Difference between revisions of "Part:BBa K1632013:Experience"

Revision as of 23:36, 18 September 2015

PBAD/araC_rbs_fimE(wild-type)

Materials and Methods

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](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

Fig. 1. Plasmids

Flow cytometer

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. 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 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

Fig. 2. The histograms of the samples measured by flow cytometer

Discussion

We tried to confirm that fim switch(wild-type) is predominantly inverted in the presence of FimE(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 1.0 % 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(wild-type) is inverted from [ON] state to [OFF] state by FimE(wild-type). From the result of the reporter cell (2), even when the expression amount of FimE(wild-type) increases, the expression amount of GFP in the reporter cell (2) does not change. From this fact, we confirmed that the fim switch(wild-type) is inverted only from [ON] state to [OFF] state sirection by FimE(wild-type). From the results of the two reporter cells (1) and (2), we successfully confirmed that FimE(wild-type) inverts the fim switch(wild-type) only from [ON] state to [OFF] state direction.

The results of Positive control 1 and Negative control 1 confirmed that the endogenous FimB and FimE did not invert our fim switch(wild-type). Also, the result of Positive control 2 and Negative control 2, indicates that the expression of FimE(wild-type) do not affect GFP expression.

Supplemental experiments

Assay protocol

Results

Fig. 3. The histograms of the samples measured by flow cytometer

Fig. 4. The histograms of the samples measured by flow cytometer

Discussion

We counted out the all colonies and those with fluorescence. In the results of the reporter cell (1), when inducing the expression of FimE(wild-type), the percentage of [ON] state decreased dramatically. On the other hand, from the results of the reporter cell (2), when inducing the expression of FimE(wild-type), the percentage of [ON] state remained being small. From the results of the two reporter cells (1) and (2), we successfully confirmed that FimE(wild-type) inverts the fim switch(wild-type) predominantly from [ON] state to [OFF] state. (Fig.3). This result was consistent with the histograms (Fig.2.).Also, we incubated the colonies with fluorescence and those without fluorescence. We minipreped cell culture. Sequence complementarity in the specific region of the switch shows intended inversion of the switch from [ON] state to [OFF] state in all sample (Fig.4.).

More information

For more information, see [http://2015.igem.org/Team:Tokyo_Tech/Project our work in Tokyo_Tech 2015 wiki].