Team:Chiba/Assay/store

Storage実験方法

Experiment:
We constructed two plasmids as shown in Fig. 1. Human ferritin genes (fth1 and ftl) are placed on the high-copy plasmid under the control of BAD promoter. By adding arabinose into the media, Human ferritin proteins are induced.E. coli strain BL21 and SHuffle® (要link) were transformed with above plasmids. The resultant “ferritin generators” were cultured in the presence of iron citrate (Fe3+) or iron ascorbate (Fe2+), and checked final cell density and colony forming efficiency. As a control, we conducted the same experiment with “sfgfp generator”.

Detailed procedure
1. E. coli strain BL21 and SHuffle® were transformed with the plasmids shown in Fig. 1.
2. All transformants were inoculated into small (2 mL) culture and was shaken for 12h (at 37°C).
3. Inoculated into flesh media (2 mL), shaken for 3 hours.
4. We added L-arabinose into each sample (final conc. 0.2%). Then cultured for another 9 hours (at 37°C).
5. After 9 hours, we measured cell density (OD600) of every sample, and put 10⁷ cell of E.coli to fresh medium containing various concentration of iron citrate or iron ascorbate.
6. Then we shaking cultured E.coli for 12 hours.
7. We got 1 mL aliquot from each sample and centrifuged with 7,000 rpm, at 25°C for 2 minutes.
8. We wasted supernatant and resuspended with 1 mL saline solution (0.9% NaClaq), and we measured cell density (OD600).
9. 5 μl of the diluted cell suspension was spotted on the LB agar medium and incubated for 12 hours. Approximately 10⁷, 10⁶, 10⁵, 10⁴, 10³, and 10² cells were contained in the spots.