Team:Chiba/Assay/store

Storage Assay

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® 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 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:
Ferritinにより鉄を取り込んだ大腸菌が磁性を持つかを確認する

Detailed procedure
使用した株(プラスミド)…BL21 (strong, middle)
※strongおよびmiddleはヒトフェリチンのO.E.を誘導するプラスミド
strongはmiddleに比べferritinを多く発現する

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、 シャーレに0.01%triton X-100溶液4mlを一面に広がるよう入れ、その上に溶液を10ml加えた。
5、 シャーレの下に300mTのドーナツ型磁石を置き、5分おきに、25分まで写真を撮影した。