Difference between revisions of "Part:BBa K1985009"

Latest revision as of 21:23, 25 October 2016

AraC pBAD mamOPXT

Sequence and Features

This is a composite part of part BBa_K1321333 (Imperial iGem, 2014), BBa_K1985006, BBa_K1985000, BBa_K1985002, BBa_K1985001 a . It was used in pSB1A3.

Usage and Biology

Usage: The mamO gene was used to initiate the formation of magnetite by "nucleating" the crystal particles, allowing further development. It was used in combination with the proposed electron transport complex of mamO, P and X in vivo to form magnetite in vivo.

BBa_K1321333 is a regulatory part is made up of the Arabinose-Inducible promoter, pBAD, and its transcriptional inhibitor/activator, AraC. It was used to give increased control over the expression of part BBa_K1985007.

The part was used in pSB1A3 rather than pSB1C3 as it was cotransformed with pec86, a cytochrome maturation factor, which was in a chloramphenicol resistant plasmid. Pec86 is a pACYC184 derivative containing the E.coli genes, ccmABCDEFGH, of the aeg operon, expressed from the tet promoter of the plasmid. The genes are essential for maturation of cytochromes c, i.e. for the covalent attachment of the heme to the protein.

Biology: For more information on the biology of mamO,P and X see parts BBa_K1985006, BBa_K1985000,BBa_K1985002 and BBa_K1985001 and for more information on the AraC pBAD promoter please see BBa_K1321333.

Validation

The part was first validated with a diagnostic restriction digest using EcoRI and PstI and agarose gel electrophoresis. The expected band sizes from the digest were: 5478 base pairs for the plasmid backbone and 2114 for the insert. A 1kB plus DNA marker was used to verify the sizes of the bands and it was confirmed that the correct plasmid had been produced.

Figure 1. Agarose gel of the restriction digest of BBa_K1985009 in pSB1A3, with EcoRI and PstI.

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 (link to parts page here?) 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 seeked to investigate whether MamO/P/X/T genes would enable magnetite biomineralization in E. coli without attempting to reconstruct 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.

Figure 2. 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. 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 2 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

Arslan, Engin et al. "Overproduction Of Thebradyrhizobium Japonicum C-Type Cytochrome Subunits Of Thecbb3oxidase Inescherichia Coli". Biochemical and Biophysical Research Communications 251.3 (1998): 744-747. Web. 17 Oct. 2016.

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.