Edinburgh iGEM 2018

General Protocols

Agarose Gel

Weigh 1 g of agarose
Add 100 mL of TAE 1X
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
While heating, prepare the bed in which the gel will polymerize. Make sure that it is well balanced and tight
When homogeneous, add 10 µL of SYBR SAFE DNA Gel Stain to the solution and mix well
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
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

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

Chemical Transformation by Heat Shock

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
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!)
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!
Incubate on ice for 30 minutes (if possible, mix once or twice by gently tapping again)
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!)
Heat shock the cells by incubating the eppis for 60 seconds in the 42 °C water bath. Do not mix or shake!
Remove the eppis from the 42 °C bath and quickly place on ice, incubate for 2 min
Add 500 μL of pre-warmed SOC medium (SOC is a rich medium; use proper sterile technique to avoid contamination)
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!
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
Wrap the plates in parafilm and incubate upside down at 37°C o.n.

Overnight Cell Incubation

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

Glycerol Stock Solution

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

Plasmid Extraction

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 )

Dissolve a single colony of Escherichia coli in 5 ml LB broth and incubate overnight at 37 °C, 220 rpm
Make up serial dilution of triclosan of desired concentration range
Check OD₆₀₀ (1 OD₆₀₀ = 109 CFU/ml) with a spectrophotometer
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)
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
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
Adjust the bacterial concentration to 1 x 107 CFU/ml with LB (1 OD₆₀₀ ~109 CFU/ml)
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
Use 180 µl LB and 20 µl triclosan dilution series for OD₆₀₀ measurement as a negative control
On last row of plate, add 200 µl LB to be used as a blank
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

The SDS electrophoresis system was set up and the glass plates were placed in their clamps.
The Running buffer was poured into the buffer tank, enough to submerge the gels which will eventually be placed in the tank.
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.
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.
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.
After gelation, the comb was removed and the gel in the casting frame was submerged in the running buffer which was in the buffer tank. A protein ladder was loaded into the left most well and the maxicell protein samples were each loaded into separate wells taking care not to let the sample overflow the well.
The lid and anode and cathode were attached to the tank and the gel was left to run for approximately 1 hour at 120 V.
Before the gels had over run the voltage was turned off and the gels were removed from the casting frames and placed into separate containers coomassie stain (enough to just cover the gel). The containers containing the gel and coomassie stain were gently agitated for approximately 1 hour (until the protein bands were clearly visible but before the whole gel was stained too much).
The gel was destained in coomassie blue destain with several washes and pictures were taken of the gel

ATP Determination Protocol

Making Reagents and Solutions:

To Determine ATP levels over Time and ATP Assay was preformed. The Kit used to preform the assay was bough at Thermofisher. A lot of the protocols used are adapted from their protocols. Only THE Strain DL2524 was used to preform the ATP assay as they were the fastest method for producing Maxicells.

Preparation of Assay Solution:

Standard assay solution was prepared as as stated in the Thermofisher ATP Determination Kit, 200-1,000 assays kit (Catalogue Number: A22066) with the addition of Triton X-100. The following were used to make the solution:

8.4 mL dH2O (deionised);
0.5 mL 20X Reaction Buffer;
0.1 mL of 0.1 M DTT (see below);
0.5 mL of 10 mM D-luciferin (see below);
2.5 µL of firefly luciferase;
0.5 mL detergent solution (8% Triton X-100, 300 mM potassium phosphate, pH 7.2).

Gently invert to mix, do not vortex; as the firefly luciferase enzyme will denatured. Keep solution protected from light. Solution may be stored at 2–6°C protected from light for several days, assay sensitivity will diminish with time. This ATP assay solution is Mix B

Add 50 µL of 20X Reaction Buffer to 950 µL of deionized water (dH2O).
To make 1 mL of a 10 mM D-luciferin stock solution,, add adding 1 mL of 1X Reaction Buffer (prepared earlier) to one vial of D-luciferin. Protect from light until use and store ≤–20°C.

Prepare a 100 mM DTT stock solution by adding 1.62 mL of dH2O to the bottle containing 25 mg of DTT. Aliquot into ten 160 µL volumes and store frozen at ≤–20°C. Thawed aliquots should be kept on ice or at 4°C until ready for use.

Prepare low-concentration ATP standard solutions by diluting the 5 mM ATP solution in dH2O. The concentrations and volumes to make depend upon the sensitivity and design of the luminometer to be used. Range used in Experiment wa from 0.5-3mM of ATP. These dilute solutions are stable for several weeks when stored at ≤–20°C. This is then used to make standard curve and convert fluorescence into mM of ATP.

Assay Protocol:

E. coli cells (DL2524 strain) were grown to the stationary phase in Luria-Bertani (LB) medium containing 2.8% glucose at 37° C for 24 h.
After 24h, cells were extracted via centrifugation (6 minutes at 10,000 rpm), and resuspended in LB + 0.2% Arabinose OR LB + 2.8 % Glucose (Depending if you are testing Maxicells or Non-Maxicells respectively). Cell allowed to incubate for an hour.
100µg/mL of Cycloserine is then added to kill live cell in Maxicell samples only. Cell allowed to incubate for another hour.
50µL of sample placed in 2mL eppendorf and centrifuged (3 minutes at 4,000rpm), sample the resuspended in 100 mM Tris-HCl buffer (pH 7.4). Cells were then subsequently washed washed with the same buffer.
Cell suspension then mixed with an equal volume of pre-treatment solution (40% [w/v] glucose, 0.8% [v/v] Triton X-100) and allowed to sit for 20 minutes at room temperature. This is Mix A.
To begin Reaction to measure fluorescence of sample: Add 10 µL of Mix A (sample) to 90 µL of Mix B (Assay solution). Invert eppendorf gently to mix and read Luminescence. Make sure to subtract background luminescence (i.e. luminescence of Mix B - assay solution).

NOTE: To make standard curve mix 10 µL of Standard ATP Stock solution (ranging from 0.5-3mM of ATP) with Mix B (assay solution) and read luminescence. Make sure to subtract background luminescence. Plot this to create standard curve of [ATP] (mM) vs Luminescence.

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