Team:Nottingham/Experiments

Experiments

Since our labs were closed during our project, we have come up with all the potential wet lab experiments we could have done if the circumstances were different.

DBHB and Control subgroup protocols:

1. Pellet 1 mL of a liquid overnight culture of the bacterial strain by centrifugation at 13,000 rpm and resuspend in 200 µL lysozyme (10 mg/ml) and incubate for 30 minutes at 37 °C.

2. Lyse the cells by the addition of 20 µL Proteinase K (20 mg/ml) and 200 µl lysis solution C and incubate for 10 minutes at 55 °C.

3. Prepare the column by adding 500 µL of the Column Preparation Solution to each pre-assembled GenElute Miniprep Binding Column in a 2 mL collection tube. Pass through by centrifugation at 13,000 rpm for 1 minute and discard eluate.

4. Add 200 µL of ethanol (95–100%) to the incubated cell lysate (from step 3), mix thoroughly and load into the prepared binding column. Centrifuge a 13,000 rpm for 1 minute and discard eluate in collection tube.

5. Wash the loaded column by adding 500 µL of Wash Solution 1, centrifuge for 1 minute at 13,000rpm. Wash for a second time by adding 500 µL of Wash Solution 2 to the column and centrifuge for 3 minutes at 13,000 rpm ensure the column is dry. Discard eluate in the collection tube after each wash step.

6. Elute the DNA by pipetting 200 µL of the Elution Solution directly onto the centre of the column, incubate for 5 minutes at room temperature and centrifuge for 1 minute at 13,000 rpm.

Table 1 - Standard PCR Reaction Setup using KOD Hot Start Master Mix. Starting template amount varies depending on template type used (B); Plasmid DNA - 10 ng, Genomic DNA – 100ng. Volume of dH20 (A) added to make reaction up to 20 µL.

Component Volume (total 20 µL reaction)
KOD Hot Smart DNA Polymerase Master Mix (0.04 U/ µL) 10 µL
Primer 1 (µM) 0.6 µL
Primer 2 (µM) 0.6 µL
Template DNA B
Deionized H2O A

Table 2 - Thermocycling Conditions. TA (annealing temperature) should be lowest primer Tm (melting temperature)

Step Target Size
<500bp 5000-1000bp 1000-3000bp >3000bp
1. Polymerase activation 95 °C - 2 mins 95 °C - 2 mins 95 °C - 2 mins 95 °C - 2 mins
2. Denaturation 95 °C – 20 s 95 °C – 20 s 95 °C – 20 s 95 °C – 20 s
3. Annealing TA °C – 10 s
4. Extension 70 °C – 20 s/kb 70 °C – 20 s/kb 70 °C – 20 s/kb 70 °C – 20 s/kb
Repeat steps 2. - 4. 40 cycles


1. Make 1% agarose (1 g agarose in 100 ml TAE buffer and heated in a microwave until clear and left in a 50 °C water bath for 30 mins to cool).

2. Add SYBR Safe (DNA stain) to the molten agarose (5 µl/100ml of agarose) and pour into a cast, inserting an appropriately sized comb.

3. Let set for around 20 minutes.

4. Remove the comb and gel in cast from its holder and place into a gel running tank with TAE buffer ensuing the entire gel in submerged.

5. Load DNA samples by pipette into the gel wells (including a DNA ladder in the first well (e.g. NEB 1 kb plus ladder).

6. Place the lid on the gel tank, attach wires to correct terminals in power pack and run at 100 Volts and 40 minutes.

7. Image gel with an imaging machine and if required cut bands using a clean scalpel for gel for purification (see protocol 4).

All centrifugation steps are carried out at 17,900 x g (13,000 rpm) in a conventional table-top microcentrifuge.

1. Excise the DNA fragment from the agarose gel with a clean, sharp scalpel.

2. Weigh the gel slice and add 3 volumes Buffer QG to 1 volume 1% agarose gel (100 mg gel ~100 µl).

3. Incubate at 50 °C for 10 min (or until the gel slice has completely dissolved). Vortex the tube every 2–3 min to help dissolve gel. After the gel slice has dissolved completely, check that the color of the mixture is yellow (similar to Buffer QG without dissolved agarose). If the color of the mixture is orange or violet, add 10 µl 3 M sodium acetate, pH 5.0, and mix. The mixture turns yellow.

