Difference between revisions of "Part:BBa K1679028"

Revision as of 03:35, 19 September 2015

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

(a) Low‐Temperature Magnetization Curves(5K) (b) Enlarge figure of the left figure, Low‐Field Magnetization Curves

(a) Magnetization Curves at 300K (b) Enlarge figure of the left figure

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 Normalized IRM acquisition and DC (Direct Current field) demagnetization (DCD), measured at 5 K

The picture show the magnetism properties of iron core of Ftna Sequence and Features