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

Revision as of 23:18, 18 September 2015

PBAD/araC_rbs_fimE(wild-type)

Design Notes

sequence confirmed

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)_rbs_gfp (pSB3K3)
(2). PBAD/araC_fimE(wild-type) (pSB6A1) + fim switch[default OFF](wild-type)_rbs_gfp (pSB3K3)
(3). pSB6A1 + fim switch[default ON](wild-type)_rbs_gfp (pSB3K3) …positive control 1
(4). pSB6A1 + fim switch[default OFF](wild-type)_rbs_gfp (pSB3K3) …negative control 1
(5). PBAD/araC_fimE(wild-type) (pSB6A1) + J23119_rbs_gfp (pSB3K3) …positive control 2
(6). PBAD/araC_fimE(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 of mass of glucose 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 of mass of glucose 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 and 30 microL of 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.)

Result

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

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

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

Source

Composite of BBa_I0500, BBa_B0034, BBa_K1632011

References

Timothy S. Ham et al. (2006) A Tightly Regulated Inducible Expression System Utilizing the fim Inversion Recombination Switch. Biotechnol Bioeng 94(1):1-4