Difference between revisions of "Part:BBa K1632002:Experience"
(→Invertion assay with FimB) |
(→Invertion assay with FimE) |
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====Invertion assay with FimE==== | ====Invertion assay with FimE==== | ||
| − | =====Construction===== | + | =====1. Construction===== |
All the samples were DH5alpha strain with antibiotic resistance to ampicillin and kanamycin.<br> | All the samples were DH5alpha strain with antibiotic resistance to ampicillin and kanamycin.<br> | ||
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(6) PBAD/''araC''_fimE(wild-type) (pSB6A1) + rbs_gfp (pSB3K3) …negative control 2 <br> | (6) PBAD/''araC''_fimE(wild-type) (pSB6A1) + rbs_gfp (pSB3K3) …negative control 2 <br> | ||
| − | =====Assay protocol===== | + | =====2. 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> | ||
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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 | + | <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 mM arabinose (final concentration of arabinose is 5 mM)<br> | |
| − | + | ||
| − | <span style="margin-left: 20px;"> | + | |
12. Incubate the samples at 37 ℃ for 6 hours, shaking at 180 rpm. (Measure the OD590 of all the samples every hour.)<br> | 12. Incubate the samples at 37 ℃ for 6 hours, shaking at 180 rpm. (Measure the OD590 of all the samples every hour.)<br> | ||
13. After the incubation, take the samples, and centrifuge at 9000x g, 1min, 4℃.<br> | 13. After the incubation, take the samples, and centrifuge at 9000x g, 1min, 4℃.<br> | ||
| Line 95: | Line 93: | ||
=====Discussion===== | =====Discussion===== | ||
We tried to confirm that'' fim'' switch(Tokyo_Tech/J23119) is unidirectionally inverted in the presence of FimE (wild-type) by using GFP as a reporter, under 2 different concentrations of arabinose. In the medium with 0 M arabinose, we supplemented the medium with 0.5 % glucose in order to repress the leakage in the PBAD/araC promoter. Fig.2 shows the histograms of the samples measured by the flow cytometer. In the results of the reporter cell (1), when the Induction of FimE(wild-type) expression increases, the fluorescence intensity decreases. From this fact, we confirmed that the ''fim'' switch (Tokyo_Tech/J23119) is inverted in the ON-to-OFF direction by FimE (wild-type). From the result of reporter cell (2), when the expression amount of FimB(wild-type) increases, the expression amount of GFP in the reporter cell (2) increases. From this fact, we confirmed that the fim switch(Tokyo_Tech/J23119) is inverted from OFF-to-ON direction by FimE(wild-type) that does not invert the fim switch(wild-type) in the OFF-to-ON direction. From this result, the inversion of fim switch (Tokyo_Tech/J23119) by FimE was not confirmed correctly. In this assay, the FimE protein inverts fim switch in the ON-to-OFF and the OFF-to-ON direction. In other words, the FimE protein works as the FimB protein. | We tried to confirm that'' fim'' switch(Tokyo_Tech/J23119) is unidirectionally inverted in the presence of FimE (wild-type) by using GFP as a reporter, under 2 different concentrations of arabinose. In the medium with 0 M arabinose, we supplemented the medium with 0.5 % glucose in order to repress the leakage in the PBAD/araC promoter. Fig.2 shows the histograms of the samples measured by the flow cytometer. In the results of the reporter cell (1), when the Induction of FimE(wild-type) expression increases, the fluorescence intensity decreases. From this fact, we confirmed that the ''fim'' switch (Tokyo_Tech/J23119) is inverted in the ON-to-OFF direction by FimE (wild-type). From the result of reporter cell (2), when the expression amount of FimB(wild-type) increases, the expression amount of GFP in the reporter cell (2) increases. From this fact, we confirmed that the fim switch(Tokyo_Tech/J23119) is inverted from OFF-to-ON direction by FimE(wild-type) that does not invert the fim switch(wild-type) in the OFF-to-ON direction. From this result, the inversion of fim switch (Tokyo_Tech/J23119) by FimE was not confirmed correctly. In this assay, the FimE protein inverts fim switch in the ON-to-OFF and the OFF-to-ON direction. In other words, the FimE protein works as the FimB protein. | ||
| + | |||
===More information=== | ===More information=== | ||
Revision as of 16:46, 18 September 2015
Materials and Methods
Invertion assay with FimB
1. Construction
All the samples were DH5alpha strain with antibiotic resistance to ampicillin and kanamycin.
