Team:Linkoping Sweden/Experiments
Experiments
Experiments
LB-Miller Agar
Agar protocol was extracted from: chsProtocols.cshlp.org
15 g agar
10 g tryptone
5 g yeast extract
10 g NaCl
Mix with 1 L of water and autoclave at 121 °C
For Low salt agar, add 0.5 g of NaCl instead of 10 g
LB-Miller
LB-Miller protocol was extracted from: USBiological
Prepare the following mixture:
10 g tryptone
5 g yeast extract
10 g NaCl
Mix with 1 L of water and autoclave at 121 °C
For Low salt LB-Miller add 0.5 g of NaCl instead of 10 g
This protocol was extracted from: Sigma-Aldrich
PCR
Q5® Hot Start High-Fidelity from New England Biolabs was used for all PCR reactions.
Prepare the following mixture in a PCR tube:
12.5 µL of Q5 master mix
1.25 µL of forward primer (10 µM)
1.25 µL of reverse primer (10 µM)
1 µL of DNA template
9 µL of water
Place the PCR tube in the thermocycler and run the following cycles:
98°C for 30 sec
98°C for 10 sec
55-72°C for 20 sec
72°C for 30 sec per kb
Repeat from step 2 12-35 times
72°C for 2 minutes
Agarose gel
Mix 1.3 g of agarose with 100 mL of TAE buffer. Microwave the mixture until boiling then cast the gel in an electrophoresis cast and add a gelcomb for wells.
Sample preparation: mix 8 µl of Loading Dye with 7 µL of DNA sample. Load a DNA ladder as well for reference. Load the gel and run it at 100 V for 60 minutes. Stain with Ethidium bromide for 5 minutes and destain with TAE for 1 minute. Place the gel on a UV table or in a UV camera.
PCR purification
Monarch® PCR & DNA Cleanup Kit was used for PCR purification. Dilute the PCR product in DNA Cleanup binding buffer (1:5) ratio. Place the mix in a spin column and centrifuge for 1 minute. Discard flow-through and add 200 µL of DNA wash buffer. Discard the flow-through and add 200 µL DNA wash buffer again. Place the spin column in new Eppendorf tubes and elute with more than 6 µL water. Analyze the samples on a Nanodrop.
Restriction digest
Restriction enzymes and protocol were provided by New England Biolabs.
Prepare the following mixture:
1 µL restriction enzyme
5 µL buffer (either 1.1, 2.1 3.1 or Cutsmart)
1 µg of DNA template
X µL water
Total volume should be set to 50 µL
Place the Eppendorf tubes containing the mixture in a heating block at 37°C for 15-60 minutes. To purify the DNA for later use, utilize a PCR purification kit.
Ligation
Ligation mixtures were provided by New England Biolabs.
Prepare the following mixture:
2 µL ligation buffer
50 ng vector
37 ng insert (or a molar ratio of 1:7 vector insert )
X µL water
1 µL ligase
Total volume should be set to 20 µL
Incubate the mixture at room temperature for 10 minutes.
Gibson Assembly
The Gibson assembly kit was provided from New England Biolabs.
Prepare the following mixture:
10 µL Master mix
Add vector and insert in a 1:2 ratio
X µL water
Total volume should be set to 20 µL
Incubate the mixture at 37°C for 15 minutes.
Chemically competent cell preparation
Culture 25 mL bacteria for 4-6 hours at 37 °C with appropriate antibiotics in a shake incubator. Harvest the cells by centrifuging for 10 minutes at 3000 g. Discard the supernatant and resuspend the pellet in 0.1 M CaCl2. Incubate the cells on ice for 20 minutes then repeat the centrifugation step. Resuspend the cells in 0.1 M CaCl2 with 15 % glycerol. Store the cells in 300 µL fractions in 1.5 mL tubes. Freeze these instantly in liquid nitrogen and store them in -80 °C.
Heatshock
Add 1-5 µL of plasmid to 50 µL of Heatshock competent cells. Incubate for 30 minutes on ice place the mix on a 42 °C heat block for 45 seconds then incubate the mix on ice for 2 minutes. Add 250 µL LB to the cells and incubate them at 37 °C for 1 hour. Take 100 µL from the mix and plate it on an agar plate with the appropriate antibiotics. Incubate the agar plate at 37 °C for 16 hours.
Electroporation
Add 1-5 µL plasmids to 50 µL of Electrocompetent cells. Place the mix in a cuvette. Make sure that the mix covers the bottom of the cuvette. Zap the bacteria with approximately 1.8 mV for approximately 5.4 ms. Add 250 µL LB to the mix and place in a new Eppendorf tube. Incubate the tube at 37°C for 1 hour. Take 100 µL from the mix and plate it on an agar plate with the appropriate antibiotics. Incubate the agar plate at 37°C for 16 hours.
Colony screening
Prepare a PCR reaction without adding the template DNA. Inoculate one colony with a pipet. Dip the inoculated colony in the PCR reaction, then gently place and scrape the tip on a new agar plate with appropriate antibiotics.
