Team:Tokyo Tech/Experiment/FimE dependent fim switch state assay
C4HSL-dependent growth assay
We have characterized previous parts.
contents
1. Introduction
2. Summary of the Experiment
3. Results
4. Discussion
5. Materials and Methods
5.1. Construction
5.2. Assay Protocol
5.2.1. Protocol1
5.2.2. Protocol2
6. Reference
1. Introduction
ここにコピペ。
2. Summary of the Experiment
ここにコピペ。
3. Results
ここにコピペ。
4. Discussion
ここにコピペ。
5. Materials and Methods
5.1. Construction
-Strain
All the samples were DH5alpha strain.
-Plasmids
Device 1: J23101 + I13504(pSB1C3)
|
|
Fig.3-7-4-1. |
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. Make a 1:100 dilution in 3 mL of fresh LB containing antibiotic, and 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. Suspend the pellet in 1 mL of LB containing Amp and Kan.
11. Add 30 microL of suspension in the following medium.
①
4.2.2. FLA analysis
1. コピペ。
2. コピペ。
3. コピペ。
4. コピペ。
5. コピペ。
6. コピペ。
7. コピペ。
8. コピペ。
9. コピペ。
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