Team:Chiba/Assay/store

From 2013.igem.org

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<h2>Storage Assay</h2>
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<h2>Sequestration Assay</h2>
<p>
<p>
<br><b>Experiment:</b>
<br><b>Experiment:</b>
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<br>Experiment: We constructed two kinds of plasmids with which RBS scores are different as shown in Fig. 2. E. coli strain BL21 and <a href="http://www.nebj.jp/products/detail/113">SHuffle®</a> 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”.     
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<br>Experiment: We constructed two kinds of plasmids with which RBS scores are different as shown in Fig. 2. <i>E. coli</i> strain BL21 and <a href="http://www.nebj.jp/products/detail/113">SHuffle®</a> 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 (Fe(III)) or iron ascorbate (Fe(II)), and checked final cell density and colony forming efficiency. As a control, we conducted the same experiment with “sfgfp generator”.     
<br>
<br>
<br><b>Detailed procedure</b>
<br><b>Detailed procedure</b>
<br>
<br>
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1.    E. coli strain BL21 and <a href="http://www.nebj.jp/products/detail/113">SHuffle®</a> were transformed with the plasmids shown in Fig. 1.
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1.    <i>E. coli</i> strain BL21 and <a href="http://www.nebj.jp/products/detail/113">SHuffle®</a> were transformed with the plasmids shown in Fig. 2.
<br>
<br>
2.    All transformants were inoculated into small (2 mL) culture and was shaken for 12h (at 37ºC).
2.    All transformants were inoculated into small (2 mL) culture and was shaken for 12h (at 37ºC).
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<h2>Experiment method of evaluation of magnetism</h2>
<h2>Experiment method of evaluation of magnetism</h2>
<p><br><b>Experiment:</b><br>
<p><br><b>Experiment:</b><br>
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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<br>
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Evaluation of whether <i>E. coli</i> have magnetism by ferritin with iron.<br>
<br>
<br>
<b>Detailed procedure</b><br>
<b>Detailed procedure</b><br>
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使用した株(プラスミド)…BL21 (strong, middle)<br>
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Plasmid:pBAD/araC-ferritin-strong(<a href="http://parts.igem.org/Part:BBa_K1057009">BBa_K1057009</a>), pBAD/araC-ferritin-mid(<a href="http://parts.igem.org/Part:BBa_K1057002">BBA_K1057002</a>)
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※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<br>
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strongはmiddleに比べferritinを多く発現する<br>
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<br>
<br>
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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。<br>
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<br>1. Introduction of pBAD/araC-ferritin-strong(<a href="http://parts.igem.org/Part:BBa_K1057009">BBa_K1057009</a>) or pBAD/araC-ferritin-mid(<a href="http://parts.igem.org/Part:BBa_K1057002">BBA_K1057002</a>) into BL21 strain. Preparation of medium(arabinose:final conc. 0.2%, ferric citrate:final conc.10mM). Applying cultured medium to be OD 10 and final volume 2 mL.
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2、 37℃で一時間、培養した。<br>
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<br>2. Culturing at 37ºC for an hour.
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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。<br>
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<br>3. After 1 hour, the solution diluted(10 fold) by LB agar medium to be final volume 10mL.
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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。<br>
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<br>4. The plate in 4ml 0.01%triton X-100 solution all over the bottom was added 10ml solution over it.<br>
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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。<br>
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<br>5. 300 mT Doughnut type magnets ware put under the plate, we take pictures at intervals of 5 minutes till 25 minutes later.<br>
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Latest revision as of 03:54, 28 September 2013

iGEM-2013 Chiba

iGEM-2013 Chiba

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 (Fe(III)) or iron ascorbate (Fe(II)), 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 10 and final volume 2 mL.
2. Culturing at 37ºC for an hour.
3. After 1 hour, the solution diluted(10 fold) by LB agar medium to be final volume 10mL.
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.