Team:NRP-UEA-Norwich/Protocols

NRP-UEA-NORWICH iGEM

  • Making an agarose gel
  • Transformation
  • Inoculation
  • Mini-prep (GenElute Plasmid MiniPrep Kit from Sigma-Aldrich)
  • Glycerol stock
  • PCR amplification
  • PCR clean up (GenElute PCR Clean-Up Kit from Sigma-Aldrich)
  • Restriction Enzyme Digest
  • Gel extraction (GenElute Gel Extraction Kit from Sigma-Aldrich)
  • Ligation
  • MyTaq colony PCR
  • Golden Gate Cloning
  • Tri-parental Conjugation with serial dilutions
  • QIAprep Spin Miniprep Kit (Qiagen Kit)
  • Preparation of liquid LB media and LB + agar
  • Preparation of stock Arabinose
  • Arabinose induced protein expression
  • SDS-PAGE gel preparation
  • Preparation of samples for SDS-PAGE gel analysis
  • SDS-PAGE gel analysis
  • SDS-PAGE gel staining
  • Electroporation preparation
  • Western Blot preparation
  • Western Blot analysis
  • Gas Chromatography Preparations
  • Gas Chromatography Readings
  • Electrochemistry
  • Appendix 1
  • Appendix 2

Protocols

    1. To 100ml of TAE (Tris base, acetic acid and EDTA) buffer 1g of agarose was added. This was heated until the agarose completely dissolved.
    2. Once cooled, 7µl of Ethidium Bromide was added to the dissolved agarose and the solution was poured into the tray, a comb added and left to set.
    1. E. coli cells were thawed on ice for 15 mins and at the same time LB medium was pre-warmed to 37°C.
    2. A 1 µl volume of standard prepared DNA was added to 25 µl of cells and mixed gently. This was then incubated on ice for 30 mins. Cells not transformed with DNA were used as a control.
    3. The samples were incubated at 42°C for 2 mins, then 250 µl of LB medium was added to each sample.
    4. The diluted transformations were left to recover in a shaker incubator at 37˚C. The recovery time changed depending on the antibiotic resistance e.g. chloramphenicol was 2h and kanamycin was 1h.
    5. A volume of 50µl of the transformed cells were plated on LB agar plate with the required selective marker. The plates were then incubated overnight at 37˚C.
    1. Under sterile conditions, single colonies were selected and then transferred into a sterilin tube containing 10ml of LB liquid medium with required antibiotic added.
    2. The inoculated medium was then incubated overnight at 37°C (for E.coli) or 30°C  (for Shewanella) with shaking.
    1. To obtain DNA from the transformed E.coli we followed the “GenElute Plasmid MiniPrep Kit” provided by Sigma-Aldrich. To elute the DNA from the spin column 50µl of molecular water was used as opposed to the elution solution provided.
    1. Under sterile conditions, 500µl of the overnight inoculated LB culture was added to a 2ml screw-cap tube. To this, 500µl of 50% sterile glycerol solution was added and the tube mixed by inverting.
    2. The tubes were flash frozen in liquid nitrogen and stored in -80˚C freezer.
    1. PCR tubes were prepared with the reaction mixture which contained: 25µl of Master Mix (Phusion High-Fidelity PCR Master Mix with HF Buffer), 2.5µl of primers (forward and reverse), 1µl of the DNA to be amplified and 21.5µl of molecular water to get the reaction mixture up to 50µl.
    2. The PCR was set to the conditions appropriate for the primers used.
    1. Amplified DNA was purified from the sample using the GenElute PCR Clean-Up Kit. See the “GenElute PCR Clean-up Kit” from Sigma Aldrich protocol for details
    1. Eppendorf tubes were labelled and the restriction digest mixture was prepared. In each tube the following was added, note the enzymes were added last.
      • • 2µl of Anza 10X Buffer. Note, if these restrictions were loaded on a gel afterwards then Anza 10X red buffer was used
      • • 1µl of the enzyme(s). In this experiment it was EcoR1 and Pst1.
