Team:Kent/Experiments

Materials


Chemical Synthesis Materials:

  • Iron(III) Chloride Hexahydrate (0.4M)
  • Ethylene Glycol (Anhydrous)
  • Iron(III) Chloride (aq) Hexahydrate (1M)
  • Iron(II) Sulphate (HCl) Tetrahydrate (1M)
  • HCl (2M)
  • Ammonia Solution (0.7M)

Antibiotics for Plates:

  • Ampicillin 100 g/L stock
  • Chloramphenicol 25 g/L stock

Protein Induction Sugar:

  • IPTG Stock (1M)

Nickel Ion Affinity Chromatography Column Buffers:

Bacterial Transformation:

Competent cells were thawed on ice until defrosted, and 30-40µl of cells were mixed with >50µg of DNA. After a 20 min incubation on ice the sample was heat shocked at 42oC for 60 sec, and placed on ice for a further 5 min. LB broth or SOC medium was added to the cells in order to create a total volume of 1ml. Finally the samples were incubated for 60 min at 37oC and then pipetted onto the relevant plate in volumes of 20µl, 200µl and 780µl and incubated overnight at 37oC. Single colonies were then chosen from these plates and plated onto quarter plates which were incubated under the same conditions overnight.

Protein Expression:

Conical flasks or baffled flasks were inoculated with overnight liquid cultures. The appropriate antibiotic was added and the conical flasks were incubated at 30oC at 180 rpm until an OD 600 reading of 0.4 was observed. IPTG was added to the conical flasks at a final concentration of 100 microM and flasks were incubated for 16 hours at 18oC at 180 rpm. Cells were then harvested at 4000rpm and 4oC for 20 min, the supernatant was discarded and the pellet frozen.

Protein purification:

Cell pellets were resuspended in nickel column binding buffer, sonicated and cell debris was removed via centrifugation at 20,000 rpm and 4oC for 20 minutes. The supernatant was then applied to a nickel ion affinity chromatography column. The first 5 ml of flow-through from each wash step was collected and stored, the eluted protein was collected in 10 x 1ml fractions which were frozen for later use.

SDS-PAGE:

The 1 ml fractions from the elution step of the purification were defrosted and resolved on a reducing 12% SDS- PAGE gel. Samples that were analysed included whole cells pre-induction, whole cells post- induction, soluble cell fraction (i.e. initial supernatant), the unbound fraction washed out with binding buffer, as well as samples eluted during the wash and final elution steps. Elutions were then desalted in a spin column and run through a buffer exchange column. UV-visible absorption spectra were recorded on the final elution fractions that tested positive using Bradford's reagent, absorption maxima were expected at approximately 407 nm for proteins predicted to bind c-type haem cofactors.

Transmission Electron Microscopy Imaging:

Reaction mixtures were prepared with final concentrations of purified protein at 5µM, 0.1M Fe 2+ and 0.2M Fe 3+ . Reactions were incubated for 1h 40 min under anaerobic conditions to prevent nonenzymatic magnetite formation. Once this incubation period had elapsed, 5µl samples were imaged using a transmission electron microscope to determine whether crystals had formed. Reactions carried out include single and triple combinations of mam P, T and X proteins, with and without synthetic magnetite crystals as a nucleating site under non-reducing conditions, and without synthetic crystals under reducing conditions. Controls under the same conditions were also carried out with pet3a (mam negative), and protein negative.

Chemical Synthesis of Magnetite Nanoparticles:

Ethylene Glycol Reduction: Iron(III) Chloride Hexahydrate (0.54g) was dissolved in deionised water (50 ml) and heated at 80oC for 24 hours. Reaction was cooled to room temperature, then centrifuged at 4000rpm for 10 min. The precipitate was then washed with deionised water and resuspended in 5 ml and stored. 500µl of precursor was added to ethylene glycol (50 ml) and refluxed at 200oC for 6 hours and cooled to room temperature. The solution was then centrifuged at 4000rpm for 10 minutes, washed with ethanol and then resuspended.

Co-Precipitation: Iron(III) Chloride (aq) (1M, 10 ml) and Iron(II) Sulphate (1M, 5 ml) in HCl (2M) was added to Ammonia Solution (0.7M, 175ml). The orange Iron(III) solution and the green Iron(II) solution upon addition to the ammonia solution formed a black gelatinous precipitate. This was then centrifuged at 4000 rpm for 10 min and resuspended in ethanol for characterisation.

In vivo protein expression and biomineralization:

Co-transformation of pSB1A3+[AraC-pBAD]+MamOPXT and pEC86 into E. coli strain BL21 DE3 was carried out in accordance with transformations protocol described previously. Transformants were plated onto double antibiotic (ampicillin and chloramphenicol) growth medium plates and subsequently incubated overnight at 37oC.

A 50mM stock solution of Ferric Citrate was prepared by mixing 50mM Trisodium Citrate with 50mM Iron (III) Chloride.

Inducing liquid media was prepared containing LB, 50µM Ferric citrate, 0.2% L-Arabinose (w/v), 1mM IPTG, 100 mg/ml ampicillin and 25 mg/ml chloramphenicol. Successfully transformed colonies were then inoculated in the inducing liquid media and incubated overnight at 37oC

Control (non-inducing) liquid cultures were set up containing only LB, 100 mg/ml ampicillin and 25 mg/ml chloramphenicol. The inoculation procedure followed the same steps taken with the inducing liquid cultures.

Transmission Electron microscopy Imaging of in vivo experiments:

Liquid cultures of pSB1A3+[AraC-pBAD]+MamOPXT and pEC86 transformants were diluted to an OD600 reading of 0.1 for sample preparation. An extra control sample was further diluted to 0.05 OD600 in order to cover a broader range. 20µl of the liquid cultures was pipetted onto TEM support grids and dried accordingly. Samples were subsequently imaged for identification of electron dense regions/particles within the transformed cells.

AFM Preparation procedure

psb1A3 VS45 cells were grown on Chloramphenicol and Amp combined plated, with 20% w/v Arabinose and 1mM IPTG plate. This plate contained both PVS72 and PVS105 cells as controls, it was then incubated for 5 days at 22oC . Once the cells grew 25µl of 1x PBS were pipetted onto a spot of bacterial cells, the PBS was pipetted up and down 5 times to resuspend the cells. 20µl of each resuspension were transferred to an Eppendorf tube. 10µl of each sample were then pipetted onto a freshly cleaved mica and left to incubate for 40 minutes. The discs were then washed with 1ml of water and dried with nitrogen. These samples were then consequently imaged.





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