0.4 Mini-prep (GenElute Plasmid MiniPrep Kit from Sigma-Aldrich)
0.5 Glycerol stock
1.1 PCR amplification
1.2 PCR clean up (GenElute PCR Clean-Up Kit from Sigma-Aldrich)
1.3 Restriction Enzyme Digest
1.4 Gel extraction (GenElute Gel Extraction Kit from Sigma-Aldrich)
1.5 Ligation
1.6 MyTaq colony PCR
1.7 Golden Gate Cloning
2.1 Tri-parental Conjugation with serial dilutions
2.2 QIAprep Spin Miniprep Kit (Qiagen Kit)
2.3 Preparation of liquid LB media and LB + agar
2.4 Preparation of stock Arabinose
2.5 Arabinose induced protein expression
2.6 SDS-PAGE gel preparation
2.7 Preparation of samples for SDS-PAGE gel analysis
2.8 SDS-PAGE gel analysis
2.9 SDS-PAGE gel staining
2.10 Electroporation preparation
2.11 Western Blot preparation
2.12 Western Blot analysis
3.1 Gas Chromatography Preparations
3.2 Gas Chromatography Readings
3.3 Electrochemistry
Appendix 1
Protocols
To 100ml of TAE (Tris base, acetic acid and EDTA) buffer 1g of agarose was added. This was heated until the agarose completely dissolved.
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.
E. coli cells were thawed on ice for 15 mins and at the same time LB medium was pre-warmed to 37°C.
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.
The samples were incubated at 42°C for 2 mins, then 250 µl of LB medium was added to each sample.
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.
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.
Under sterile conditions, single colonies were selected and then transferred into a sterilin tube containing 10ml of LB liquid medium with required antibiotic added.
The inoculated medium was then incubated overnight at 37°C (for E.coli) or 30°C (for Shewanella) with shaking.
Overnight recombinant E.coli cells were pelleted by centrifugation at maximum speed (≈ 14,000 rpm) for 1 minute in a 1.5ml Eppendorf. The supernatant was then discarded and the processes repeated until enough cells were harvested.
The bacterial pellet was thoroughly re-suspended in 200 µl of Cell Resuspension Solution by vortexing or pipetting up and down. To this, 200µl of Cell Lysis solution was added and gently mixed by inverting the tube 4-6 times. This was left at room temperature until the solution became clear and viscous, but it was not allowed to exceed 5minutes.
Subsequently, 350µl of Neutralising Buffer was added and mixed gently by inverting the tube 6-8 times. The cell debris was pelleted by centrifugating at maximum speed for 10 minutes.
Meanwhile, 500µl of Column Preparation solution was added to a GenElute MiniPrep Binding Column which was inserted into the provided microcentrifuge collection tube. This was centrifuged at maximum speed for 1 minute, the flow through was discarded.
The cleared lysate was added to the binding column and centrifuged for 1 minute at maximum speed. The flow through was discarded. Then, 500µl of Wash Solution 1 was added and centrifuged for 1 minute at maximum speed. The flow through liquid was discarded.
A volume of 750µl of diluted Wash Solution 2 was added and centrifuged at maximum speed for 1 minute. The flow through was discarded. The spin column was centrifuged again without any additional Wash Solutions for 2 minutes at maximum speed to remove excess ethanol.
The binding column was transferred to a fresh collection tube. The DNA was eluted by adding 50µl of molecular water, which was incubated for 2 minutes before centrifuged at maximum speed for 1 minute.
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.
The tubes were flash frozen in liquid nitrogen and stored in -80˚C freezer.
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.
The PCR was set to the conditions appropriate for the primers used.
Spin column A was placed in a collection tube and 500µl of binding buffer A was added to the spin column
Up to 50µl of the PCR mixture was added to spin column A and was mixed by pipetting gently. This was spun at 10, 000 x g for 2min.
The collection tube was discarded and Spin Column A was placed in a 1.5 elution tube. A volume of 10-20µl of elution buffer was directly added to the spin column membrane. This was incubated at room temperature for 1min.
The tube was spun at 6000 x g for 1min to elute PCR product.
1. If frozen, DNA samples were thawed on wet ice.
2. 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.
3. The tubes were prepared accordingly and left to incubate in a 37C water bath for 1-2h and then frozen.
Excise gel band from agarose gel
Weigh gel in Eppendorf tube after having zeroed the scale with an empty Eppendorf
For every 100mg of gel add 300 µl of gel solubiliser solution.
Incubate at 50-60’C for 10 min (or until completely dissolved). Vortex every 2-3minutes
Prep binding column by adding 500 µl of column preparation solution to the spin column (in collection tube). Centrifuge for 1m and discard flow-through.
