General Protocols

Agarose Gel

1. Weigh 1 g of agarose
2. Add 100 mL of TAE 1X
3. Heat up until the solution is homogeneous, avoiding boiling. If it boils, move away from the heat until it “calms down” and put it back on the heat until the agarose is completely dissolved
4. While heating, prepare the bed in which the gel will polymerize. Make sure that it is well balanced and tight
5. When homogeneous, add 10 µL of SYBR SAFE DNA Gel Stain to the solution and mix well
6. Pour the solution into the bed and clear all its bubbles with a pipette tip. Add in the comb and make sure it is secure
7. Mix the samples with loading dye in a 5:1 ratio. Put the samples into the wells, as well as 5 µL of DNA ladder into the first well

LB Medium (1 litre liquid)

• 10 g tryptone
• 10 g NaCl
• 5 g yeast extract
• Water
• For selective medium, supplement with antibiotic as appropriate (kanamycin 50 µg/ mL and 100 µg/mL for chloramphenicol or ampicillin)

LB Medium (1 litre solid / 50 plates)

• 15 g agar agar
• 10 g tryptone
• 10 g NaCl
• 5 g yeast extract
• Water
• For selective medium, supplement with antibiotic as appropriate (kanamycin 50 µg/ mL and 100 µg/mL for chloramphenicol or ampicillin)

Chemically Competent Cells: CaCl2 Method

1. Inoculate a single colony of appropriate cells into 10ml LB. Add antibiotic if needed and culture o/n at 37°C, 220rpm
2. Inoculate 100ml LB with 1 ml o/n culture
3. Incubate at 37 °C, 220rpm until OD600 = 0.3-0.6 (approx. 2 hrs)
4. Transfer to 2 x 50 ml Falcon and leave on ice for 30 mins
5. Centrifuge at 400 x g, 5 mins, 4 °C
6. Resuspend pellet GENTLY in 25 ml ice cold 0.1 M MgCl2
7. Incubate on ice for 30 min
8. Centrifuge at 4000 xg, 5 min, 4 °C
9. Resuspend pellet GENTLY in 25 ml ice cold 0.1 M CaCl2
10. Incubate on ice for 30 min
11. Centrifuge at 4000 xg, 5 min, 4 °C
12. Resuspend pellet GENTLY in 1.25 ml ice cold CaCl2/Glycerol solution (1.7 ml 0.1 M CaCl2, 0.3 ml 100 % glycerol)
13. Aliquot 100ul and flash freeze on dry ice. Store at – 80 °C

Chemical Transformation by Heat Shock

1. Place a small glass bottle with sterile SOC (10 ml) in the 37 °C incubator to prewarm. Also prewarm at this point the plates for spreading out the transformation
2. Thaw on ice one eppi (200 µl) of self-made competent Top10 cells per ligation to be transformed (Keep the competent cells as much as possible on ice!)
3. Add the entire ligation (20 µl) to the cells (or 0.1 to 1 µl for plasmids to be re-transformed) and mix by tapping gently. Do not mix cells by pipetting, their membrane is fragile and shear force can kill cells at this point!
4. Incubate on ice for 30 minutes (if possible, mix once or twice by gently tapping again)
5. Switch on the water bath immediately after adding the DNA to the cells and set to 42 °C (check temperature with a thermometer, sometimes the setting needs to be adjusted to achieve measured 42 °C. In our lab, set to 39.5 °C to achieve 42 °C!)
6. Heat shock the cells by incubating the eppis for 60 seconds in the 42 °C water bath. Do not mix or shake!
7. Remove the eppis from the 42 °C bath and quickly place on ice, incubate for 2 min
8. Add 500 μL of pre-warmed SOC medium (SOC is a rich medium; use proper sterile technique to avoid contamination)
9. Secure the eppis in a small microcentrifuge rack with tape and place in the 37°C shaking incubator for 60- 90 min (the rack should stick down with its short side so that the eppis lie parallel to the platform for maximum aeration). Warning: if you label only the lids, the tape will remove the labelling when you strip it off after incubation. Label the side of the Eppendorf and secure the label with masking tape!
10. Plate 50- 100 µl of the cells on one selective prewarmed plate. Spin down the remainder of the cells at 3800 rpm for 3 min at RT and pour off the SN until about 50 µl of liquid remain in the tube. Resuspend the pellet in this remaining liquid by pipetting up and down with a 200 µl pipette and plate the entire resuspension onto a second plate
11. Wrap the plates in parafilm and incubate upside down at 37°C o.n.

