Experiments

Day 1: Transformation in DH5α Escherichia coli
Day 2: Preparation of overnight culture

• Pick clones with pipette tip and inoculate culture tube with 5 mL LB medium + 100 µg/mL ampicillin.
• Incubate culture at 37 °C overnight.

Day 3: Miniprep

• Follow Miniprep Kit protocol ROTI®Prep Plasmid MINI-XL kit, Carl Roth.

Protocol for 25 µL sample.

• Prepare mix with with the following substances:

Substance	Volume
10 mM FW Primer	0,5 µL
10 mM Rev Primer	0,5 µL
OneTaq® 2X Master Mix	12 µL
Nucelase free Water	11,5 µl

• For information about primers used in this protocol see chapter primers.
• Pick one colony with a pipette tip and mix with sample solution
• Run reaction as following:

Step	Temperature	Time
initial denaturation	95 °C	5 min
35 cycles	95 °C	10 sec
	60,5 °C	10 sec
	72 °C	1 min
final extension	72 °C	5 min
	4 °C	hold

We purified DNA by centrifugation by using the Wizard® SV Gel and PCR Clean-Up System kit, Promega.

• Thaw 100 µL of competent cells on ice for 20 min.
• Add 100 ng/µL plasmid to aliquot and resuspend carefully.
• Incubate the cell-plasmid suspension on ice for 30 min.
• Incubate solution at 42 °C for exactly 45 seconds.
• After incubation, transfer cells instantly on ice and incubate for 5 min.
• Add 400 µL of SOC medium at room temperature to the cells.
• Incubate cells at 37 °C and 300 rpm for 1 hour.
• Plate the sample on LB-Agar containing 100 µg/mL ampicillin.
• Incubate at 37 °C overnight.

• Components:

Substance	Volume
DNA	0,3 ng
SapI	1 µL
BsaI-HF-v2	1 µL
rCutSmart buffer	5 µL
H20	up to 50 µL

• Incubate at 37 °C for 1 hour

Kinase, Ligase and DpnI digest to circularize PCR products.

1. Prepare buffer (for 5 reactions):
• 5 µL T4 DNA Ligase Buffer 10x
• 2.5 µL ddH₂0
2. Reaction mixture:

   Substance	Volume
   Buffer	1.5 µL
   DNA (PCR Product)	5.5 µL
   T4 Polynucleotidkinase (PNK)	1.0 µL
   DpnI	1.0 µL
   Total	10 µL

3. Incubate for 2 hours at room temperature (20 °C).
4. Store DNA at -20 °C.

For further information see KLD Enzyme Mix Reaction Protocol.

Use template before PCR reaction as control.

Protocol adapted from group Prof. Dr. Tobias Beck.
Used bacterial strain: BL21 (DE3) Star

Day 1: Transformation via heat stock method

• Thaw 100 µL of competent BL21 (DE3) Star cells on ice for 20 minutes.
• Transfer 50 µL of the cells in eppi tubes.
• Add 5 µL of DNA.
• Incubate samples on ice for 30 minutes.
• Heat cells at 42 °C for exactly 45 seconds.
• Place samples directly on ice and incubate them for another 5 minutes.
• Add 450 µL of preheated SOC-medium to the sample.
• Incubate cells for 1 h at 37 °C and 300 rpm.
• Centrifuge cells for 1 min at room temperature and 16,000 g.
• Discard 400 µL of the supernatant and resuspend the cells in the remaining medium.
• Plate cells out on LB Agar with ampicillin (100 µg/mL) plates and store overnight at 37 °C.

Day 2: Pre-culture preparation

• Pick colonies and add them into TB-Media containing 100 µg/mL ampicillin.
• Incubate cells at 37 °C and 180 rpm.

Day 3: Induction of the main culture

• Prepare a flask with a size of at least 5 times of the required volume of main culture.
• Prepare TB medium containing 100 µg/mL ampicillin.
• Add 2 mL of pre-culture into the TB medium.
• Incubate at 37 °C and 200 rpm shaking until OD600 0.6-0.7 is reached.
• Induce with 0.25 mM IPTG and incubate for 48 h at 18 °C and 200 rpm shaking.

Day 4: Harvest

• Spin down cells for 20 min and 5000 g at 4 °C.
• Discard supernatant and resuspend the pellet in 40 mL storage buffer (50 mM Tris, 0.30 M NaCl, pH 7.5).
• Transfer the suspension into a 50 mL falcon and spin down at 5000 g, 4 °C for 20 min.
• Discard supernatant and freeze pellet with liquid nitrogen.
• Store at -80 °C until lysis day.

