Team:Chiba/Assay/uptake

Uptake Assay

Expreriment:

    BL21 strains introduced plasmids shown in Fig.1 and Fig. 2 were cultured in the presence of arabinose and aTc. After induction of ferritin and fur and fieF knockdown, these strain were cultured in the presence of ferric citrate. We measure concentration of iron remaining in medium(Fig. 3).

Experiment:

    E. coli stains BL21 and SHuffle® were transformed by Plasmid shown in Fig. 1 and Fig. 2. Then we cultured all transformants with arabinose and aTc.  Arabinose and aTc were added to express ferritin or to knock down fur and fieF.  After that we cultured it in the presence of ferric citrate, and measured the density of iron that weren't taken in to E. coli and remained in the culture by measuring Absorbance (Abs 756) with color reagent(Fig. 3).

Detailed procedure

1.E. coli strains BL21 and SHuffle® were transformed with the plasmids shown in Fig. 1 and Fig.2.
2.All transformants were inoculated into culture(100 mL TB medium) and was shaken for 12h (at 37ºC).
3.We centrifuged samples with 3,750 rpm, at 4ºC for 25 minutes and resuspended with 5 mL LB medium.
4.We added L-arabinose into each sample (final conc. 0.2%). Then cultured for another 9 hours (at 37ºC).
5.We diluted samples by LB medium and got 500 uL of the diluted cell suspension. Approximately, 2×10⁹, 10⁹, 5×10⁸ cells were contained in it.
6.We added 5 uL from 10 mM iron citrate(III) and 5uL from 10 mM iron ascorbate(II) to the sample, and we shake the sample for 1h (at 37ºC) to charge iron inside E.coli.
7.We centrifuged the sample with 19,600 rpm, at 37ºC for 1 minutes and got supernatant(60 mL).
8.We added color reagent, Tris buffer solution, Reducing Agent to the sample and measured absorbance (Abs756) to determine the density of iron.