4. Add 1 gel volume isopropanol to the sample and mix.

5. Place a QIAquick spin column in a provided 2 ml collection. To bind DNA, apply the sample to the QIAquick column and centrifuge for 1 min.

6. Discard flow-through and place the QIAquick column back into the same tube. For sample volumes >800 µl, load and spin/apply vacuum again.

7. To wash, add 750 µl Buffer PE to QIAquick column and centrifuge for 1 min or apply vacuum. Discard flow-through and place the QIAquick column back into the same tube.

8. Place QIAquick column into a clean 1.5 ml microcentrifuge tube.

9. To elute DNA, add 30 µl water to the center of the QIAquick membrane, let the column stand for 1 min and centrifuge the column for 1 min.

1. Clean the upper and lower optical surfaces of the microvolume spectrophotometer with clean deionized water and a clean, dry, lint-free lab wipe.

2. Open the NanoDrop software and select the double stranded nucleic acid application.

3. Perform a blank measurement by dispensing 1 µL of clean deionized water (same as used to eluted kit column) onto the lower optical surface. Lower the lever arm and select "Blank" in the Nucleic Acid application.

4. Once the blank measurement is complete, clean both optical surfaces with a clean, dry, lint-free lab wipe.

5. Dispense 1 µL of nucleic acid sample onto the lower optical pedestal and close the lever arm. Select "Measure" and the software will automatically calculate the nucleic acid concentration and purity ratios which is displayed on equipment’s screen.

Table 3 – Standard HiFi DNA assembly reaction setup

2-3 Fragment assembly 4-6 Fragments assembly
DNA Molar Ratio vector : insert = 1:2 vector : insert = 1:1
Total amount of Fragments X µL (0.03-0.2 pmols) X µL (0.2-05 pmols)
NEBuilder HiFi DNA Assembly Master Mix 10 µL 10 µL
Deionized H20 10- X µL 10- X µL
Total reaction volume 20 µL 20 µL*

1. Setup the reaction above on ice. (pmols = (weight in ng) x 1,000 / (base pairs x 650 daltons)) *- If greater numbers of fragments are assembled, increase the volume of the reaction, and use additional NEBuilder HiFi DNA Assembly Master Mix.

2. Incubate samples in a thermocycler at 50 °C for 15 minutes (when 2 or 3 fragments are being assembled) or 60 minutes (when 4–6 fragments are being assembled). Following incubation, store samples on ice or at –20 °C for subsequent transformation.

1. Thaw chemically competent cells on ice and transfer 50 µL of competent cells to a 1.5 ml microcentrifuge tube (if necessary).

2. Add 2 µL of assembled product to NEB competent cells and mix gently by pipetting up and down or flicking the tube 4–5 times. Place the mixture on ice for 30 minutes. Do not mix.

3. Heat shock at 42 °C for 30 seconds. Do not mix.

4. Transfer tubes on ice for 2 minutes.

5. Add 950 µL of room temperature SOC media to the tubes and incubate at 37 °C for 60 minutes, shake vigorously (250 rpm).

6. Warm selection plates (with appropriate antibiotics) to 37 °C and spread 100 µL of the cells onto the plates with.

7. Incubate plates at 37 °C until colonies appear (usually after 24 hours).

1. Label approximately 20 plates per 400 mL bottle of agar media.

2. Melt a 400 mL Duran bottle of agar media in a microwave on low power (for 10-15 minutes) with the cap unscrewed.

3. Leave the bottle in a 50 °C water bath for around 30 mins to allow the media to cool.

4. Under a bunsen burner (to keep aspectic) add required volume of antibiotic(s)/supplements and pour media into labelled plates until the surface is covered.