(1) PBAD/araC_fimB(wild-type) (pSB6A1) + fim switch[default ON](Tokyo_Tech/J23119)_gfp (pSB3K3)
(2) PBAD/araC_fimB(wild-type) (pSB6A1) + fim switch[default OFF](Tokyo_Tech/J23119)_gfp (pSB3K3)
(3) pSB6A1 + fim switch[default ON](Tokyo_Tech/J23119)_gfp (pSB3K3) …positive control 1
(4) pSB6A1 + fim switch[default OFF](Tokyo_Tech/J23119)_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
2. Assay protocol
1. Prepare overnight cultures for the 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 incubation, take 1 mL of the samples, and centrifuge at 5000x g, 1 min, 25 ℃
5. Remove the supernatant.
6. Suspend the pellet in 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. Suspend the pellet in 1 mL 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 of mass of glucose 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)
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
We tried to confirm that fim switch(Tokyo_Tech/J23119) is bidirectically inverted in the presence of FimB (wild-type) by using GFP as a reporter, under 4 different concentrations of arabinose. In the medium with 0 M arabinose, we supplemented the medium with 0.5 % glucose in order to repress the leakage in the PBAD/araC promoter. Fig. 2 shows the histograms of the samples measured by the flow cytometer. In the results of the reporter cell (1), when the Induction of FimB(wild-type) expression increases, the fluorescence intensity decreases. From this fact, we confirmed that the fim switch(Tokyo_Tech/J23119) is inverted from ON to OFF by FimB (wild-type). From the result of the reporter cell (2), when the expression amount of FimB(wild-type) increases, the expression amount of GFP in the reporter cell (2) increases. From this fact, we confirmed that the fim switch(Tokyo_Tech/J23119) is inverted from OFF to ON by FimB(wild-type). From the results of the two reporter cells (1) and (2), we successfully confirmed that FimB (wild-type) inverts the fim switch from ON to OFF and from OFF to ON.
The result of the reporter cell (2) shows that when the concentration of arabinose is increased to 0〜20 microM, the fluorescence intensity increases. This shows the function of FimB (wild-type) inverting the fim switch (wild-type) from OFF to ON. However, when the arabinose concentration is excess amount (5mM), the fluorescence intensity decreases. According to [1], this is caused by the excess increase of the inversion rate of the fim switch. When the inversion rate is too high, there is not enough time for transcription initiation. Consequently, the GFP expression decreases.
Invertion assay with FimE
1. Construction
All the samples were DH5alpha strain with antibiotic resistance to ampicillin and kanamycin.
(1) PBAD/araC_fimE(wild-type) (pSB6A1) + fim switch[default ON](Tokyo_Tech/J23119)_gfp (pSB3K3)
(2) PBAD/araC_fimE(wild-type) (pSB6A1) + fim switch[default OFF](Tokyo_Tech/J23119)_gfp (pSB3K3)
(3) pSB6A1 + fim switch[default ON](Tokyo_Tech/J23119)_gfp (pSB3K3) …positive control 1
(4) pSB6A1 + fim switch[default OFF](Tokyo_Tech/J23119)_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
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 mM arabinose (final concentration of arabinose is 5 mM)
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
We tried to confirm that fim switch(Tokyo_Tech/J23119) is unidirectionally inverted in the presence of FimE (wild-type) by using GFP as a reporter, under 2 different concentrations of arabinose. In the medium with 0 M arabinose, we supplemented the medium with 0.5 % glucose in order to repress the leakage in the PBAD/araC promoter. Fig.2 shows the histograms of the samples measured by the flow cytometer. In the results of the reporter cell (1), when the Induction of FimE(wild-type) expression increases, the fluorescence intensity decreases. From this fact, we confirmed that the fim switch (Tokyo_Tech/J23119) is inverted in the ON-to-OFF direction by FimE (wild-type). From the result of reporter cell (2), when the expression amount of FimB(wild-type) increases, the expression amount of GFP in the reporter cell (2) increases. From this fact, we confirmed that the fim switch(Tokyo_Tech/J23119) is inverted from OFF-to-ON direction by FimE(wild-type) that does not invert the fim switch(wild-type) in the OFF-to-ON direction. From this result, the inversion of fim switch (Tokyo_Tech/J23119) by FimE was not confirmed correctly. In this assay, the FimE protein inverts fim switch in the ON-to-OFF and the OFF-to-ON direction. In other words, the FimE protein works as the FimB protein.
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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