Difference between revisions of "Part:BBa K1632008:Experience"
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=====Assay protocol===== | =====Assay protocol===== | ||
| − | 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> | ||
| Line 31: | Line 31: | ||
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 | + | <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> | ||
<span style="margin-left: 20px;">④ 3 mL of LB containing Amp, Kan and 30 microL of 500 mM arabinose (final concentration of arabinose is 5 mM)<br> | <span style="margin-left: 20px;">④ 3 mL of LB containing Amp, Kan and 30 microL of 500 mM arabinose (final concentration of arabinose is 5 mM)<br> | ||
| Line 66: | Line 66: | ||
=====Assay protocol===== | =====Assay protocol===== | ||
| − | 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 1.0 %) 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 1.0 %).<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> | ||
| Line 77: | Line 77: | ||
10. Suspend the pellet in 1mL of LB containing Amp and Kan.<br> | 10. Suspend the pellet in 1mL of LB containing Amp and Kan.<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 and 30 microL | + | <span style="margin-left: 20px;">① 3 mL of LB containing Amp, Kan, glucose (final concentration is 1.0 %) and 30 microL sterile water<br> |
<span style="margin-left: 20px;">② 3 mL of LB containing Amp, Kan and 30 microL of 500 microM arabinose (final concentration of arabinose is 5 microM)<br> | <span style="margin-left: 20px;">② 3 mL of LB containing Amp, Kan and 30 microL of 500 microM arabinose (final concentration of arabinose is 5 microM)<br> | ||
<span style="margin-left: 20px;">③ 3 mL of LB containing Amp, Kan and 30 microL of 1 mM arabinose (final concentration of arabinose is 10 microM)<br> | <span style="margin-left: 20px;">③ 3 mL of LB containing Amp, Kan and 30 microL of 1 mM arabinose (final concentration of arabinose is 10 microM)<br> | ||
Revision as of 02:44, 18 September 2015
Contents
Materials and Methods
Invertion assay with FimB
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)_gfp (pSB3K3)
(2) PBAD/araC_fimB(wild-type) (pSB6A1) + fim switch[default OFF](wild-type)_gfp (pSB3K3)
(3) pSB6A1 + fim switch[default ON](wild-type)_gfp (pSB3K3) …positive control 1
(4) pSB6A1 + fim switch[default OFF](wild-type)_gfp (pSB3K3) …negative control 1
(5) PBAD/araC_fimB(wild-type) (pSB6A1) + J23119_gfp(pSB3K3) …positive control 2
(6) PBAD/araC_fimB(wild-type) (pSB6A1) + rbs_gfp(pSB3K3) …negative control 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.)
Results
Discussion
Invertion assay with FimE
Construction
All the samples were DH5α strain with antibiotic resistance to ampicillin and kanamycin.
(1) PBAD/araC_fimE(wild-type) (pSB6A1) + fim switch[default ON](wild-type)_gfp (pSB3K3)
(2) PBAD/araC_fimE(wild-type) (pSB6A1) + fim switch[default OFF](wild-type)_gfp (pSB3K3)
(3) pSB6A1 + fim switch[default ON](wild-type)_gfp (pSB3K3) …positive control 1
(4) pSB6A1 + fim switch[default OFF](wild-type)_gfp (pSB3K3) …negative control 1
(5) PBAD/araC_fimE(wild-type) (pSB6A1) + J23119_gfp (pSB3K3) …positive control 2
(6) PBAD/araC_fimE(wild-type) (pSB6A1) + rbs_gfp (pSB3K3) …negative control 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 1.0 %) at 37 ℃ for 12h.
2. Make a 1:100 dilution in 3 mL of fresh LB containing Amp, Kan and glucose (final concentration is 1.0 %).
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. Take the samples, and centrifuge at 5000x g, 1 min, 25 ℃
9. Remove the supernatant.
10. Suspend the pellet in 1mL of LB containing Amp and Kan.
11. Add 30 microL of suspension in the following medium.
① 3 mL of LB containing Amp, Kan, glucose (final concentration is 1.0 %) and 30 microL sterile water
② 3 mL of LB containing Amp, Kan and 30 microL of 500 microM arabinose (final concentration of arabinose is 5 microM)
③ 3 mL of LB containing Amp, Kan and 30 microL of 1 mM arabinose (final concentration of arabinose is 10 microM)
④ 3 mL of LB containing Amp, Kan and 30 microL of 2 mM arabinose (final concentration of arabinose is 20 microM)
※ As for C and D, the suspension were added only in medium ① and ④.
12. Incubate the samples at 37 ℃ for 6 hours, shaking at 180 rpm. (Measure the 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.)
Results
Discussion
More information
For more information, see [http://2015.igem.org/Team:Tokyo_Tech/Project our work in Tokyo_Tech 2015 wiki].
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