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

 
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__NOTOC__
 
__NOTOC__
 
<partinfo>BBa_K1632001 short</partinfo>
 
<partinfo>BBa_K1632001 short</partinfo>
  
 
<partinfo>BBa_K1632001 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K1632001 SequenceAndFeatures</partinfo>
−
 
  
 
===Design Notes===
 
===Design Notes===
−
1
 
  
 +
sequence confirmed
 +
 +
===Materials and Methods===
 +
 +
====Invertion assay with FimB====
 +
 +
=====1. Construction=====
 +
 +
All the samples were DH5alpha strain with antibiotic resistance to ampicillin and kanamycin.<br>
 +
 +
(1) PBAD/''araC''_''fimB''(wild-type) (pSB6A1) + ''fim'' switch[default ON](Tokyo_Tech/J23119)_''gfp'' (pSB3K3) <br>
 +
(2) PBAD/''araC''_''fimB''(wild-type) (pSB6A1) + ''fim'' switch[default OFF](Tokyo_Tech/J23119)_''gfp'' (pSB3K3) <br>
 +
(3) pSB6A1 + ''fim'' switch[default ON](Tokyo_Tech/J23119)_''gfp'' (pSB3K3) …positive control 1<br>
 +
(4) pSB6A1 + ''fim'' switch[default OFF](Tokyo_Tech/J23119)_''gfp'' (pSB3K3) …negative control 1<br>
 +
(5) PBAD/''araC''_''fimB''(wild-type) (pSB6A1) + J23119_''gfp''(pSB3K3) …positive control 2 <br>
 +
(6) PBAD/''araC''_''fimB''(wild-type) (pSB6A1) + rbs_''gfp''(pSB3K3) …negative control 2 <br>
 +
 +
=====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.<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>
 +
4. After the incubation, take 1 mL of the samples, and centrifuge at 5000x g, 1 min, 25 ℃ <br>
 +
5. Remove the supernatant.<br>
 +
6. Suspend the pellet in 1 mL of LB containing Amp and Kan, and centrifuge at 5000x g, 1 min, 25 ℃ <br>
 +
7. Remove the supernatant.<br>
 +
8. Suspend the pellet in 1 mL of LB containing Amp and Kan, and centrifuge at 5000x g, 1 min, 25 ℃ <br>
 +
9. Remove the supernatant.<br>
 +
10. Suspend the pellet in 1 mL of LB containing Amp and Kan.<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, glucose (final concentratio 0.5 %) and 30 microL of sterile water<br>
 +
<span style="margin-left: 20px;">② 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;">③ 3 mL of LB containing Amp, Kan and 30 microL of 20 mM arabinose (final concentration of arabinose is 200 microM)<br>
 +
<span style="margin-left: 20px;">④ 3 mL of LB containing Amp, Kan and 30 microL of 500 mM arabinose (final concentration of arabinose is 5 mM)<br>
 +
12. Incubate the samples at 37 ℃, shaking at 180 rpm for 6.5 hours. (Measure OD590 of all the samples every hour.)<br>
 +
13. After the incubation, take the samples, and centrifuge at 9000x g, 1min, 4℃.<br>
 +
14. Remove the supernatant.<br>
 +
15. Add 1 mL of filtered PBS (phosphate-buffered saline) and suspend. (The ideal of OD is 0.3)<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>
 +
 +
=====3. Results=====
 +
 +
[[Image:Tokyo_Tech_fim_switch_TT_FimB_assay_Results.png |thumb|center|700px|<b>Fig. 1. </b>The histograms of the samples with FimB measured by flow cytometer]]<br>
 +
<br><br><br><br><br>
 +
 +
====Invertion assay with FimE====
 +
 +
=====1. Construction=====
 +
 +
All the samples were DH5alpha strain with antibiotic resistance to ampicillin and kanamycin.<br>
 +
 +
(1) PBAD/''araC''_''fimE''(wild-type) (pSB6A1) + ''fim'' switch[default ON](Tokyo_Tech/J23119)_''gfp'' (pSB3K3) <br>
 +
(2) PBAD/''araC''_''fimE''(wild-type) (pSB6A1) + ''fim'' switch[default OFF](Tokyo_Tech/J23119)_''gfp'' (pSB3K3) <br>
 +
(3) pSB6A1 + ''fim'' switch[default ON](Tokyo_Tech/J23119)_''gfp'' (pSB3K3) …positive control 1<br>
 +
(4) pSB6A1 + ''fim'' switch[default OFF](Tokyo_Tech/J23119)_''gfp'' (pSB3K3) …negative control 1<br>
 +
(5) PBAD/''araC''_''fimE''(wild-type) (pSB6A1) + J23119_''gfp'' (pSB3K3) …positive control 2 <br>
 +
(6) PBAD/''araC''_''fimE''(wild-type) (pSB6A1) + rbs_''gfp'' (pSB3K3) …negative control 2 <br>
 +
 +
=====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.<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>
 +
4. After the incubation, take 1 mL of the samples, and centrifuge at 5000x g, 1 min, 25 ℃ <br>
 +
5. Remove the supernatant.<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>
 +
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>
 +
10. Suspend the pellet in 1mL of LB containing Amp and Kan.<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, glucose (final concentration is 0.5 %) and 30 microL of sterile water<br>
 +
<span style="margin-left: 20px;">② 3 mL of LB containing Amp, Kan and 30 microL of 500 mM arabinose (final concentration of arabinose is 5 mM)<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>
 +
14. Remove the supernatant.<br>
 +
15. Add 1 mL of filtered PBS (phosphate-buffered saline) and suspend. (The ideal of OD is 0.3)<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>
 +
 +
=====3. Results=====
 +
 +
[[Image:Tokyo_Tech_fim_switch_TT_FimE_assay_Results.png |thumb|center|700px|<b>Fig. 2. </b>The histograms of the samples with FimE measured by flow cytometer]]<br>
 +
 +
===More information===
  
 +
For more information, see [[http://2015.igem.org/Team:Tokyo_Tech/Project Our work in Tokyo_Tech 2015 wiki]],  [[http://2015.igem.org/Team:Tokyo_Tech/Experiment/ssrA_tag_degradation_assay About ssrA-tag]],  [[http://2015.igem.org/Team:Tokyo_Tech/Experiment/Overview_of_fim_inversion_system About ''fim'' inversion system]]
  
 
===Source===
 
===Source===
  
−
1
+
PCR after the invertion experiment of BBa_K1632000
  
 
===References===
 
===References===
 +
Ian C. Blomfield <i>et al</i>. (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 <i>et al</i>. (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 <i>et al</i>. (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

Latest revision as of 03:52, 19 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.)

3. Results
Fig. 1. The histograms of the samples with FimB measured by flow cytometer






Invertion assay with FimE

1. 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.)

3. Results
Fig. 2. The histograms of the samples with FimE measured by flow cytometer

More information

For more information, see http://2015.igem.org/Team:Tokyo_Tech/Project Our work in Tokyo_Tech 2015 wiki, http://2015.igem.org/Team:Tokyo_Tech/Experiment/ssrA_tag_degradation_assay About ssrA-tag, http://2015.igem.org/Team:Tokyo_Tech/Experiment/Overview_of_fim_inversion_system About ''fim'' inversion system

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