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)

M9 Medium (1 litre liquid)

• 1% casamino acids
• 0.2% glucose
• 2 mL 1M MgSO4
• 0.1 µg/ml thiamine (B1)
• 0.1 ml 1M CaCl2
• 60 g Na2HPO4
• 30 g KH2PO4
• 5 g NaCl
• 10 g NH4Cl
• Bring to final volume of 1l with distilled water. Autoclave.

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

Maxicell Protocols

UV Induced production

For all strains that are not uvrA^-, all steps during and after UV exposure must be done in the dark. Store cells in a light proof container (i.e. brown flask). Photoreactivation peaks at 384 nm.

Day 1:

1. Start overnight culture in 5 ml culture medium + antibiotic. Incubate at 37 °C, 220 rpm

Day 2:

1. Dilute o.n. culture approximately 1/60 in 60 ml of medium plus appropriate concentration of antibiotic in a 250mL flask. This will yield a 60 mL culture with an OD₅₅₀ ~0.05
2. Incubate 37 °C with agitation. Take OD₅₅₀ readings every hour or less and plot a growth curve for future reference.
3. At OD₅₅₀ = 0.4, transfer 10 mL of the culture into a glass petri dish and irradiate with UV light on a shaking platform. Shaking is essential for even exposure. Time of exposure dependent on strain – a good starting point is to expose strains for 20 seconds, 40 seconds, 60 seconds and 80 seconds. Negative control = 0 min exposure, positive control = 4 min exposure
4. Transfer to a 50 mL dark falcon tube, incubate 37 °C, 220 rpm for 45 minutes
5. Add 200 µg/ml cycloserine (1/500 dilution of stock) and continue gentle shaking 37° incubation overnight. Don’t reuse cycloserine stocks too many times, as it becomes unstable.

Day 3:

1. Centrifuge maxicells 6 mins at 6000x g (7,000 rpm in an SS34)
2. Resuspend the pellet in 5 ml of media + appropriate concentration of antibiotic
3. Centrifuge maxicells 6 mins at 6000x g
4. Resuspend the pellet in 1 ml of media + appropriate concentration of antibiotic
5. Cells can then be frozen in glycerol for future use

I-SceI Induced production

Using the strain BW27784, with DL name DL2524, acquired from the Leach Lab (MG1655 recA::CmR araB::PBAD-I-SceI lacZ::I-SceIcs)

Day 1:

1. 5 ml overnight culture in LB + 25 μg/ml chloramphenicol + 0.5% glucose, in 50 ml falcon tube, vigorously shaking at 37 °C

Day 2:

1. Measure the OD₆₀₀ in the morning
2. Dilute the culture approximately 1/60 in LB + 0.2% glucose + 25 μg/ml chloramphenicol (CAM) to OD₆₀₀ 0.025
3. Grow for 80 minutes at 37 °C
4. Measure the OD - should be 0.1. Dilute part of the culture 1/10 in LB + 0.2% glucose + CAM (negative control) and another part in LB + 0.5% arabinose + CAM
5. Incubate 37 °C, 220 rpm O/N

Day 3:

1. Centrifuge maxicells 6 mins at 6000 x g (7,000 rpm in an SS34)
2. Resuspend the pellet in 5 ml of media + appropriate concentration of antibiotic
3. Centrifuge maxicells 6 mins at 6000 x g
4. Resuspend the pellet in 1 ml of media + appropriate concentration of antibiotic
5. Cells can then be frozen in glycerol for future use

sbcDC Induced Production

Using the strain BW27784, with DL name DL3355, acquired from the Leach Lab (MG1655 lacIq lacZχ- ParaBAD-sbcDC lacZ::pal460 recA::Cm). Follow protocol for I-SceI induced production (above).

DAPI Staining

If using UV maxicells, do this 15-18 hours post-maxicell-induction. Maxicells cannot be used after DAPI staining so do not stain whole stock.

1. Measure the OD₆₀₀ - aim for 0.1 – 0.4. Add DAPI. Incubate 15 minutes at 37 °C under agitation.
2. Dissolve agarose in 25 ml distilled water in microwave 1-2 minutes. This can be kept in liquid state at 55 °C.
3. Pipette two 150 μl agarose spots onto the glass slide and immediately place a coverslip on the spots and flatten gently. Leave 10 minutes.
4. Remove coverslips - airdry for at least 5 minutes
5. Transfer 10μl sample onto the agarose spots and place coverslip on top

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.
6. 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.
7. 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.
8. 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).
9. The gel was destained in coomassie blue destain with several washes and pictures were taken of the gel

ATP Determination Protocol

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.

Making Reagents and Solutions:

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:

1. 8.4 mL dH2O (deionised);
2. 0.5 mL 20X Reaction Buffer;
3. 0.1 mL of 0.1 M DTT (see below);
4. 0.5 mL of 10 mM D-luciferin (see below);
5. 2.5 µL of firefly luciferase;
6. 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

Reagent Preparation:

1. Add 50 µL of 20X Reaction Buffer to 950 µL of deionized water (dH2O).
2. 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.
3. 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.
4. 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:

1. 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.
2. After 24h, cells were extracted via centrifugation (6 minutes at 10,000 rpm). Then Induction (or no indcution for control - similar to WT) needs to be made (i.e. cells need to be converted to Maxicells). Therefore, cells were either re suspended in LB + 0.2% Arabinose OR LB + 2.8 % Glucose (Depending if you are testing Maxicells or Non-Maxicells respectively). Cells are then allowed to incubate for an hour.
3. 100µg/mL of Cycloserine is then added to kill live cell in Maxicell samples only. Cell allowed to incubate for another hour.
4. An hour after the cycloserine is added, the time of induction is zero (see below). Measurements were taken at every 2 hour intervals (at times labs were open). Two inductions were made one at the beginning of the day and one at the end of the day to get allow all possible timepoints to be measured (again, see below).
5. 50µL of sample placed in 2mL eppendorf and centrifuged (3 minutes at 4,000rpm), sample then re-suspended in 100 mM Tris-HCl buffer (pH 7.4). Cells were then subsequently washed washed with the same buffer, again.
6. 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.
7. 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.

Protocol for Freezing Maxicells:

DL2524 was induced with arabinose and stored in a freezer (<-20°C) to test the effect of freezing Maxicells after induction. This was done to see how ell these cells can be transported (after induction). The Protocol for Freezing Maxicells is as follows:

1. Prepare an overnight Culture of DL2524 with LB + 2.8% Glucose
2. Next day Spin cells down in centrifuge (6 minutes at 10,000rpm)
3. Cells then Re-suspended in either LB + 0.2% Arabinose for Maxicells OR LB + 2.8% glucose for Non-Maxicells. Cell allowed to incubate for 1 hour.
4. After an hour add 100μg/mL of cycloserine, to Maxicell sample only, to kill any living cells. Leave cells to incubate for an hour.
5. Add equal volume of 50% [v/v] glyerol stock solution to the samples (both Maxicells and Non-Maxicells)
6. Samples then placed in <-20°C freezer.
7. To get range of hours; sample was taken out of freezer at 2 timepoitns during testing which occured 2 weeks after freezing (once at beginning of day (11am) and second at end of the day (5pm) this is to mirror the other ATP assays).
8. ATP assay protocol was followed from step 5 onwards to measure ATP conc. (Sample was stored at room temp in between tests after initial de-thaw).