Team:Tokyo Tech/Experiment/FimE dependent fim switch state assay
FimE dependent fim switch state assay
contents
1. Introduction
2. Summary of the Experiment
3. Results
3.1. Arabinose-dependent FimB (wild-type) expression
3.2. FLA analysis
4. Discussion
5. Materials and Methods
5.1. Construction
5.2. Assay Protocol
5.2.1 Arabinose dependent FimB expression
5.2.2. FLA analysis
6. Reference
1. Introduction
To confirm the function of fim switch in the presence of FimE(wild-type), we constructed two Biobrick parts, BBa_K1632013 and BBa_K1632002(Fig. 3-5-1-1). BBa_K1632013 enables arabinose-inducible expression of the FimE (wild-type). In BBa_K1632013 and BBa_K1632002, either the fim switch [default ON] or the fim switch [default OFF] is placed upstream of the GFP coding sequence.
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Fig.3-5-1-1. New plasmids we constructed to confirm the function of fim switch |
2. Summary of the Experiment
(1) PBAD/araC_fimE (pSB6A1)+ fim switch[default ON](wild-type)_GFP (pSB3K3)
(2) PBAD/araC_fimE (pSB6A1) + fim switch[default OFF](wild-type) _GFP (pSB3K3)
(3)Positive control 1: (pSB6A1)+ fim switch[default ON](wild-type) _GFP (pSB3K3)
(4)Negative control 1: (pSB6A1)+ fim switch[default OFF](wild-type) _GFP (pSB3K3)
(5)Positive control 2: PBAD/araC_fimE (wild-type) (pSB6A1)+Pcon_GFP (pSB3K3)
(6)Negative control 2: PBAD/araC_fimE (wild-type) (pSB6A1)+promoter less_GFP (pSB3K3)
Fig.3-4-2-1. Plasmids for the experiment of FimB dependent fim switch state assay |
3. Results
3.1. Arabinose-dependent FimB (wild-type) expression
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. 3-5-3-1 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-tyoe). 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.
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Fig. 3-4-3-1. Histogram of the samples measured by flow cytometer |
3.2. FLA analysis
写真とシークエンスデータ
4. Discussion
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. |
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Fig.3-4-4-1. Histogram of reporter cell (2) |
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Even though there is no fim switch (wild-type) in the plasmid of positive control 2, similar increase of fluorescence intensity dependent on the expression of FimB (wild-type) was found in our positive control 2 (Fig.3-5-4-1) This unpredictable increase of fluorescence intensity is caused by the decrease of dilution rate of proteins inside cells. The FimB (wild-type) expression, depending on the arabinose induction, inhibits cell division that decreases protein concentration inside the individual cells. Therefore, the concentration of GFP in individual cell increases. |
Fig.3-4-4-2. The histogram of positive control 2 |
4. Materials and Methods
4.1. Construction
-Strain
All the samples were DH5alpha strain.
-Plasmids
A. Pbad/araC_fimE(wild-type) (pSB6A1)+ fim switch[default ON](wild-type)_gfp (pSB3K3)
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Fig. 3-5-4-1. |
B. Pbad/araC_fimE(wild-type) (pSB6A1)+ fim switch[default OFF](wild-type)_gfp (pSB3K3)
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Fig. 3-5-4-2. |
C. promoter less M256ICysE(pSB6A1)+ fim switch[default ON](wild-type)_gfp(pSB3K3)…Positive control 1
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Fig. 3-5-4-3. |
D. promoter less M256ICysE(pSB6A1)+ fim switch[default OFF](wild-type)_gfp(pSB3K3)…Negative control 1
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Fig. 3-5-4-4. |
E. Pbad/araC-fimE (pSB6A1) +J23119 promoter_gfp (pSB3K3)…Positive control2
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Fig. 3-5-4-5. |
F. Pbad/araC-fimE (pSB6A1) +promoter less gfp (pSB3K3)…Negative control2
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Fig. 3-5-4-6. |
4.2. Assay Protocol
4.2.1. Arabinose dependent FimE expression
1. Prepare overnight cultures for the each sample in 3 ml LB medium, containing ampicillin (50 microg / mL), kanamycin (30 microg / mL) and glucose (final concentration of mass of glucose is 1.0 percent) at 37 ℃ for 12h.
2. Make a 1:100 dilution in 3 mL of fresh LB containing Amp, Kan and glucose (final concentration of mass of glucose is 1.0 percent).
3. Grow the cells at 37 ℃ 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 by using P1000 pipette.
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 by using P1000 pipette.
8. Take the samples, and centrifuge at 5000x g, 1 min, 25 ℃.
9. Remove the supernatant by using P1000 pipette.
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 and 3 microL sterile water
② 3 mL of LB containing Amp, Kan and 30 microL of 500μM arabinose (final concentration of arabinose is 1 microM)
③ 3 mL of LB containing Amp, Kan and 30 microL of 1 mM arabinose (final concentration of arabinose is 2 microM)
④3 mL of LB containing Amp, Kan and 30 microL of 2 mM arabinose (final concentration of arabinose is 5 microM)
※ As for C and D, the suspension were added only in medium ① and ④.
12. Grow the samples at 37 ℃ for 6 hours.
13. Measure OD590 of all the samples every hour.
14. Start preparing the flow cytometer 1 h before the end of incubation.
15. After the incubation, take the samples, and centrifuge at 9000x g, 1min, 4℃.
16. Remove the supernatant by using P1000 pipette.
17. Add 1 mL of filtered PBS (phosphate-buffered saline) and suspend. (The ideal of OD is 0.3)
18. Dispense all of each suspension into a disposable tube through a cell strainer.
19. Use flow cytometer to measure the fluorescence of GFP. (We used BD FACSCaliburTM Flow Cytometer of Becton, Dickenson and Company.)
4.2.2. FLA analysis
1. After the assay of “Arabinose dependent FimB expression”, miniprep cell culture (A,B,
,C and D) of leftover as here.(http://parts.igem.org/Help:Protocols/Miniprep)
2. Turn on water bath to 42℃.
3. Take competent DH5alpha strain from -80℃ freezer and leave at rest on ice.
4. Add 3 µl of each plasmids in a 1.5 ml tube.
5. Put 25 µl competent cell into each 1.5 ml tubes with plasmid.
6. Incubate on ice for 15 min.
7. Put tubes with DNA and competent cells into water bath at 42℃ for 30 seconds.
8. Put tubes back on ice for 2 minutes.
9. Add 125 µl of SOC medium. Incubate tubes for 30 minutes at 37℃.
10. Make a 1:5 dilution in 150µl of fresh SOC medium.
11. Spread about 100 µl of the resulting culture of LB plate containing kanamycin.
12. Incubate LB plate for 14-15 hours at 37℃.
5. Reference
1. Timothy S. Ham et al. (2006) A Tightly Regulated Inducible Expression System Utilizing the fim Inversion Recombination Switch. Biotechnol Bioeng 94(1):1-4