Team:Kent/Results

SDS-PAGE images revealed bands in all gels that were not present in the before induction samples and the control. For MamP (Figure 1-2), the band indicated by the arrow equates to the, 26 kDa, soluble MamP protein with his-tag. For the soluble MamT protein with his-tag (Figure 3-4) the band also appears at the predicted size of the protein, 17 kD. Soluble MamX with his-tag also displays a new band at 24 kDa as expected for this protein (Figure 5-6). These SDS results validated that the proteins were successfully expressed within E. coli cells compared to the controls (Figures 7-9)

Fig 1: Reducing 12% SDS-PAGE of soluble mamP including his-tag, PM is protein marker, BI is before induction, AI is after induction, SN is supernatant flow through fraction, and BB is binding buffer flow through fraction. Arrow indicates suspected protein.

Fig 2: Reducing 12% SDS-PAGE of soluble mamP including his-tag, PM is protein marker, purified refers to the protein sample that was used for the absorbance spectrum and the in vitro iron induction. Arrow indicates suspected protein.

Fig 3: Reducing 12% SDS-PAGE of soluble mamT including his-tag, PM is protein marker, BI is before induction, AI is after induction, SN is supernatant flow through fraction, and BB is binding buffer flow through fraction. Arrow indicates suspected protein.

Fig 4: Reducing 12% SDS-PAGE of soluble mamT including his-tag, PM is protein marker, purified refers to the protein sample that was used for the absorbance spectrum and the in vitro iron induction. Arrow indicates suspected protein.

Fig 5: Reducing 12% SDS-PAGE of soluble mamX including his-tag, PM is protein marker, BI is before induction, AI is after induction, SN is supernatant flow through fraction, and BB is binding buffer flow through fraction. Arrow indicates suspected protein.

Fig 6: Reducing 12% SDS-PAGE of soluble mamX including his-tag, PM is protein marker, purified refers to the protein sample that was used for the absorbance spectrum and the in vitro iron induction. Arrow indicates suspected protein.

Fig 7: Reducing 12% SDS-PAGE of control with pet3a, PM is protein marker, BI is before induction, AI is after induction, SN is supernatant flow through fraction, and BB is binding buffer flow through fraction. Arrow indicates suspected protein.

Fig 8: Reducing 12% SDS-PAGE of control with pet3a, PM is protein marker, BI is before induction, AI is after induction, SN is supernatant flow through fraction, and BB is binding buffer flow through fraction.

Fig 9: Reducing 12% SDS-PAGE of control with pet3a, PM is protein marker.

Analysis of the UV-visible absorption spectra for the purified protein samples, revealed absorption maxima at 407 nm for all proteins (figures 10-12). Out of the three proteins MamX displayed a significant peak at this point while all the other proteins displayed 407 nm peaks at a similar magnitude to the control experiment where proteins were purified from cells lacking a Mam expression vector, presumably due to low protein concentration. The presence of a 407 nm peak in the control experiment is probably due to the ccm helper plasmid facilitating the overproduction of native periplasmic c-type cytochromes, which are binding nonspecifically to the resin.

Chemical synthesis of magnetite nanoparticels

Magnetite (Fe₃O₄) nanoparticles were synthesised through a simple co-precipitation of a base to ferric and ferrous salts. This method used a 2:1 ratio of ferrous to ferric ions; the size, shape and composition of the magnetite depends heavily on Fe²⁺/Fe³⁺ ratio, reaction temperature, pH value and ionic strength. ¹ If all conditions are standardised this method is completely reproducible but tends to produce rather polydisperse nanoparticles. To produce monodisperse nanoparticles there must be a quick nucleation followed by a slow controlled growth of crystals, which is absent from this method as a gelatinous precipitate of crystals is formed immediately.1 Despite this methodological shortcoming, the black magnetic particles formed in the precipitate and were imaged by EM.

Figure 13: EM images of Magnetite Nanoparticle.

Figure 13 shows various images taken on EM, they show spherical polydispersed nanoparticles of approximately 10-20nm. This is an average size of nanoparticles however these nanoparticles are not regulated diameter. However, there was considerable variability in diameter, and there are alternative methods where the particle sizes are more uniform.² The second image shows where the individual nanoparticles have aggregated forming what appears to be a cuboctahedral geometric shape; this is one of the most common geometric shapes of magnetite nanoparticles.³ This implies that the cuboctahedral geometry is most stable with {100} and {111} miller planes. This shape of nanoparticle has a large surface area which would make it more reactive, however this is at odds with our observations that these nanoparticles are very stable. These nanoparticles were used in subsequent protein experiments to test whether the Mam proteins is able to alter the structure and shape of these presynthesised particles.

A H Lu, E L. Salabas, F Schüth, Angew. Chem. Int. Ed, 2007, Vol 46, Pages 1222 – 1244
S Sun, H Zeng, D B. Robinson, S Raoux, P M. Rice, S X. Wang, G Li, J. Am. Chem. Soc., 2004, Vol 126, Pages 273–279
D A. Bazylinski, R B. Frankel, Nature Reviews Microbiology, 2004, Vol 2, Pages 217-230

Visualisation of Magnetite nanoparticles

Using the reaction performed under zero oxygen conditions multiple EM images were obtained figures 14-16 (red circles) which are consistent with the modulation of the size and shape of magnetite nanoparticles. However, these samples contained too much protein material and it was therefore difficult to identify magnetite particles. Future experiments with a higher concentration of each protein, using more highly purified protein samples or separating the proteins entirely from the magnetite crystals will improve the validation of the function of the proteins. In addition, experiments would need to be run of solutions containing double enzymes in order to verify the relationship between each of the enzymes.

Fig 10: UV-visible absorption spectrum of soluble his-tagged MamP with a maxima observed of 0.0119 at the 407nm peak

Fig 11: UV-visible absorption spectrum of soluble MamX with his-tag displaying an extremely high peak at 407 nm showing a maxima of 0.538

Figure 12: UV-visible absorption spectrum of soluble his-tagged MamT showing its 407nm peak with an absorbance of 0.0127

Figure 14: Electron Microscopy image of triple protein incubated with synthetic crystals

Figure 15: Electron Microscopy image of MamP incubated with synthetic crystals

Figure 16: Electron Microscopy image of pet3a control incubated with synthetic crystals