Protocols adapted from group Prof. Dr. Tobias Beck.

1. Preparation of gels and SDS sample buffer
1. Prepare seperation gels
• For each gel (12 %) aliquot (5 mL gel volume):

  Substance	Volume
  H20	1.5 mL
  30 % acrylamide mix	2.0 mL
  Tris -HCl (1.5 M, pH 8.8)	1.3 mL
  SDS (10 %)	50 µL
  10 % ammonium persulfate	50 µL
  TEMED	2 µL

• For each gel (12 %) aliquot (5 mL gel volume):

Substance	Volume
H20	1.1 mL
30 % acrylamide mix	2.5 mL
Tris-HCl (1.5 M, pH 8.8)	1.3 mL
SDS (10 %)	50 µL
10 % ammonium persulfate	50 µL
TEMED	2 µL

2. Pour gel mix in gel casting form and leave about 2 centimeters below the top of the comb for stacking gel.
3. Pour a layer of isopropanol on top of the gel.
4. Leave gel at room temperature to polymerize.
5. Prepare stacking gel.
• For each (5 %) stacking gel (1 mL gel volume):

  Substance	Volume
  H20	0.68 mL
  30 % acrylamide mix	170 µL
  Tris-HCl (1.0 M, pH 6.8)	130 µL
  SDS (10 %)	10 µL
  10 % ammonium persulfate	10 µL
  TEMED	1 µL

6. Remove isopropanol.
7. Pour stacking gel in gel casting form on top the cast gels and insert comb.
8. Leave gel at room temperature to polymerize.
9. For storage, wrap the gel in moistened paper towel and store at 4 °C for one week maximum.
10. Prepare 4X SDS sample buffer stock.
• For 10 mL:

  Substance	Volume
  100 % Glycerol	4 mL
  Tris-HCl (1 M, pH 6.8)	2.4 mL
  SDS	0.8 g
  Bromphenol blue	4 mg
  ß- Mercaptoethanol	0.5 mL
  H2O	3.1 mL

2. Sample preparation
1. Dilute the protein sample to desired concentration.
2. Mix 4 X SDS-Samplebuffer with protein extract.
3. Incubate mix at 95 °C for 10 min.
3. Running the gel
1. Put gel into gel box and pour SDS-PAGE running buffer into the chamber.
2. Remove comb.
3. Wash gel pockets with SDS running buffer.
4. Pipette samples into the gel pockets.
5. Pipette molecular weight standards in one lane to determine the molecular weight of the sample.
6. Run the gel at 160 V until lowest molecular weight lane of the standard is down to 0.5 cm, stop electrophoresis.
7. Staining
• Remove gel from casting form and put gel in a staining box.
• Add Quick Coomassie Stain (by ProteinArk) to the gel.
• Incubate the gel at least for 15 minutes.
• Remove staining solution and wash the gel for 2 times with water.
• Incubate the gel for 2 h in water to remove background.

Used kit: Trans-Blot® Turbo™ Ready-to-Assemble (RTA) Kit

• Soak both stacks and membrane in transfer buffer.
• Place the first stack on blotting station, put the soaked membrane on top, carefully place SDS-Gel with protein on the membrane and place the last stack on top.
• With a roller, carefully roll over each part to remove air.
• Remove supernatant transfer buffer with a tissue.
• Start blocking program with 1.3 As and 25 V for 7 min.
• After blotting place membrane in a clean box.
• (If required, stain membrane with Ponceau S for 5 min to check if blotting have been successfull, after washing membrane with water to remove supernant staining, image the membrane.)
• Wash membrane 2 times for 5 min with TBS-T on a shaker.
• Place membrane in blocking buffer and incubate for 1 h on a shaker.
• Wash membrane 3 times for 10 min with TBS-T.
• Incubate membrane in antibody solution using a 1:12,000 dilution of 1µg/µL Ferritin Antibody with conjugated horseradish peroxidase for 1 hour at room temperature on a shaker or at 4 °C overnight.
• Wash membrane 3 times for 10 min with TBS-T on a shaker.
• Incubate membrane in substrate solution (1:1 HLP and luminol enhancer) for 1 min.
• Place membrane in foil to prevent it from drying out.

