dissolved gel mixture or prepared PCR product into the Spin Column
• Incubate for 1 min at RT
• Centrifuge at top speed for 1 min
• Discard flow through
• Add 700 µl Washing Solution
• Centrifuge at top speed for 1 min
• discard flow through
• add 500 µl Wash Solution
• centrifuge at top speed for 5 min
• discard flow through
• centrifuge for another 5 min to get rid of ethanol
• transfer Spin Column to a new reaction tube
• add 20 – 50 µl nuclease free water
• incubate for 1 min at RT
• centrifuge at top speed for 1 min

Cultivation of Escherichia coli in M9-Minimal-Media

Add appropriate selection marker to all media involved.

• Pick a colony from an agar-plate and inoculate LB-Media with it, incubate at 28°C/37°C overnight (ONC).
• Centrifuge down the cells, remove media and resuspend in M9-minimal-media with yeast extract. Incubate overnight at 28°C/37°C.
• Centrifuge down the cells, remove media and resuspend in M9-minimal-media. Incubate overnight at 28°C/37°C.

Inducing expression of fluorescence proteins for detection

Alkaline induction of alx promoter

• ONC in LPM with pH 7.0
• Inoculate 20 ml of pH 7.0, 8.0 and 8.5
• After 20 and 40 minutes take 1 ml of each culture
• Measure OD600 and fluorescence (mNeonGreen excitation 490 nm, absorbance 520 nm)
• Divide fluorescence by OD600 to standardize the values

Acid induction of asr promoter

• ONC in LPM with pH 7.0
• Inoculate 100 ml of LPM wit pH 7.0, 5.0 and 4.5 to an OD600 of 0.2
• After 1, 1.5 and 2 hours take 3 OD unit samples of each culture

Westernblot

SDS-Page

• assemble electrophoresis chamber and fill it with MES buffer
• load standard and samples
• run SDS-page at 180V for approximately 45 min

Blotting

• fill up the blotting chamber with Transferbuffer (20% Methanol) and soak sponges
• assemble blotting chamber with SDS-gel, nitrocellulose membrane, filters and sponges
• blot at 230 mA for 75 min

Detection

• block membrane overnight with TBST milk (5%) at 4°C
• wash 3 times with TBST (0,05% Tween20) for about 5 min each time
• incubate membrane with peroxidase linked antibody for 2 h
• wash 3 times with TBST (0,05% Tween20) for about 5 min each time
• incubate with peroxidase substrate for about 1 min
• detect substrate (choose detection method suitable for specific substrate)