5. Let plates solidify on the bench and before use dry plates at 37 °C for ~40 minutes. Store any extra plates at 4 °C upside down to ensure condensation doesn’t collect on the agar surface.

1. Pick a single colony using a small sterile 1 µL inoculation loop and resuspend in 10 µL of dH2O.

2. Use 1 µL of this colony suspension in the reaction setup outlined below (table 4) and transfer tubes to thermocycler following the steps in table 5 – this includes a 10 minute denaturation step to lyse the cells.


Table 4 -Standard PCR Reaction Setup using Dreamtaq™ Green PCR Master Mix for colony PCR.

Component Volume (total 20 µL reaction)
DreamTaq™ Green PCR Master Mix (2X) 10 µL
Primer 1 (10 µM) 1 µL
Primer 2 (10 µM) 1 µL
Template DNA 1 µL colony suspension
Deionized H2O 7 µL

Table 5 -Thermocycling Conditions for colony PCR. TA (annealing temperature) should be lowest primer Tm (melting temperature)

Step Temperature Time Number of cycles
Initial denaturation 95 10 mins 1
Denaturation 95 30 s 40
Annealing TA 30 s
Extension 72 1 min/kb
Final extension 72 10 mins 1

1. Pellet 1-2 mL of an overnight bacterial liquid culture by centrifugation at maximum speed (~13,000 rpm) and remove the supernatant.

2. Resuspend pellet in 250 µL P1 (Resuspension Buffer) and vortex.

3. Lyse cells by adding 250 µL of P2 (Lysis Buffer) and mix by inverting the tube 4-6 times. Do not vortex.

4. Neutralize the lysate by adding 350 µL of N3 (Neutralization Buffer) and mix thoroughly by inverting the tube 4–6 times.

5. Centrifuge for 10 minutes at maximum speed.

6. Transfer the supernatant from step 4 to a QIAprep 2.0 spin column, centrifuge for 1 minute and discard flow-through.

7. Wash the column by adding 500 µL Buffer PB (wash Buffer 1), centrifuge for 1 min, discard flow-through and repeat this step with 750 µL of Buffer PE (wash buffer 2). Discard all flow-through and centrifuge at max speed for an additional 1 minute to remove residual wash buffer.

8. Place spin column in a clean 1.5 mL microcentrifuge tube. To elute plasmid DNA add 50 µL of dH2O to the centre of the column leave to stand 1 minute and centrifuge for 1 minute.

1. Thaw the electrocompetent E. coli cells to be used on ice – in 50 µL aliquotes.

2. Add ~200-500 ng of plasmid DNA into the tube with the competent cells and transfer the mixture to a 0.2 cm Bio-Rad electroporation cuvette.

3. Transfer the cuvette on ice to the electroporator and set to the appropriate setting.

4. Load the cuvette into the electroporator, pulse (with a read out >5.0), retrieve cuvette and immediately add 400 µL of SOC media (Invitrogen™). Transfer the whole mixture to a labelled microcentrifuge tube and incubate at 30 °C, shaking at 250 rpm, for at least 1 hour to recover.

5. Plate a suitable volume (200 µL) onto the appropriate agar selection plates and incubate at 30 °C until colonies appear (usually after 24 hours).

1. Inoculate vectors transformed into donor E. coli CA434 in 5 ml of LB (+2.5 µL Chloramphenicol) at 30 °C shaking at 250 rpm overnight. In the anaerobic cabinet also inoculate 1 mL of reduced TYG with C. sporogenes and leave to incubate overnight.

2. In the morning pellet the E. coli by centrifuging 1 mL of this overnight culture at 4000 rpm for 1 minute.

3. Wash pellet by resuspending in 400 µL of PBS and centrifgue at 4000 rpm for 1 minute. Discard supernatant and repeat one more wash.

4. Transfer the washed E. coli donor to the anaerobic cabinet.

5. Gently resuspend the E. coli pellet in 200 µL of the C.  sporogenes overnight culture and plate the mixture onto a TYG plate supplemented with glucose (0.5% v/v final concentration) in 5 or 6 dots. Leave the bacteria to mate for at least 7 hours or overnight.

