Difference between revisions of "Part:BBa K1632010:Design"
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<b>2. Assay protocol</b><br> | <b>2. Assay protocol</b><br> | ||
− | 1. Prepare overnight cultures for | + | 1. Prepare overnight cultures for each sample in 3 mL of LB medium containing ampicillin (50 microg / mL), kanamycin (30 microg / mL) and glucose (final concentration is 0.5 %) at 37 ℃ for 12h.<br> |
− | 2. Make a 1:100 dilution in 3 mL of fresh LB containing Amp, Kan and glucose (final concentration | + | 2. Make a 1:100 dilution in 3 mL of fresh LB containing Amp, Kan and glucose (final concentration is 0.5 %).<br> |
− | 3. | + | 3. Incubate the cells at 37 ℃, shaking at 180 rpm until the observed OD590 reaches 0.4 (Fresh Culture).<br> |
− | 4. After incubation, take 1 mL of the samples, and centrifuge at 5000x g, 1 min, 25 ℃ <br> | + | 4. After the incubation, take 1 mL of the samples, and centrifuge at 5000x g, 1 min, 25 ℃ <br> |
5. Remove the supernatant.<br> | 5. Remove the supernatant.<br> | ||
6. Add 1 mL of LB containing Amp and Kan, and centrifuge at 5000x g, 1 min, 25 ℃ <br> | 6. Add 1 mL of LB containing Amp and Kan, and centrifuge at 5000x g, 1 min, 25 ℃ <br> | ||
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10. Add 1 mL of LB containing Amp and Kan, and suspend.<br> | 10. Add 1 mL of LB containing Amp and Kan, and suspend.<br> | ||
11. Add 30 microL of suspension in the following medium.<br> | 11. Add 30 microL of suspension in the following medium.<br> | ||
− | <span style="margin-left: 20px;">① 3 mL of LB containing Amp, Kan, glucose (final concentration | + | <span style="margin-left: 20px;">① 3 mL of LB containing Amp, Kan, glucose (final concentration is 0.5 %) and 30 microL of sterile water<br> |
<span style="margin-left: 20px;">② 3 mL of LB containing Amp, Kan and 30 microL of 2 mM arabinose (final concentration of arabinose is 20 microM)<br> | <span style="margin-left: 20px;">② 3 mL of LB containing Amp, Kan and 30 microL of 2 mM arabinose (final concentration of arabinose is 20 microM)<br> | ||
<span style="margin-left: 20px;">③ 3 mL of LB containing Amp, Kan and 30 microL of 20 mM arabinose (final concentration of arabinose is 200 microM)<br> | <span style="margin-left: 20px;">③ 3 mL of LB containing Amp, Kan and 30 microL of 20 mM arabinose (final concentration of arabinose is 200 microM)<br> |
Revision as of 01:23, 18 September 2015
fimB (wild-type)
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
Design Notes
sequence confirmed
Materials and Methods
1. Construction
All the samples were DH5α strain with antibiotic resistance to ampicillin and kanamycin.
(1) PBAD/araC_fimB(wild-type) (pSB6A1) + fim switch[default ON](wild-type)_rbs_gfp (pSB3K3)
(2) PBAD/araC_fimB(wild-type) (pSB6A1) + fim switch[default OFF](wild-type)_rbs_gfp (pSB3K3)
(3) pSB6A1 + fim switch[default ON](wild-type)_rbs_gfp (pSB3K3) …positive control 1
(4) pSB6A1 + fim switch[default OFF](wild-type)_rbs_gfp (pSB3K3) …negative control 1
(5) PBAD/araC_fimB(wild-type) (pSB6A1) + J23119_rbs_gfp(pSB3K3) …positive control 2
(6) PBAD/araC_fimB(wild-type) (pSB6A1) + rbs_gfp(pSB3K3) …negative control 2
2. Assay protocol
1. Prepare overnight cultures for each sample in 3 mL of LB medium containing ampicillin (50 microg / mL), kanamycin (30 microg / mL) and glucose (final concentration is 0.5 %) at 37 ℃ for 12h.
2. Make a 1:100 dilution in 3 mL of fresh LB containing Amp, Kan and glucose (final concentration is 0.5 %).
3. Incubate the cells at 37 ℃, shaking at 180 rpm until the observed OD590 reaches 0.4 (Fresh Culture).
4. After the incubation, take 1 mL of the samples, and centrifuge at 5000x g, 1 min, 25 ℃
5. Remove the supernatant.
6. Add 1 mL of LB containing Amp and Kan, and centrifuge at 5000x g, 1 min, 25 ℃
7. Remove the supernatant.
8. Suspend the pellet in 1 mL of LB containing Amp and Kan, and centrifuge at 5000x g, 1 min, 25 ℃
9. Remove the supernatant.
10. Add 1 mL of LB containing Amp and Kan, and suspend.
11. Add 30 microL of suspension in the following medium.
① 3 mL of LB containing Amp, Kan, glucose (final concentration is 0.5 %) and 30 microL of sterile water
② 3 mL of LB containing Amp, Kan and 30 microL of 2 mM arabinose (final concentration of arabinose is 20 microM)
③ 3 mL of LB containing Amp, Kan and 30 microL of 20 mM arabinose (final concentration of arabinose is 200 microM)
④ 3 mL of LB containing Amp, Kan and 30 microL of 500 mM arabinose (final concentration of arabinose is 5 mM)
※ As for C and D, the suspension were added only in medium ① and ④.
12. Incubate the samples at 37 ℃, shaking at 180 rpm for 6.5 hours. (Measure OD590 of all the samples every hour.)
13. After the incubation, take the samples, and centrifuge at 9000x g, 1min, 4℃.
14. Remove the supernatant.
15. Add 1 mL of filtered PBS (phosphate-buffered saline) and suspend. (The ideal of OD is 0.3)
16. Dispense all of each suspension into a disposable tube through a cell strainer.
17. Use flow cytometer to measure the fluorescence of GFP. (We used BD FACSCaliburTM Flow Cytometer of Becton, Dickenson and Company.)
Source
PCR from MG1655
References
Timothy S. Ham et al. (2006) A Tightly Regulated Inducible Expression System Utilizing the fim Inversion Recombination Switch. Biotechnol Bioeng 94(1):1-4
Hung M. et al. (2014) Modulating the frequency and bias of stochastic switching to control phenotypic variation. Nat Commun 5:4574. doi:10.1038/ncomms5574