Check colour of gel when dissolved. If red rather than usual yellow then add 10 µl of 3M sodium acetate buffer pH5.2. add in increments until yellow.
Add 1 gel volume of 100% isopropanol and mix until homogenous. (gel with agarose concentration greater than 2% then use 2 gel volumes of 100% isopropanol)
Load solubilised gel solution from previous step into the prepped column. If the volume of gel mixture is >700 µl load sample onto column in 700 µl portions. Centrifuge for 1m after each load, discarding the flow-through.
Add 700 µl of Wash solution to binding column. Centrifuge for 1min. Discard flow-through. Centrifuge for 1 min to remove excess ethanol (residual wash solution will not be completely removed unless the flow-through is discarded before the final centrifugation).
Elute DNA: transfer column to fresh collection tube. Add 50 µl of elution solution to the centre membrane and incubate for 1 min. centrifuge for 1min. (preheat elution solution to 65’C for greater plasmid recovery. To increase concentration or eluted DNA, volume of elution solution can be reduced to 25 µl. However this will lead to a loss in yield)
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).
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.
Gently mix reaction by pipetting up and down or centrifuge briefly. This was left to Incubate overnight at 16˚C.
Prior to use for downstream application, the reaction mixture was heat inactivated for 10 min at 65˚C.
When using a colony: an inoculation loop was used to extract one colony from a plate. It was then resuspended in 10µl of nuclease free water and boiled for 5min, for gram-negative bacteria. Alternatively, when using DNA: a volume of 50µl of fresh bacterial culture was centrifuged for 3min at 13000rpm and the supernatant was dissolved. The pellet was dissolved in 100µl of nuclease free water and boiled for 5min. The solution was then centrifuged for 3min at 13000rpm and the supernatant was transferred into a new tube for storage.
Using the aforementioned DNA a PCR reaction was set up with the following volumes, 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)
The PCR reactions were set to cyclic conditions appropriate for the primers, DNA template and enzymes
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.
The vector and ligation ratios were determined by using the equation in appendix 1.
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.
Afterwards, the PCR product was directly used to transform competent E.coli cells to observe correct ligations.
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
1 ml of each culture was taken and each centrifuged at max speed for 1 minute.
Each pellet was re-suspended in separate 1 ml volumes of fresh LB media.
These were then spun at max speed for 1 min and the supernatant was then discarded.
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.
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).
The lawn of bacteria was scraped off the plate and re-suspended in 1 ml of fresh LB liquid media.
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)
100µl of each dilution was spread onto LB+Kanamycin (50µg/ml) + Carbenicillin (100µg/ml) plates. These were then grown overnight at 30°C.
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.
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
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.
1.5g of Arabinose (L-(+)-Arabinose) was dissolved into 10ml of distilled water. Giving 1 M stock solution
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.
Arabinose was added to cultures when the OD reached 600, and cultures were incubated for a further 4 hours at 30℃
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)
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)
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.
Each pellet was then re-suspended in 1ml of lysis buffer (consisting of: 20mM HEPES, 0.01mg DNase, 0.01mg lysozyme, pH7).
Samples were prepared at a 1:1 ratio with protein sample buffer
The samples were then denatured by incubating at 100°C for 10min
A 1x SDS Buffer (25 mM Tris, 192 mM glycine, 0.1% SDS) was prepared for running the gel.
The gel was run at 115V, 40mA for 1h 15min.
Coomassie Blue Dye was used to stain the gel for 10min
After unplugging and dismantling the machine, the gel was carefully placed into a square tray and for all Protein analysis bands:
• Coomassie Blue Dye was poured over the top of gel, the tray was sealed and placed onto a rocking platform for 10 mins until the dye turned an almost clear bright blue.
The dye was poured away and the gels in the trays were then viewed under a light box to compare bands (the gel image bands appeared stronger and bolder when left overnight).
1 mol of 50 ml Sorbitol was made up (dissolved 9.1085 g in 50 ml of distilled water, then filtered using blue circle filter and syringe) and was put on ice.
Required golden gate vector was nano-dropped to ensure ideal concentration for electroporation (100-500ng), if not it was adjusted.
Inoculated each of the required number of 100 ml LB media flasks (with no antibiotic), with 2 ml of each required Shewanella culture
These flasks were then incubated in the 30°C room for 2 hrs
The first set of Optical Density (OD) readings were measured (using fresh LB as a blank), and immediately put on ice once the culture had reached an OD between 0.4 – 0.6.
Each 100ml culture was split into two 50 ml falcon tubes and spun down for 10 mins at max speed to form a pellet.
The supernatant was fully discarded (using a pipette to remove everything as high salt concentration is not good for electroporation) the pellets were then re-suspended in 8ml of Sorbitol.