      • • 16µl of DNA and Nuclease Free water. Water was to be added when the volume of DNA was less than 16µl.
      • The samples were left to incubate in a 37˚C water bath for 1-2h and then analysed using an agarose gel.
    1. See the “Sigma-Aldrich GenElute Gel Extraction Kit” protocol. To elute the DNA from the spin column, 50µl of molecular water was used as opposed to the provided elution solution.
    1. Set up the reaction mixture as required, see appendix 1 (for different ligation reactions, optimum insert: vector volume ratio varies so try multiple for each sample, e.g. 1:1 and 1:3).
    2. A total reaction volume of 10µl or 20µl, depending on the ligation rations required with: 10X ligase buffer at a tenth volume of the total reaction volume, nuclease free water to make it up to the total reaction volume and 1µl T4 DNA ligase.
    3. Gently mix reaction by pipetting up and down or centrifuge briefly. This was left to Incubate overnight at 16˚C.
    4. Prior to use for downstream application, the reaction mixture was heat inactivated for 10 min at 65˚C.
    1. A single  colony was picked from a plate. It was then resuspended in 10µl of nuclease free water and heated to 100˚C for 5 min.
    2. Using the boiled bacterial colony as the DNA, a PCR reaction was set up with the following volumes, to give total volume of 25µl
      • • 12.5µl of My Taq red Mix Bioline
      • • 1µl of forward primer, 10µM
      • • 1µl of reverse primer, 10µM
      • • 1µl bacterial DNA or colony
      • • 9.5µl of water
      • • Sometimes addition of DMSO, up to 5% concentration (1.25µl in a total volume of 25µl)
    3. The PCR reactions were set to cyclic conditions appropriate for the primers, DNA template and enzymes
    1. A PCR tube containing a total reaction volume of 15µl was set up containing: 1.5µl ligase buffer, 1.5µl Cut buffer, 1µl BsaI, 1µl T3 DNA ligase and 10µl of the vector and insert being used.
    2. The vector and ligation ratios were determined by using the equation in appendix 1.
    3. Consequently, a PCR reaction was performed under the following conditions: 25 cycles of 37˚C for 3min and 16˚C for 4min. Followed by 50˚C for 5min and 80˚C for 5min.
    4. Afterwards, the PCR product was directly used to transform competent E.coli cells to observe correct ligations.
    1. Overnight cultures of the following were set up (using 1 ml to 1 µl, LB to antibiotic ratio):
      • • Carbenicillin selected Shewanella oneidensis MR-1 in LB + Carbenicillin 100 µg/ml
      • • PRk2013 containing E. coli cells on LB + Kanamycin 50 µg/ml.
      • • Transformed E. coli cells on LB + Kanamycin 50 µg/ml
    2. 1 ml of each culture was taken and each centrifuged at max speed for 1 minute.
    3. Each pellet was re-suspended in separate 1 ml volumes of fresh LB media.
    4. These were then spun at max speed for 1 min and the supernatant was then discarded.
    5. Each pellet was re-suspend sequentially (from transformed E.coli to PRk2013 to S. oneidensis) in 1 ml LB so that all were suspended in the same Eppendorf tube before spinning for a further 1 min at max speed.
    6. The mixed pellet was then re-suspended in 100 µl LB and transferred to the middle of an LB plate (this was not spread and no selective antibiotics were present in the media), this was then grown overnight at 30°C (the optimum temperature for Shewanella oneidensis MR-1).
    7. The lawn of bacteria was scraped off the plate and re-suspended in 1 ml of fresh LB liquid media.
    8. This cell suspension was serially diluted by taking 100 µl of the culture and adding it to 900 µl of LB media. This was done sequentially until a suitable dilution had been reached (range down to ≈ 10-6)
    9. 100µl of each dilution was spread onto LB+Kanamycin (50µg/ml) + Carbenicillin (100µg/ml) plates. These were then grown overnight at 30°C.
