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.

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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

Design of Mag-Nano-Tite Biobrick device

It was decided by the team that MamO; MamP; MamX; MamT should all feature on this ultimate device as each gene codes proteins which we believe to be crucial for magnetite nanoparticle formation. MamO is involved in nucleation of magnetite biomineralization therefore serves a crucial role. MamP/X/T have been proposed to be cytochrome proteins which are thought to work together in the biomineralization process of magnetite within magnetotactic bacteria. As such, this ultimate device features all four said Mam genes in a single plasmid which will enabled us to execute in vivo experiments in E. coli pSB1A3+[AraC-pBAD]+MamOPXT device for In Vivo experiments was constructed by firstly ligating AraC-pBAD promoter (Imperial College 2014: BBa_K1321333) into pSB1A3 vector. The MamO gene was then subsequently Gibson assembled into pSB1A3+[AraC-pBAD]. The final construct was achieved by ligating MamP/X/T genes from corresponding BioBricks into pSB1A3+[AraC-pBAD]+MamO. All ligations and assemblies utilised Methods section.

Figure 17. Plasmid map of ultimatefrom BBa_K1321333) to enable control over expression of Mam genes. Ribosome Binding Sites are upstream of each coding region to ensure complete expression of desired genes. Ampicillin resistance gene and its corresponding promoter are also encoded within the construct.

Figure 18. Diagnostic restriction digest for verification of pSB1A3+[AraC-pBAD]+mamOPXT. Two undigested plasmid samples were run on a 1% agarose gel along with two single digests using EcoRI and PstI respectively; single digest fragments show a base pair length consistent with the size of the entire plasmid (7492 bp) . A single double restriction digest (EcoRI and PstI) of the plasmid is also shown; the corresponding fragments were shown to have the expected base pair lengths of 5378 bp and 2114 bp. The results obtained from this diagnostic digest suggest that the desired plasmid has indeed been successfully purified and mamP/X/T genes have been successfully ligated to form the final construct.

Functional validation of BioBrick device

The formation of magnetosome compartments and thus the biomineralization of magnetite nanoparticles in E. coli has not been previously achieved. The in vivo construct pSB1A3+[AraC-pBAD]+MamOPXT in conjunction with pEC86 (encoding cytochrome C maturation genes) was co-transformed into BL21 DE3 E. coli cells and then incubated in an inducing liquid growth medium containing ferric citrate to enable iron uptake into the cells. Following overnight incubation the transformants were imaged using Transmission Electron Microscopy.

This experiment informed whether MamO/P/X/T genes would enable magnetite biomineralization in E. coli without reconstruction the entire magnetosome compartment. The Mam genes present within the construct are the wild type; their membrane anchors have not been cleaved unlike those used for our parallel in vitro investigations. Magnetosome magnetite (Fe₃O₄) crystals have been described to have a typical length of 35-120 nm in diameter [1].

Figure19: Electron micrograph of E. coli cells containing pEC86 and BioBrick device encoding MamOPXT. Electron dense regions can be observed within the cells, in particular small spherical shapes in close proximity to the cell membranes (A,B,C) which appear to resemble magnetite nanoparticles. Diameters of these shapes are (A)120.4 nm; (B) 88.5 nm; (C) 165.8 nm as measured from the micrographs using image analysis software ImageJ.

Results show that cells have successfully taken up Iron in the form of ferric citrate which was present in the inducing growth medium; staining was not carried out during sample preparation on EM grids . A and B electron dense spherical shapes have diameters which lay within the expected range of magnetosome magnetite nanoparticles. However, the diameter of C is considerably larger.

Biogenesis of magnetosome compartments in native magnetotactic bacteria involves the invagination of the cytoplasmic membrane [2]. The BioBrick device contains wild type versions of the Mam genes, therefore it was hypothesized that successfully expressed proteins would be targeted to the cytoplasmic membrane of E. coli. The presence of said electron dense spherical regions somewhat supports this notion as they are in close proximity to the cytoplasmic membrane; It could be proposed that if magnetite biomineralization has occurred within E. coli, the nanoparticles would be situated close to the site where Mam proteins are present: the cytoplasmic membrane.

It is worth highlighting that in Figure 17, it can be observed that only single electron dense spherical shapes are found close to the membranes in the cells that do contain them. Hypothetical reasoning would postulate that upon successful expression of MamO/P/X/T proteins and their correct targeting to the cytoplasmic membrane, multiple sites on the cytoplasmic membrane would be present and available for biomineralization. Thus it was expected that multiple sites would yield multiple electron dense sites like A,B,C. As such the reasoning for single sites observed in our data is unclear.

Control samples which were grown in non-inducing liquid and in the absence of ferric citrate did not appear when imaged with TEM, this could be attributed to the lack of staining upon sample preparation. Thus due to the lack of iron, there was insufficient electron dense matter to render the control cells visible.

The findings from our in vivo study suggests a potential leap forward with regards to the biomineralization of magnetite in a foreign organism. The observations presented are however not fully conclusive, thus further characterisation of the electron dense matter is indeed required for a clearer insight. The investigations carried out studied the effect of only four Mam genes originating from magnetotactic bacterium and their behaviour in E. coli; future endeavours could build on these findings by adding further Mam genes to the BioBrick device.

References

D. A. Bazylinski and R. B. Frankel, “Magnetosome formation in prokaryotes,” Nat. Rev. Microbiol., vol. 2, no. 3, pp. 217–230, 2004.
I. Kolinko, A. Lohße, S. Borg, O. Raschdorf, C. Jogler, Q. Tu, M. Pósfai, E. Tompa, J. M. Plitzko, A. Brachmann, G. Wanner, R. Müller, Y. Zhang, and D. Schüler, “Biosynthesis of magnetic nanostructures in a foreign organism by transfer of bacterial magnetosome gene clusters.,” Nat. Nanotechnol., vol. 9, no. 3, pp. 193–7, 2014.