6. After the mating time harvest all the cells with a sterile 10 µL loop and gently resuspend in 500 µL of reduced PBS in a microcentrifuge tube.

7. Plate 100 µL this PBS mixture onto pre-reduced TYG -Thiamphenicol – D-Cylcoserine selective plates (with final concentrations of thiamphenicol - 15 µg/mL, D-Cylcoserine - 250 µg/ml) and leave to incubate in the anaerobic cabinet.

8. After 48 hours trans-conjugant colonies should appear.

1. Inoculate 10 mL of reduced TYG broth only in the anaerobic cabinet with a confirmed transconjugant mutant.

2. Leave the culture for 48 hours to give the bacteria time to lose the plasmid.

3. Serially dilute the culture down to 10-4 in reduced PBS and spread on a reduced TYG only agar plate to obtain single colonies.

4. Patch plate these single colonies onto first TYG only plate and then TYG plate supplemented with Thiamphenicol (15 µg/mL) and leave to incubate overnight.

5. Colonies that have lost the plasmid will only be able to grow on the TYG only agar plates.

The following step were followed when designing our primers

1. Each primer was at least 18bp (and less then 100 bp) long and an annealing temperature (TA) ensured to be between 55 °C and 72 °C.

2. A G-C clamp was desirable when looking for primer sequences - the last base of the primer (at the 3’ end) is a G/C base pair.

3. The primer melting temperature (Tm) was checked online by a Tm calculator- this shows the temperature that duplex will melt at. The TA is typically 5 °C lower than Tm calculator value.

4. Secondary structures were checked online at the mfold web server- ensuring the delta G values are positive (spontaneously secondary structures will not happen at the desired temperature). If secondary structures predicted made sure the Tm value of the secondary structures is significantly lower than your TA.

5. To amplify DNA primers were designed in pairs, with the forward and reverse primers annealing to the opposite DNA strand.

6. The NEB assembly Tool was used to design HiFi primers. This programme added complementary overhangs to the 5’end of a primer to enable HiFi assembly.

1. Inoculate reduced (left in an anaerobic cabinet for at least 4 hours to allow for all the oxygen to be lost) liquid media with the bacterial strain from a frozen stock. Adding appropriate antibiotic at the required concentration if needed. Leave to growth overnight.

2. Dilute the overnight culture 1:100 into fresh liquid media – this is t = 0 hours.

3. Pipette a 200 µL sample of this culture into well of a 96 well plate (in triplicate). Put the 96 well plate into a microplate absorbance reader and measure at OD600.

4. Incubate the culture for usually 72 hours, taking samples (as described for t=0) every 2 hours from t=0 to t=12 and then at t=24 hours, t=48 and t=72 hours. When the OD of the culture reaches >0.8, dilute the samples by a factor of 10 (20 µL of sample, 180 µL TYG) in the plate well.

5. These recorded OD values can then be plotted as a ‘growth’ curve, with time on the x-axis and OD on the y-axis.

1. Aliquot a 1 mL of each sample culture into a respectively labelled 1.5 mL microcentrifuge tube and centrifuge for 1 minute at 16,000 rpm.

2. Pipette the supernatant into fresh labelled 1.5 mL microcentrifuge tubes, taking care not to disturb the pellet which has formed.

3. Store the resultant supernatant samples -80 °C until all timepoint samples have been collected.

4. Take samples every 2 hours from inoculation of culture (t=0) to t = 12 and then at t=24 hours and t=48 hours.

5. Once all samples have been taken, thaw all samples and follow the standard GC protocol which is used at your institute.

Routes of Administration subgroup protocols:

1. Set up a culture of C. sporogenes (TYG medium, grow at 37 °C in the anaerobic cabinet).

2. Leave culture in the cabinet for at least 5 days to induce sporulation by starvation.

3. Heat-treat culture (80 °C, 20 min) to kill any remaining vegetative cell (spores survive the heat treatment). After heat-treatment, allow culture to cool down.