    10. Single colonies were picked from the plates that visually appeared to be Shewanella (red) rather than E.coli (white). These were then grown overnight in LB and Kanamycin.
    1. The QIAprep Spin MiniPrep Kit from Qiagen was used (see provided protocol). A volume of 50µl of molecular water was used as opposed to the provided elution buffer
    1. To make fresh LB media: 25g of LB high salt powder (consisting  of: 10g tryptophan, 5g yeast extract and 10g of sodium chloride), was dissolved in 1L of distilled water.
    1. 1.5g of Arabinose (L-(+)-Arabinose) was dissolved into 10ml of distilled water. Giving 1 M stock solution
    1. Cultures were inoculated into fresh 100ml LB with Carbenicillin and Kanamycin (for the control culture no Kanamycin was added), 100µl/mL and 50µl/mL respectively. These were incubated at 30°C until an OD600 was reached.
    2. Arabinose was added to cultures when the OD reached 600, and cultures were incubated for a further 4 hours at 30℃
    1. Running Gel Recipe: Acrylamide: 4ml Sol (1M tris pH 8.8) 2: 2.5ml H2O: 3.5ml SDS 10%: 100µl TEMED: 10µl 10% APS: 100µl (added last, as gel will begin to set) (to prepare dissolve 0.05g APS in 500µl distilled water)
    2. Stacking gel recipe Acrylamide: 0.75ml Sol (Tris 0.5M pH 6.4) 3: 1.25ml H2O: 3ml SDS 10%: 50µl TEMED: 5µl 10% APS: 50µl (added last, as gel will begin to set)
    1. The 10ml overnight cultures were poured into falcon tubes and centrifuged for 5min at max speed to pellet the cells. The supernatant was then discarded.
    2. Each pellet was then re-suspended in 1ml of lysis  buffer (consisting of: 20mM HEPES, 0.01mg DNase, 0.01mg lysozyme, pH7).
    3. Samples were prepared at a 1:1 ratio with protein sample buffer
    4. The samples were then denatured by incubating at 100°C for 10min
    1. A 1x SDS Buffer (25 mM Tris, 192 mM glycine, 0.1% SDS) was prepared for running the gel.
    2. The gel was run at 115V, 40mA for 1h 15min.
    1. Coomassie Blue Dye was used to stain the gel for 10min
    1. 50 ml of 1 M Sorbitol was put on ice.
    2. Required golden gate vector was nano-dropped to ensure ideal concentration for electroporation (100-500ng), if not it was adjusted.
    3. Inoculated each of the required number of 100ml LB media with 2ml of each required Shewanella culture.
    4. These were then incubated in the 30°C room for 2h.
    5. OD was monitored and samples were placed on ice when OD was reached
    6. Each 100ml culture was centrifuged for 10min at max speed to form a pellet.
    7. The supernatant was fully removed via pipetting ( as high salt concentration is not good for electroporation) the pellets were then re-suspended in 8ml of Sorbitol.
    8. Samples were then all spun down for a further 10min at max speed to form a pellet.
    9. The supernatant was fully discarded and the pellets were then re-suspended in 2ml of Sorbitol.
    10. SOC media (from Sigma-Aldrich) was filtered using a blue circle filter and syringe into a clean sterile tube.
    11. 400µl of each culture was pipetted into six separate electroporation cuvettes.
    12. To each electroporation cuvette 5µl of the relevant GG Vector was added.
    13. The cuvettes were then tapped to circulate the DNA.
    14. A current was applied to the cuvette and the contents transferred into 800µl SOC.
    15. All electroporated samples were incubated for 2h in the shaking incubator at 30°C.
    16. 200µl of each sample was plated onto LB + Km 20µg/ml and left to grow overnight at 30°C.
