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Part:BBa_K1632008:Experience

Designed by: Riku Shinohara   Group: iGEM15_Tokyo_Tech   (2015-09-10)
Revision as of 08:51, 18 September 2015 by YuiKora (Talk | contribs) (→‎Construction)

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

Fig. 1. Plasmids

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

Discussion

We tried to confirm that fim switch 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. 3-4-3-1 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 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 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 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 negative control 2, indicates that the expression of FimB (wild-type) do not have effects on the gfp expression. The reason the fluorescence intensity of the positive control 2 is increasing in proportion to the arabinose concentration is described in 4. Discussion section.




When the concentration of FimB (wild-type) increased by increasing the concentration of arabinose, we confirmed that the fluorescence intensity decreased in both ON to OFF process and OFF to ON process.

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 (Fig.3-4-4-1). 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

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

Fig. 3. Plasmids

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
Fig. 2. Histogram of the samples measured by flow cytometer

Discussion

We tried to confirm that fim switch is unidirectionally 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. 3-6-3-1 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 to OFF 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 to OFF 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 only from ON to OFF.

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 have effects on the GFP expression.

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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