Difference between revisions of "Part:BBa K1632002:Design"

(→‎2. Assay protocol)
(→‎2. Assay protocol)
Line 64: Line 64:
 
=====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 of mass of glucose is 1.0 %) 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 1.0 %) at 37 ℃ for 12h.<br>
−
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 %).<br>
+
2. Make a 1:100 dilution in 3 mL of fresh LB containing Amp, Kan and glucose (final concentration is 1.0 %).<br>
−
3.Grow 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 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>
 
5. Remove the supernatant.<br>
 
5. Remove the supernatant.<br>
 
6. Add 1 mL of LB containing Amp and Kan, and centrifuge at 5000x g, 1 min, 25 ℃ <br>
 
6. Add 1 mL of LB containing Amp and Kan, and centrifuge at 5000x g, 1 min, 25 ℃ <br>
Line 75: Line 75:
 
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>
 
11. Add 30 microL of suspension in the following medium.<br>
 
11. Add 30 microL of suspension in the following medium.<br>
−
<span style="margin-left: 20px;">① 3 mL of LB containing Amp, Kan and 30 microL of sterile water<br>
+
<span style="margin-left: 20px;">① 3 mL of LB containing Amp, Kan, glucose (final concentration is 1.0 %) and 30 microL sterile water<br>
−
<span style="margin-left: 20px;">② 3 mL of LB containing Amp, Kan and 30 microL of 500 microM　arabinose (final concentration of arabinose is 5 microM)<br>
+
<span style="margin-left: 20px;">② 3 mL of LB containing Amp, Kan and 30 microL of 500 microM arabinose (final concentration of arabinose is 5 microM)<br>
 
<span style="margin-left: 20px;">③ 3 mL of LB containing Amp, Kan and 30 microL of 1 mM arabinose (final concentration of arabinose is 10 microM)<br>
 
<span style="margin-left: 20px;">③ 3 mL of LB containing Amp, Kan and 30 microL of 1 mM arabinose (final concentration of arabinose is 10 microM)<br>
 
<span style="margin-left: 20px;">④ 3 mL of LB containing Amp, Kan and 30 microL of 2 mM arabinose (final concentration of arabinose is 20 microM)<br>
 
<span style="margin-left: 20px;">④ 3 mL of LB containing Amp, Kan and 30 microL of 2 mM arabinose (final concentration of arabinose is 20 microM)<br>
−
<span style="margin-left: 20px;">※ As for C and D, the suspension were added only in medium ① and ④. <br>
+
<span style="margin-left: 20px;">※ As for (3) and (4), the suspension were added only in medium ① and ④. <br>
 
12. Incubate the samples at 37 ℃ for 6 hours, shaking at 180 rpm. (Measure the OD590 of all the samples every hour.)<br>
 
12. Incubate the samples at 37 ℃ for 6 hours, shaking at 180 rpm. (Measure the OD590 of all the samples every hour.)<br>
 
13. After the incubation, take the samples, and centrifuge at 9000x g, 1min, 4℃.<br>
 
13. After the incubation, take the samples, and centrifuge at 9000x g, 1min, 4℃.<br>
Line 86: Line 86:
 
16. Dispense all of each suspension into a disposable tube through a cell strainer.<br>
 
16. Dispense all of each suspension into a disposable tube through a cell strainer.<br>
 
17. Use flow cytometer to measure the fluorescence of GFP. (We used BD FACSCaliburTM Flow Cytometer of Becton, Dickenson and Company.)<br>
 
17. Use flow cytometer to measure the fluorescence of GFP. (We used BD FACSCaliburTM Flow Cytometer of Becton, Dickenson and Company.)<br>
 +
 
===Source===
 
===Source===
  

Revision as of 16:17, 18 September 2015

fim switch[default ON](Tokyo_Tech/J23119)_rbs_gfp


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 345
    Illegal NheI site found at 368
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 374
    Illegal BamHI site found at 333
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 1102


Design Notes

sequence confirmed

Materials and Methods

Invertion assay with FimB

1. 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](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 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.
2. Make a 1:100 dilution in 3 mL of fresh LB containing Amp, Kan and glucose (final concentration is 0.5 %).
3.Grow the cells at 37 ℃, shaking at 180 rpm 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.
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. 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 concentration of mass of glucose 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.)

Invertion assay with FimE

1. 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](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 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 (3) and (4), 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.)

Source

Composite of BBa_K1632000, BBa_I13500

References

[1]Hung M. et al. (2014) Modulating the frequency and bias of stochastic switching to control phenotypic variation. Nat Commun 5:4574. doi:10.1038/ncomms5574 [2]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

[3]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

[4]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