Bacterial culturing
Take 100 µL from a glycerol stock and plate it on an agar plate with the appropriate antibiotics. Incubate the plate overnight.
Add 1 L LB to a culture flask and add the appropriate antibiotics.
Add 20 mL LB to the plate and resuspend the bacteria in it. Add the bacteria mix to the culture flask and incubate it until OD is between 0.4-0.6. Induce the bacteria and place the culture flask in 16°C for 16 hours.
Centrifuge the cells at 3000 g for 30 minutes and extract the supernatant. Resuspend the pellet in 30 mL water and pour the mix in a 50 mL tube. Freeze it in liquid nitrogen and store it at -80°C.
Plasmid Preparation
Monarch® Plasmid DNA Miniprep Kit Protocol was used for Plasmid Preparation.
Add 5 mL LB-Miller to a 50 mL tube then add the appropriate antibiotics. Inoculate a colony and add it to the mix. Incubate overnight.
Harvest the cell by centrifugation at 3000 g for 5-10 minutes. Extract the supernatant and replace it with 200 µL Plasmid resuspension buffer. Move the mixture to an Eppendorf tube and add 200 µL Plasmid lysis buffer. Mix the sample by inverting it 5-6 times. After 1 minute, add 400 µL Plasmid neutralization buffer then invert the tube 5-6 times and let the samples incubated for 2 minutes at room temperature. Centrifuge the mix for 5 minutes at 16 000 g. Extract the supernatant, place it in a spin column and centrifuge at 16 000 g for 1 minute. Discard the flow through and add 200 µL Plasmid wash buffer 1. Repeat the centrifugation step, then add 400 µL of Plasmid wash buffer 2 and repeat the centrifugation step. Place the spin column in an eppendorf tube and add 30 µL water. Repeat the centrifugation step. Measure the DNA concentration in a Nanodrop and run the sample on a gel electrophoresis to confirm the size.
Bacterial lysis
There are several different ways to conduct bacterial lysis. We used two types of lysis methods during this project; freeze thaw and sonication. We found varying success, but the most effective for us was sonication.
Sonication
Take a pellet of harvested cells and resuspend them in the appropriate buffer for your protein. Place the sonication probe in the mix. Run the sonication on 30 % amplitude with 30 seconds on 30 seconds of. Then centrifuge at 12000 g for 20 minutes. Save the supernatant or the insoluble fraction (pellet) if working with insoluble proteins.
Freeze thaw
Take a pellet of harvested cells and resuspend them in the appropriate buffer for your protein. Place the sample in liquid nitrogen for 1 minute then place the sample on a heat block set to 42 °C for 1 minute. Vortex the sample and repeat all the steps five times.
CBD binding purification
The CBD cellulose binding purification can be conducted in several ways. Either it can be done in a PD-10 column, in a Falcon tube or on a cellulose bandage.
-PD10 column purification
Add 1 mL of protein sample to the column then incubate it on a end-to-end rotator for 1 hour. Wash it with 8 mL of ethanol 1 mL at a time. Elute the sample in 8 mL of water 1 mL at a time. Save all the elution fractions for SDS-PAGE confirmation of purity.
-50 mL tube purification
Add cellulose to a protein solution and incubate it on an end-to-end rotator for 1 hour. Add 20 mL of ethanol and incubate it. Then centrifuge the sample att 3000 g for 5 minutes. Extract the ethanol, repeat the centrifugation and extract the rest of the ethanol. Repeat the ethanol wash steps 3 times. Add 5 mL ddH20 and incubate the sample at 37°C for 1 hour. Centrifuge the sample 3000 g for 5 minutes. Extract the supernatant. Run the sample on an SDS-PAGE to confirm the results.
IMAC Purification
Proteins that are tagged with a His-tag can be purified using IMAC purification. Add 1 mL nickel agarose to a PD10 column. Add the wash the agarose with 10 mL wash buffer. Add the protein sampel to the column. Wash with 10 mL wash buffer. Add 10 mL elution buffer 1 mL at a time and save all the fractions for gel electrophoresis confirmation.
Buffers
Buffer recipe for IMAC Purification was extracted from this articleEquilibration: Low Imidazole
50 mM Tris
300 mM NaCl
500 mM Imidazole
Set the pH to 8
Elution: High Imidazole
50 mM Tris
300 mM NaCl
500 mM Imidazole
Set the pH to 7
Bradford assay
Bradford assay protocol was extracted from: Bio-Rad
Make a dilution series of BSA.
Take 5 µL sample and add 250 µL Bradford reagents and place the stained sample in a 96 well plate and measure the absorbance at 594 nm. Compare the absorbance to the standard curve and calculate the amount of protein.