4. Divide culture into several tubes and spin. Discard supernatant.

5. Wash each pellet with water and spin. Discard supernatant.

6. Wash each pellet with ethanol and spin (ethanol should kill remaining vegetative cells but not spores). Discard supernatant.

7. Wash again with water to remove ethanol residues. Spin and discard supernatant.

8. Resuspend final pellet in water.

9. Pour sucrose solution (50% w/v) into Falcon tubes.

10. Gently pipette spore suspension on top of the sucrose.

11. Gently place tubes in centrifuge and spin. The vegetative cells and debris will be collected at the interface and in the gradient, while the spores will move through the gradient and form a pellet at the bottom of the tube.

12. Gently remove the cell debris from the interface and then the rest of the solution, leaving the spore pellet.

13. Wash spore pellets with water. Spin and discard supernatant.

14. Resuspend spores in water.

1. Take pure culture of Bacillus coagulant.

2. Incubate to sterilise seed medium.

3. Confirm the purity through microscopy.

4. Add water, supernatant of Corn Steep Powder, MnSO4, CaCO3, Dextrose and Ammonium Sulphate.

5. Adjust pH with Ortho-Phosphoric Acid.

6. Sterilise for 20-25 mins at 1.5 ± 0.2 bars.

7. Cool the media to 37 °C - 29 °C.

8. Inoculate with the seed inoculum.

9. Agitate and provide sterile aeration to the fermentation medium.

10. Incubate at 37 °C - 39 °C for 35-37 hrs.

11. Centrifuge at 13,000 - 15,000 rpm.

12. Add sterile water in 1:1 ratio.

13. Filter the slurry through SS sterile mesh.

14. Spray dry the filtered slurry at 115 °C inlet temperature and 55 °C outlet temperature.

15. Dialate using Maltodextrin

1. Inoculate sterile, reduced seed medium with C. sporogenes.

2. Incubate for 24 hours under anaerobic conditions, at 37 °C. Confirm purity of the culture through microscopy.

3. Prepare appropriate medium (previously selected by performing sporulation assays and evaluating spore formation), sterilise it and reduce it.

4. Inoculate medium with pure C. sporogenes from the seed culture.

5. Incubate under anaerobic conditions, at 37 °C, for 5 days (or the optimal time previously determined by performing sporulation assays).

6. Harvest cells by centrifugation.

7. Add sterile water and filter the slurry through SS sterile mesh (previous assays would have to be performed to determine if the mesh would also allow the purification of C. sporogenes spores).

8. Spray dry the filtered slurry and use the concentrated spay dried powder in the formation of the pill.

Note: sub-culturing ensures that there are no spores at the beginning of the assay (time point 0h), only vegetative cells.


1. Set up C. sporogenes overnight culture (TYG broth).

2. Sub-culture into fresh TYG (1:100) and incubate for 3h.

3. Sub-culture again into fresh TYG (1:100).

4. Immediately take 0h sample and divide in 2 aliquots.

5. Heat-treat one of the aliquots (80 °C, 20 min).

6. Serially dilute both aliquots (heat treated, and non-heat treated).

7. Plate appropriate dilutions onto TYG plates.

8. Repeat steps 4 to 7 for each time point (24 h, 48 h, 72 h, 96 h and 120 h).

9. For each time point, count colonies after approx. 24 h of growth.

10. Calculate CFU/mL:

CFU/mL = N x D x 1000 µL / V(µL)

  1. N: number of colonies
  2. D: dilution factor
  3. V: volume plated

1. Set up C. sporogenes overnight culture (TYG broth).

2. Sub-culture into fresh TYG (1:100) and incubate for 120 h to allow spore formation.

3. Heat-treat culture (80 °C, 20 min) to kill vegetative cells.

4. Purify the spores (follow spore production and purification protocol, using sucrose gradient).

5. At time point 0 h, resuspend the spores in the germinant being tested to an OD600 of 1.0.

6. Monitor OD600 at several time points: 0 h, 2 min, 5 min, 10 min, 15 min, 30 min, 45 min, 60 min and 90 min. This can be done in the CLARIOstar plate reader.