    17. The remaining sample was used to inoculate 10ml LB overnight cultures at 30°C.
    1. Western Blot preparation Recipes- Alkaline phosphatase buffer:
      • NaCl: 100mM
      • MgCl2: 5mM
      • Tris. HCl: 100mM at pH 9.5
      • NBT: 66µl in 15ml of the alkaline phosphatase buffer
      • BCIP: 33µl
      • Transfer Buffer:
      • Tris. HCl: 20mM at pH 8.3
      • Glycine: 192mM
      • Methanol: 20 %
      • Tris Buffer Saline (TBS):
      • NaCl: 140mM
      • Tris. HCl: 20mM at pH 7.5
      • TBS + TWEEN 20 (TBST):
      • TBS + 0.1 % TWEEN 20 at pH 7.5
      • Blocking solution: TBS + 5 % skimmed milk powder (w/v) made fresh
    1. An SDS-PAGE gel was set-up and run following protocol 2.6 with samples and 8 µl of a marker.
    2. Six sheets of blotting paper were soaked in transfer buffer and laid onto the semi dry blotter.
    3. The PVDF membrane was soaked in methanol and then washed in transfer buffer. This was then placed on top of the blotting paper.
    4. The SDS-PAGE protein gel was then carefully removed from the plates, washed in transfer buffer and placed directly on top of the PVDF membrane.
    5. Six more sheets of blotting paper were soaked in transfer buffer and placed on top of the SDS-PAGE protein gel.
    6. The blotter was run at 20mA per gel for 45 – 60min at a voltage below 5V.
    7. The membrane blot was blocked with 15 – 20 ml of blocking solution at 4°C, and left for 1 hr.
    8. The membrane blot was then washed with 20 ml of TBST and placed on a rocking platform for 5 mins. This was done three times, discarding the previous wash each time.
    9. 15 ml of TBST + 1% milk powder solution with addition of the primary antibody was then added to the membrane blot, and left at 4°C for 1 hr.
    10. The membrane blot was then washed with 20 ml of TBST + 1% milk powder and placed on a rocking platform for 5 mins.
    11. The membrane blot was then washed with 20 ml of TBST and placed on a rocking platform for 5 mins. This was done five times, discarding the previous wash each time.
    12. 15 ml of TBST + 1% milk powder solution with addition of the secondary antibody was then added to the membrane blot, and left at 4°C for 1 hr.
    13. The membrane blot was then washed with 20 ml of TBST + 1% milk powder and placed on a rocking platform for 5 mins.
    14. The membrane blot was then washed with 20 ml of TBST and placed on a rocking platform for 5 mins. This was done three times, discarding the previous wash each time.
    15. After discarding the TBS from the membrane blot the alkaline phosphatase buffer was added and left at room temperature for 15 – 30 mins or longer, to allow the blot to develop.
    16. The reaction was then stopped by washing the membrane blot twice with distilled water and then drying it.
    1. Additional Growth Addition quantities
      • Lactate  - 0.84 mL
      • HEPES - 0.893 g
      • NiCl2- 44.75 mg
      • Fumarate - 0.3 g
    1. Prepared the AGA according to the quantities listed above.
    2. Dissolved in a minimum amount of water (15 - 20 mL, magnetic stirrer bead & stirrer plate).
    3. pH was set to approximately 7.80 - 7.90 using concentrated NaOH
    4. AGA was added to M72 and mix then 10 ml of new solution was added to the hungate tube, making sure the lid was the correct orientation when removed.
    5. 200 μL of the desired liquid was added to overnight bacterial culture to the tube and sterilised at the end of the tube over a Bunsen flame, the lid was closed and inverted once. Thi was repeated for each hungate tube (3 each of wild type, knockout and growth medium only).
    6. Oxygen was purged from headspace with N2 for 5 minutes.
    7. We checked the optical density of the stock bacterial solutions. The blank was 800 μL of water, and the samples were 200 μL of bacteria in 600 μL of water. The true optical density is therefore 4 times the value shown.
    8. Optical density readings of the hungate bacterial cultures were taken every hour to produce a growth curve while kept in a 30 degree incubator for 24 hours.
