Difference between revisions of "Part:BBa K1679028"

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T5 promoter + RBS + 6x His Tag + ftnA + λ T0 Terminator

The design is composed of a T5 promoter regulated by double lacI repressor-binding sites, a RBS, a 6x His tag, a ftnA coding sequence and a λ T0 terminator. We created it for expression of ftnA in E.coli for a long time.

Ferritin

FtnA is a bacterial ferritin with a protein shell is assembled from 24 identical 19.4 kDa FtnA monomers. Its central cavity is around 7.5 nm in diameter and can be loaded with iron when cells grow under iron-rich conditions[1]. The iron is stored in the form of ferrihydrite iron cores normally that with superparamagnetic properties[2]. The iron contained ferritin can generate heat in response to electromagnetic signal[3]. For the reasons above, we design it as our magnetic receiver which can turn electromagnetic signal into heat.

[1]Smith J L. The physiological role of ferritin-like compounds in bacteria[J]. Critical reviews in microbiology, 2004, 30(3): 173-185. [2] Papaefthymiou G C, Viescas A J, Devlin E, et al. Electronic and magnetic characterization of in vivo produced vs. in vitro reconstituted horse spleen ferritin[C]//MRS Proceedings. Cambridge University Press, 2007, 1056: 1056-HH03-27. [3] Stanley S A, Sauer J, Kane R S, et al. Remote regulation of glucose homeostasis in mice using genetically encoded nanoparticles[J]. Nature medicine, 2015, 21(1): 92-98.

Fig.1. Schema of ferritin

Experiment

We purified the ferritin overexpressed in E.coli through Ni-chelating affinity chromatography and highly concentrated it and then do SDS-PAGE. The position of clearly targeted band(about 19.4kDA) on the gel was consistent with the size of ferritin monomer fused with tag on plasmid, explaining that the ftnA expression is successful.

Figure 2. SDS-PAGE shows the expected protein band.

Gel was stained with (A) potassium ferrocyanide and (B) Coomassie Brilliant Blue R250. lane 1, concentrated mineralized ferritin purified product; lane 2, sediment of bacteria with mineralization; lane 3, concentrated unmineralized ferritin purified product ; lane 4, sediment of bacteria without mineralization.

Figure 3. Native-PAGE analysis of mineralized FtnA

Fig.1. Schema of ferritin

Fig.4.(left) Low‐Temperature Magnetization Curves(5K) (right) Enlarge figure of the left figure, Low‐Field Magnetization Curves

Fig.5.(left) Magnetization Curves at 300K (right) Enlarge figure of the left figure

Fig.6.(left)Decay curves of saturation isothermal remanent magnetization(IRM) acquired in a 2.5 T field after zero‐field cooling (ZFC) and field‐cooling (FC) treatments (right) Normalized IRM acquisition and DC (Direct Current field) demagnetization (DCD), measured at 5 K

For the Fig.6.(left),Vertical axis means normalized remanent magnetization, its unit:emu/g. Horizontal axis stand for temperature. The remanent magnetization decreased rapidly with increasing temperature. This trend is consists with the property of superparamagnetism. Superparamagnetic particles are essentially single-domain particles, means that magnetization of the nanoparticles is a single giant magnetic moment. When its volume is sufficiently small, its magnetization can randomly flip direction under the influence of temperature without external field. This is the state of superparamagnetism. And that means superparamagnetic particles may show superparamagnetism in room temperature, while it is single domain particles in low temperature such as 5K, and can have remance in this state. Our measurement process is different from typical ZFC/FC magnetization curves:

Process of typical ZFC/FC magnetization curves

Process of our decay curves

Fig.6.(left) just shows the warming process, the remance decreased rapidly with increasing temperature. This trend is consists with the property of superparamagnetism. For superparamagnetic particle, its magnetization should be zero in room temperature without external magnetic field. In Fig.6.(left), the remanent magnetization didn’t decrease to zero, which may result from two cases: First, superparamagnetic particles gathered together and generate magnetostatic interactions, this kind of superparamagetic particles can have remance in room temperature. Second, there are some big single domain particles or multi-domain particles in the E. coli. Sequence and Features