Sample preparation

• Glow-discharge of a carbon-coated copper grid with GloQube(R) Plus air chamber for 30 s using negative polarity at 25 mA intensity.
• Negatively charged grid was incubated with protein solution (4 µL, 0.036 µL/mL) in buffer based by pH 7.5 for 30 s and washed 2x with MilliQ water (20 µL).
• Negative staining with uranyl acetate solution (4 µL, 2%).
• Liquid was carefully removed by side-blotting the grid.

Talos L120C TEM

Electron microscope from Thermo Fisher Scientific at cryoEM facility of Centre for Strucutral Systems Biology (CSSB) with 120 kV accelerating voltage, a CETA camera, with manual side entry, mainly for negative stain samples, but also capable of cryo-EM using holders from Gatan. Software options include SerialEM and Velox.

• Reagents:
• p15A backbone (40 ng/µL)
• Fragment 1 (50 ng/µL)
• Fragment 2 (50 ng/µL)
• Fragment 3 (23 ng/µL)
• T4 DNA ligase buffer (10x)
• NEB GGA Mix BsaI
• MiliQ water
• Mix reagents as shown and gently mix by pipetting up and down 4 times.
• Briefly microcentrifuge to bring material to the bottom of the tube.
• Transfer to thermocycler.
• Program:

  	37 °C	5 min
  29 cycles of step 1 and 2	16 °C	5 min
  	60 °C	5 min
  Hold	4 °C

For further informaton see Golden Gate Assembly Protocol for Using NEBridge ® Golden Gate Assembly Kit (BsaI-HF®v2) (E1601).

• Mix components.
• Incubate at 37 °C for 5-15 minutes.

Preparation for the filters:

• Punch holes in filter paper.
• Prepare the concentrations of flavone, quercetin, rutin and ethylparaben individually and mixed in dishes with ethanol.
• 100 µg/mL antibiotic substance
• 50 µg/mL antibiotic substance
• 10 µg/mL antibiotic substance
• 1 µg/mL antibiotic substance
• 100 µg/mL ampicillin (as positive control)
• Ethanol (as negative control)
• Pipette 10 µL of the substance into filter paper circles (let it dry in between application).

Preparation of the plates:

• Take prepared LB-Agar plates.
• Plate 100 µL of BL21 (DE3) Star cell culture.
• Place the filter with antibiotic substances about 1.5 cm from plate border.
• Incubate plates at 37 °C for about 24 hours.

1. Preparation of solutions
• Fixation solution: 4 % formalin in 1X PBS
• DNA staining solution: 1:4000 DAPI in 1X PBS (DAPI stock solution: 1 mg/mL)
2. Overnight culture of E. coli DH5α
• Pick colony from agar plate and inoculate 20 mL LB medium.
• Incubate overnight at 120 rpm and 37 °C.
3. Subculture and washing
• Inoculate 20 mL fresh LB medium with 1,000 µL overnight culture.
• Incubate at 120 rpm and 37°C until OD600 0.7-0.8 is reached.
• Harvest 1 mL culture for each assay in Eppendorf tubes.
• Spin at 5200 g, 4 °C for 5 min, wash two times with 400 µL PBS, and resuspend in 500 µL PBS.
4. Penetration
• Add 10 µg of CPP-eGFP fusion protein or eGFP (negative control).
• Incubate for 1 hour at 37 °C and 150 rpm (mantle tubes in parafilm and fix horizontally in incubator).
5. Fixation
• Spin at 5200 g, 4 °C for 5 min, wash two times with 400 µL PBS, and discard supernatant.
• Add 700 µL 4 % formaldehyde in PBS and incubate 20 min at room temperature.
• Spin at 5200 g, 4 °C for 5 min, wash two times with 400 µL PBS, and discard supernatant.
6. DNA staining
• Add 200 µL of DNA staining solution per pellet and incubate for 10 min at 150 rpm shaking and room temperature in the dark (cover tubes in aluminium).
• Spin at 5200 g, 4 °C for 5 min, wash two times with 400 µL PBS, and discard supernatant.
• Resuspend pellets in 10 µL PBS and pipette up and down several times.
7. Mounting
• Label glass slides for each assay.
• Load 4 µL mowiol mounting medium to glass slides.
• Add 2.5 µL bacterial suspension on mowiol droplet.
• Cover with cover slip, after 1 min press cover slip gently to the glass slide and let them dry at room temperature overnight in the dark.