SDS-PAGE
Sample preparation and running the gel
Place two gels in the gel electrophoresis tray and add SDS-PAGE running buffer between the gels until it overflows. Then add SDS-PAGErunning buffer in the tray to the appropriate mark. Mix 10 µL protein sample with 10 µL of 2X sample preparation buffer, add 1 µL 2-mercaptoethanol and mix the sample. Incubate the sample at 95 °C for 5 minutes then load the sample on the SDS-PAGE. Set the voltage to 100 V and run the page until all protein samples have wandered into the gel, the increase the voltage to 200 V. Run the gel until the loading dye has reached the bottom.
Staining
Dismantle the SDS-PAGE mold and place the gel in a tray. Rinse the gel with water. Extract all the water and add the staining solution. Incubate the SDS-PAGE with low rotation for 15 minutes. Remove the staining solution, add Destaining solution and incubate the SDS-PAGE with low rotation for 15 minutes. Remove the Destaining solution and add new. Repeat this step until the proteins are seen as visible bands on the gel.
Polyacrylamide gel recipe
Prepare the polyacrylamide mold.
For separation gel mix:
2 mL 33 % acrylamide
0.7 mL 1 % Bis-acrylamide
1 mL 2.122 M Tris-HCl buffer pH 9.18
0.7 mL water
0.1 mL 0.22 M ammoniumperoxodisulfate
Let the mixture degas for 5 minutes, then add 11.5 µL TEMED. Quickly cast the gel in the gel mold. Add isopropanol to the gel and let it sit until it solidifies, then extract the isopropanol.
For stacking gel mix:
0.625 mL 33 % acrylamide
1.25 mL 1 % Bis-acrylamide
0.625 mL 0.541 M Tris-HCl buffer pH 6.1
2.4 mL water
0.1 mL 0.22 M ammoniumperoxodisulfate
Let the mixture degas for 5 minutes, then add 10 µL TEMED. Quickly cast the gel in the gel mold. Add the gel combs and let the gel sit until it solidifies.
Running buffer 10X
The protocol was extracted from: CSHProtocols
Prepare the following mix:
30 g Tris
144 g glycine
10 g SDS
1 L ddH20
When using the running buffer, dilute it to 1 X with ddH20
Laemmli Sample preparation buffer
The protocol was extracted from: USBiological
Prepare the following mix:
4 % SDS
20 % glycerol
0,004 % bromphenol blue
0.125 M Tris-Cl, pH 6.8
Staining solution stock
Buffer recipe for Staining solution stock was extracted from this protocol
Mix the following chemicals:
1 g of Coomassie R250
100 mL of Glacial acetic acid
400 mL methanol
500 mL ddH2O
Destaining solution stock
Mix the following chemicals:
100 mL of Glacial acetic acid
400 mL methanol
500 mL ddH2O
CBD binding capability assay
Take 6 mL purified fusion protein containing one carbohydrate binding domain and one part fluorescent protein. The sample need to be in PBS. place a Epiprotect bandage in the solution. Incubate the sample in 37 °C. Take a sample from the supernatant and measure the absorbance at 485 nm. Repeat this every 25 minutes. When no more absorbance is lost from the supernatant the bandage is saturated.
Thrombin cleavage assay
The protocol was extracted from: Novagen
For a total of 50 μL reaction volume mix:
5 μL 10X Thrombin Cleavage Buffer (200 mM Tris-HCl, 1.5 M NaCl, 25 mM CaCl2, pH 8.4)
X μL of target protein
1 μL of 0.5 U thrombin
x μL deionized water
Incubate overnight in either R.T or 37 °C on 200 rpm. (End-to-end rotator works as well).
Antimicrobial plate reader assay
Dilute bacteria stocks in low salt LB to appropriate concentrations. Add the antimicrobial peptides in respective well. Fill the well to 200 µL with the LB bacteria mix. Place a cover slip over the plate and puncture one hole in the slip for each well. Place the 96 well plate in a plate reader with the incubation temperature 37 °C. Measure OD600 every 15 minutes and shake the plate 1 minute prior to this.
Bandage activity assay
This assay is carried out to determine the effect of the bandage on bacteria. Prepare the bandage by dipping in cell lysate that contains the CBD fusion protein of your choice. Incubate it for 30 minutes in 4 °C. Remove the lysate and add 70 % ethanol, incubate it for 30 minutes in 4 °C. Repeat this ethanol washing step 3 times. Cut the bandage and place it in a 96 well plate. Add the target bacteria, thrombin cleavage buffer and thrombin. Incubate the plate at 37 °C with shaking in a 96 well plate reader. Measure the absorbance at 600 nm every hour.
CFU counting
Take 20 µL from the initial stock and dilute it in 180 µL LB. Take 20 µL from the diluted bacteria and add it in 180 µL LB. Repeat this for a series of 10 tubes. Plate 100 µL of bacteria from each dilution on an agar plate with appropriate antibiotics. Incubate the plates at 37 °C for 16 hours. Count the colonies on the first countable plate and the plate after. This will tell you the concentration of the stock.
