Part:BBa_K1679031

ftnA , RNA thermometer and RFP

This device is consist of ftnA which is our magnetic receiver, RNA thermometer which is our thermo-regulator and a RFP in the role of reporter.

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. Control, HFn; 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

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 752
  Illegal NheI site found at 775
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 133
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 1401
  Illegal AgeI site found at 1513
  
• 1000
  COMPATIBLE WITH RFC[1000]