Team:Chiba/Assay/uptake

uptake実験方法

1.E.coli strain BL21 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 37℃).
3.We centrifuged samples with 3,750 rpm, at 4℃ 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 37℃).
5.We diluted samples by LB medium and got 500 uL of the diluted cell suspension. Approximately, 2×10^9, 10^9, 5×10^8 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 37℃) to charge iron inside E.coli.
7.We centrifuged the sample with 19,600 rpm, at 37℃ 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.
絵とかあるといいよね！！