They were then all spun down for a further 10 mins at max speed to form a pellet.
The supernatant was fully discarded (using a pipette to remove everything as high salt concentration not good for electroporation) and the pellets were then re-suspended in 2 ml of Sorbitol.
Already prepared SOC media was filtered using a blue circle filter and syringe into a clean sterile tube.
6 x 800 µl of SOC media was pipetted into 6 sterile Eppendorf tubes in preparation for electroporation later
400 µl of each culture (six falcon tube cultures re-suspended in 2 ml sorbitol) was pipetted into six separate electroporation cuvettes.
To each electroporation cuvette 5 µl of the relevant GG Vector was added.
The cuvettes were then tapped to circulate the DNA.
Each cuvette was zapped using the electroporation machine after which the contents in all the cuvettes were quickly transferred into the corresponding SOC (800 µl) Eppendorf prepared earlier.
All electroporated Eppendorf samples were left for 2 hrs in the shaking incubator at 30°C.
After 2 hrs, 200 µl of each sample was plated onto LB + Km 20 µg/ml and left to grow overnight at 30°C.
The remaining volume of each Eppendorf sample was used to inoculate corresponding 10 ml LB overnight cultures at 30°C.
200 µl of the inoculates from electroporation were plated onto LB + Km 30 µg/ml plates the following day and left to grow overnight at 30°C.
1. Western Blot preparation
Alkaline phosphatase buffer:
• NaCl: 100 mM
• MgCl2: 5 mM
• Tris. HCl: 100 mM at pH 9.5
• NBT: 66 µl in 15 ml of the alkaline phosphatase buffer
• BCIP: 33 µl
Transfer Buffer:
• Tris. HCl: 20 mM at pH 8.3
• Glycine: 192 mM
• Methanol: 20 %
Tris Buffer Saline (TBS):
• NaCl: 140 mM
• Tris. HCl: 20 mM at pH 7.5
• Methanol: 20 %
TBS + TWEEN 20 (TBST):
• TBS + 0.1 % TWEEN 20 at pH 7.5
Blocking solution:
• TBS + 5 % skimmed milk powder (w/v) made fresh
An SDS-PAGE gel was set-up and run as normal with samples and 8 µl of a marker.
Six sheets of blotting paper were soaked in transfer buffer for a few minutes and laid onto the semi dry blotter.
The PVDF membrane was soaked in methanol for a few seconds and then washed in transfer buffer. This was then placed on top of the blotting paper.
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.
Six more sheets of blotting paper were soaked in transfer buffer and placed on top of the SDS-PAGE protein gel.
The lid was then placed onto the blotter and connected to a power pack.
The blotter was run at 20 mA per gel for 45 – 60 mins at a voltage below 5 V.
When the blot was complete, the membrane was removed and placed into a plastic tray.
The membrane blot was blocked with 15 – 20 ml of blocking solution at 4°C, and left for 1 hr.
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 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.
The membrane blot was then washed with 20 ml of TBST + 1% milk powder and placed on a rocking platform for 5 mins.
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.
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.
The membrane blot was then washed with 20 ml of TBST + 1% milk powder and placed on a rocking platform for 5 mins.
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.
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.
The reaction was then stopped by washing the membrane blot twice with distilled water and then drying it.
Additional Growth Addition quantities
Lactate - 0.84 mL
HEPES - 0.893 g
NiCl2- 44.75 mg
Fumarate - 0.3 g
Prepared the AGA according to the quantities listed above.
Dissolved in a minimum amount of water (15 - 20 mL, magnetic stirrer bead & stirrer plate).
pH was set to approximately 7.80 - 7.90 using concentrated NaOH
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.
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).
Oxygen was purged from headspace with N2 for 5 minutes.
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.
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.
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.
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.
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.
The syringe was purged between each sample.
Made 2L of M72 media with AGA additions - as described in 3.1.
Prepared four 500mL durans of this media which were then autoclaved.
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).
Sparged the headspace with nitrogen for approximately 20min.
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.
Basal media equilibrated in electrochemistry glovebox for at least 12 hours.
Cultures were then taken into glovebox and aliquoted into 50ml falcon tubes to maintain anaerobic environment.
Samples Removed from glove box and pellet cells, 5100rpm, 15/20min – red pellet should be visible at the bottom.
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.
Samples spun at 5100rpm, 15 minutes.
Repeated wash step twice more, thus a total of three HEPES buffer washes.
Final resuspension of cell pellets in 2ml of HEPES buffer - pipetted each strain into a smaller screw top tube.
OD600 was measured for all strains and diluted to normalise, this allows for a valid comparative study.
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.
