Difference between revisions of "Part:BBa K1632001:Design"

(→‎1. Construction)
(→‎2. Assay protocol)
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=====2. Assay protocol=====
 
=====2. Assay protocol=====
  
−
1. Prepare overnight cultures for the 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.<br>
+
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.<br>
−
2. Make a 1:100 dilution in 3 mL of fresh LB containing Amp, Kan and glucose (final concentration is 0.5 %).<br>
+
2. Make a 1:100 dilution in 3 mL of fresh LB containing Amp, Kan and glucose (final concentration is 0.5 %).<br>
 
3.Incubate the cells at 37 ℃, shaking at 180 rpm until the observed OD590 reaches 0.4 (Fresh Culture).<br>
 
3.Incubate the cells at 37 ℃, shaking at 180 rpm until the observed OD590 reaches 0.4 (Fresh Culture).<br>
 
4. After the incubation, take 1 mL of the samples, and centrifuge at 5000x g, 1 min, 25 ℃ <br>
 
4. After the incubation, take 1 mL of the samples, and centrifuge at 5000x g, 1 min, 25 ℃ <br>
Line 69: Line 69:
 
6. Suspend the pellet in 1mL of LB containing Amp and Kan, and centrifuge at 5000x g, 1 min, 25 ℃ <br>
 
6. Suspend the pellet in 1mL of LB containing Amp and Kan, and centrifuge at 5000x g, 1 min, 25 ℃ <br>
 
7. Remove the supernatant.<br>
 
7. Remove the supernatant.<br>
−
8. Take the samples, and centrifuge at 5000x g, 1 min, 25 ℃ <br>
+
8. Suspend the pellet in 1mL of LB containing Amp and Kan, and centrifuge at 5000x g, 1 min, 25 ℃ <br>
 
9. Remove the supernatant.<br>
 
9. Remove the supernatant.<br>
 
10. Suspend the pellet in 1mL of LB containing Amp and Kan.<br>
 
10. Suspend the pellet in 1mL of LB containing Amp and Kan.<br>

Revision as of 20:04, 18 September 2015

fim switch[default OFF](Tokyo_Tech/J23119)


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 98
    Illegal NheI site found at 121
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 92
    Illegal BamHI site found at 133
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]

Design Notes

sequence confirmed

Materials and Methods

Invertion assay with FimB

1. Construction

All the samples were DH5alpha strain with antibiotic resistance to ampicillin and kanamycin.

(1) PBAD/araC_fimB(wild-type) (pSB6A1) + fim switch[default ON](Tokyo_Tech/J23119)_gfp (pSB3K3)
(2) PBAD/araC_fimB(wild-type) (pSB6A1) + fim switch[default OFF](Tokyo_Tech/J23119)_gfp (pSB3K3)
(3) pSB6A1 + fim switch[default ON](Tokyo_Tech/J23119)_gfp (pSB3K3) …positive control 1
(4) pSB6A1 + fim switch[default OFF](Tokyo_Tech/J23119)_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

2. 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. 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 1 mL 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 concentratio 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)
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.)

Invertion assay with FimE

1Italic text. 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](Tokyo_Tech/J23119)_gfp (pSB3K3)
(2) PBAD/araC_fimE(wild-type) (pSB6A1) + fim switch[default OFF](Tokyo_Tech/J23119)_gfp (pSB3K3)
(3) pSB6A1 + fim switch[default ON](Tokyo_Tech/J23119)_gfp (pSB3K3) …positive control 1
(4) pSB6A1 + fim switch[default OFF](Tokyo_Tech/J23119)_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

2. 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. Suspend the pellet in 1mL of LB containing Amp and Kan, and centrifuge at 5000x g, 1 min, 25 ℃
7. Remove the supernatant.
8. Suspend the pellet in 1mL 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 0.5 %) and 30 microL of sterile water
② 3 mL of LB containing Amp, Kan and 30 microL of 500 mM arabinose (final concentration of arabinose is 5 mM)
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.)

Source

PCR after the invertion experiment of BBa_K1632000

References

Ian C. Blomfield et al. (1997) Integration host factor stimulates both FimB- andFimE-mediated site-specific DNA inversion that controlsphase variation of type 1 fimbriae expression in Escherichia coli. Molecular Microbiology 23(4), 705–717

John M. Abraham et al. (1985) An invertible element of DNA controls phase variation of type 1 fimbriae of Escherichia coli. Proc Natl Acad Sci U S A 82(17):5724-7

Matthew P. McCusker et al. (2008) DNA sequence heterogeneity in Fim tyrosine-integrase recombinase-binding elements and functional motif asymmetries determine the directionality of the fim genetic switch in Escherichia coli K-12. Molecular Microbiology 67(1): 171–187