Team:Chiba/Assay/store

Sequestration Assay

Experiment:
Experiment: We constructed two kinds of plasmids with which RBS scores are different as shown in Fig. 2. E. coli strain BL21 and SHuffle® were transformed with above plasmids. Human ferritin genes (fth1 and ftl) are placed on the high-copy plasmid under the control of BAD promoter. To express Human ferritin proteins, arabinose was added into media(final conc. 0.2%). 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. 2.
2. All transformants were inoculated into small (2 mL) culture and was shaken for 12h (at 37ºC).
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 37ºC).
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 25ºC 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.

Experiment method of evaluation of magnetism

Experiment:
Evaluation of whether E. coli have magnetism by ferritin with iron.

Detailed procedure
Plasmid:pBAD/araC-ferritin-strong(BBa_K1057009), pBAD/araC-ferritin-mid(BBA_K1057002)

1. Introduction of pBAD/araC-ferritin-strong(BBa_K1057009) or pBAD/araC-ferritin-mid(BBA_K1057002) into BL21 strain. Preparation of medium(arabinose:final conc. 0.2%, ferric citrate:final conc.10mM). Applying cultured medium to be OD 10and final volume 2 mL.
2.Culturing at 37ºC for an hour. 1, 上に示す2つのプラスミドを導入した大腸菌を、それぞれ試験管に、OD10、終濃度0.2% arabinose、かつ終濃度10mM ferric citrateになるよう、2mlの溶液を調整した。
2, 37℃で一時間、培養した。
3, 一時間後、溶液を、LB培地により10倍に希釈し、20mlの溶液を調整した。
4, The plate in 4ml 0.01%triton X-100 solution all over the bottom was added 10ml solution over it.
5, 300 mT Doughnut type magnets ware put under the plate, we take pictures at intervals of 5 minutes till 25 minutes later.