    1. We Injected 150μL (0.15mL) of air into the chromatograph, checked the graph and recorded the results (retention time and area), ignoring the first peak. Then injected on the long beep (after 5 seconds) and kept a firm grip on the plunger to prevent back-flow.
    2. The syringe was purged with N2: the needle was placed into the N2 tap and this was filled/emptied repeatedly. 150μL was left in the syringe.
    3. 150μL N2 was injected into the hungate tube to equilibrate the gases. 150μL was withdrawn from the tube and injected, results were recorded as before.
    4. The syringe was purged between each sample.
    1. Made 2L of M72 media with AGA additions - as described in 3.1.
    2. Prepared four 500mL durans of this media which were then autoclaved.
    3. Inoculated 5 mL of double KO (LS473) and WT (MR1) to two 500mL durans of M72, incubated for 12 hours at 30 degrees without agitation (anaerobic growth).
    4. Sparged the headspace with nitrogen for approximately 20min.
    5. Prepared basal media - consisting of 0.46g/L NH4Cl, 0.225g/L K2HPO4, 0.225g/L KH2PO4, 0.117g/L MgSO4 and 23.63g/L HEPES. Filter sterilised and made up using analytical grade water.
    6. Basal media equilibrated in electrochemistry glovebox for at least 12 hours.
    7. Cultures were then taken into glovebox and aliquoted into 50ml falcon tubes to maintain anaerobic environment.
    8. Samples Removed from glove box and pellet cells, 5100rpm, 15/20min – red pellet should be visible at the bottom.
    9. Samples returned  to glovebox, pellets combined for each strain (DKO and WT) into two falcon tubes, resuspend in 50 ml of 100mM HEPES buffer pH 7.2.
    10. Samples spun at 5100rpm, 15 minutes.
    11. Repeated wash step twice more, thus a total of three HEPES buffer washes.
    12. Final resuspension of cell pellets in 2ml of HEPES buffer - pipetted each strain into a smaller screw top tube.
    13. OD600 was measured for all strains and diluted to normalise, this allows for a valid comparative study.
    14. Weight out Methyl Viologen (MV) to transport to chemistry - 5mg of methyl viologen, when dissolved in 2ml basal media, gives 10 mM solution - store powder in small glass screw top container and dissolve anaerobically in electrochemistry glovebox.
    15. Day before - All graphite working electrodes were sonicated and polished (with a small amount of aluminium oxide on cotton wool) 3 times, each sonication step for 40seconds.
    16. Cells were washed with 100% ethanol thoroughly then left to dry. Then an appropriate number of counter-electrodes were prepared (platinum wire in 200µl tip).
    17. 6 cells were prepared in the glove box with Ag/AgCl reference electrode, platinum counter electrode and graphite working electrode. Any other screw fittings were sealed with suba seals and parafilm.
    18. 30 ml of basal media was added to each cell
    19. The working electrode was poised at a reducing potential, using DropSens multichannel potentiostat. -0.76 vs AgCl, (-0.56 vs SHE)
    20. Whilst recording current add bacterial cells, it was monitored for 10min - add using syringe and needle by injecting through one of the suba seal fittings.
    21. Whilst still recording the current the MV was added, (50µl of 10 mM stock) and current was monitored 10mins.
    22. After measuring current,  final samples were taken from the headspace using a gas tight hamilton.
    23. A sample of culture from the bioreactor was also taken for determining the protein concentration in each experiment as another way of normalising results, this was done using a standard bradford assay according to the manufacturer's instructions.
    • Ligation reaction equation can be used to calculate the minimum reaction volumes needed for a 1:1 ratio between vector and insert. This method takes into account the relative concentrations of the insert and vector.
    • (ng vector) × (kb insert)=(ng insert)× (kb vector)
    • (ng vector × kb insert)/kb vector =ng insert
    • ng insert/concentration of insert  (ngµl-1) = volume of insert (µl)
    • ng vector/concentration of insert  (ngµl-1) =volume of vector (µl)

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