Difference between revisions of "Part:BBa K1679029"

Latest revision as of 02:13, 22 October 2019

ftnA

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

(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.

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

Characterization by UM_MACAU 2019

Overview of the characterization

Our team has characterized this part by giving protein expression levels in different strains under the promoter aRABAD. For our characterization, we first did western blotting of different strains of bacteria which have been transformed with this FtnA gene.

Protein expression level in different strains

Figure 1: Western blotting protein expression band signal results from different strains of E.coli

Figure 2: The bar chart represents the different strains and their relative protein expression levels

Different E.coli strain types can influence FtnA protein expression levels. To investigate which E.coli strain is the best strain for Ftna protein expression, we ran western blot with the extracted protein samples from 6 different kinds of bacteria strains (Figure 1). We used rpoB (DNA-directed RNA 9polymerase subunit beta) as an internal control for our western blot analysis. Band signal’s intensity from the membrane were analyzed using Image Studio. After being normalized against the internal control (rpoB), we can see that different protein expression levels could be observed in different bacteria strains and all BL-21 derivatives strains which includes Rosetta, Star, pLys and DE3 showed a much higher level of protein expression compared to wild-type BL-21 strain (WT) as well as Stbl3 and DH5α strains.

Based on the results above, by using BL-21(DE3) we then proceeded to investigate the relative protein expression of this part in different arabinose concentrations.

Protein expression level in different Arabinose concentrations

Figure 3: Western blot result containing our protein bands which we later analyzed

Figure 4: Bar chart to represent the relative expression level and arabinose concentration

To investigate whether concentrations of arabinose will affect FtnA protein expression level, we ran western blot with bacteria lysis sample from BL-21 (DE3) bacteria incubated with 6 different concentrations of arabinose: 0-13300uM (Figure 3). Band signal’s intensity from the membrane were analyzed using Image Studio. Normalized with internal control rpoB (which the protein size is 150kDa), we can see that FtnA protein expression level rises when the arabinose concentrations are increased (Figure 4). Therefore, it indicates that the concentrations of arabinose can affect the FtnA protein expression level in the same bacteria strain. We have used both of these results for the characterization of the part number BBa_K1679029 by OUC China 2015.

Functional magnetization tests

Figure 4: Bar chart to represent the relative expression level and arabinose concentration

After testing the protein expression level, we also want to know whether those expressed proteins are functional. A functional FtnA protein would increase the magnetization efficiency; we expect that after 1 hr, there would be significant migration of the bacteria toward the magnet. Therefore, we did a magnetization test: The pBAD24-FtnA construct was transformed respectively into the six different strains which are BL21 (Rosetta), BL-21 (Star), Stbl3, BL-21 (pLys), DH5 alpha and BL-21(DE3). Our negative control is the wild type BL-21 DE3 which is without the construct. Overnight cultures of 5 mL were made and after overnight culture they were all induced by 0.02% arabinose and cultured overnight. The next day, 1ml of 100mM ferric ammonium citrate for 24 hours was added. After incubation, we centrifuged the bacteria culture, resuspended it with autoclaved water and poured 2ml into the small dish. A round shape magnet was set under each small dish during the 1hour magnetization. In figure 4, we can see that the strains, which includes BL-21 Rosetta, BL-21 Star, BL-21 pLys and BL-21 DE3 showed a clear bacteria migration towards the shape of the magnet after 1 hour. Meanwhile, no significant change can be observed in the wild type BL-21 strain (DE3) as well as Stbl3 and DH5α strains. This functional test results shows consistent results with our protein expression test result in Figure 4, and it also can demonstrate that our FtnA overexpression system can work in these strains that we would recommend using in the future.

These results would be a valuable characterization contribution to the said part for future teams who wishes to use the part and express the protein under the araBAD promoter.

Sequence and Features

Functional Parameters