Overnight Cell Incubation

1. Add 5 ml LB into 50 ml falcon tube
2. Inoculate specific antibiotic
3. Scrape/pick glycerol stock surface/colony and transfer into falcon tube
4. Incubate at 37 °C overnight

Glycerol Stock Solution

1. Cultures plated on LB Agar + antibiotic and grown at 37°C overnight
2. A 5ml LB culture in LB + antibiotic inoculated from a single, freshly growing colony
3. Cultivate for 16h at 37 °C, 220 rpm
4. 0.5ml of culture inoculated into sterile Eppendorf
5. Add 0.5ml of 80% glycerol
6. Vortex
7. Spin down
8. Freeze at -80 °C

Plasmid Extraction

1. Plasmid extraction was carried out following QIAprep Spin Miniprep Kit protocol, found here: https://www.qiagen.com/us/resources/download.aspx?id=56b0162c-23b0-473c-9229- 12e8b5c8d590&lang=en

Triclosan Protocols

Minimal Inhibitory Concentration (MIC) Assay for Escherichia coli (adapted from Bio-protocol.org )

1. Dissolve a single colony of Escherichia coli in 5 ml LB broth and incubate overnight at 37 °C, 220 rpm
2. Make up serial dilution of triclosan of desired concentration range
3. Check OD₆₀₀ (1 OD₆₀₀ = 109 CFU/ml) with a spectrophotometer
4. Dilute the bacterial solution with LB broth to get 0.1 OD₆₀₀ suspension and incubate at 37 °C, 220 rpm till mid-log phase (~2 h)
5. Put 1 ml mid-log phase bacterial solution in 1.5 ml Eppendorf tube, centrifuge at 6,000 rpm for five min, and wash with 1 ml PBS. Repeat the washing procedure twice. Dissolve the bacterial pellet with 1 ml LB
6. Get 100 µl the above bacterial solution and mix it with 900 µl PBS, then check OD₆₀₀ with a spectrophotometer. The bacterial concentration can be deduced from the measured value x 10
7. Adjust the bacterial concentration to 1 x 107 CFU/ml with LB (1 OD₆₀₀ ~109 CFU/ml)
8. In a well of a 96-well plate, mix 10 µl adjust E. coli bacterial solution (1x107 CFU/ml), 170 µl LB and 20 µl of serial diluted triclosan. Repeat for each well for range of triclosan concentrations
9. Use 180 µl LB and 20 µl triclosan dilution series for OD₆₀₀ measurement as a negative control
10. On last row of plate, add 200 µl LB to be used as a blank
11. Incubate at 37 °C, 220rpm in a plate reader, taking OD₆₀₀ measurements over a period of 20-24 h

Triclosan Preparation

• Triclosan (5-chloro-2-(2,4-dichlorophenoxy)phenol) was ordered in powdered form from Alfa Aesar
• 58 mg powdered triclosan was dissolved in 500 µl dimethyl sulfoxide (DMSO) to make 400 mM stock solution
• 400 mM stock solution was diluted 1/1000 in DMSO to make 400 µM stock
• Add 125 µl of 400 µM stock to LB to a final volume of 5 ml for overnight culture at 10 µM triclosan
• For 1 µM triclosan agar plates, add 125 µl of 400 µM stock to a 50 ml falcon tub. Pour melted agar into falcon tube, mix and pour into 2 empty plates

SDS PAGE of Proteins in Maxicells

E. coli cells of strain DL2524 were made into maxicells using the UV maxicell protocol 48 hours, 24 hours and 0 hours (see maxicell producing protocol for details) and stored at 37 °C, 25 °C and 4°C (each timepoint had samples stored at each temperature) before being used as samples in the following protocol:

(A Bio-rad brand SDS page electrophoresis system was used for this experiment)

Mixtures

Running Buffer

The following was added to a beaker to make 2x running buffer:

• 6 g of Tris base
• 28.8 g of glycine
• 2 g of SDS
• Added enough distilled water to make it to 1 liter

The buffer was then diluted 1 to 1 buffer to distilled water to make it 1x running buffer.

Separating Gel:

First the following was added to a beaker:

• 3.35 ml of Distilled Water
• 2.5 ml of 1.5M Tris pH 8.8
• 0.1 of 10% SDS
• 4 ml of 30% acrylamide

Immediately before transferring into the casting frames/gel mould the following was added to the beaker:

• 50 μl of 10% APS
• 5 μl of TEMED

Stacking Gel:

First the following was added to a beaker:

• 6.1 ml of Distilled Water
• 2.5 ml of 0.5 M Tris pH 6.8
• 100 μl of 10% SDS
• 1.3 ml of 30% acrylamide

Immediately before transferring into the casting frames/gel mould the following was added to the beaker:

• 50 μl of 10% APS
• 10 μl of TEMED

Sample Buffer

The sample buffer was prepared and was:

• 10% w/v SDS
• 0.2 M Tris-HCl, pH 6.8
• 20% w/v Glycerol
• 10 mM beta-mercapto-ethanol
• 0.05 % w/v Bromophenolblue

Protocol

1. The SDS electrophoresis system was set up and the glass plates were placed in their clamps.
2. The Running buffer was poured into the buffer tank, enough to submerge the gels which will eventually be placed in the tank.
3. The separating gel was pipetted into the gap between the glass plates of the casting frame and a small amount if isopropanol was pipetted on top to level out the separating gel. The gel was left to set for 20 mins.
4. The isopropanol was discarded from the casting frame leaving only the separating gel. The stacking gel was then pipetted between the glass plates off the casting frame (on top of the separating gel) until it reached the top of the casting frame then a well forming comb was inserted into the top of the casting frame taking care not to trap any air bubbles. The stacking gel was left for 20 mins to set.
5. The O.D. 600 nm of the maxicell samples were taken and the samples were diluted till they all had the same O.D. 600 nm as the lowest sample and the total volume was 500 μl. 500 μl of sample buffer was added to the microfuge tube and then the tube was boiled at 95 – 100 °C for 7 mins.

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