Difference between revisions of "Part:BBa K1632005:Design"

 
(9 intermediate revisions by 2 users not shown)
Line 15: Line 15:
 
=====1. Construction=====
 
=====1. Construction=====
  
All the samples were DH5α strain with antibiotic resistance to ampicillin and kanamycin.<br>
+
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](wild-type)_gfp (pSB3K3) <br>
+
(1) PBAD/''araC''_''fimB''(wild-type) (pSB6A1) + ''fim'' switch[default ON](wild-type)_''gfp'' (pSB3K3) <br>
(2) PBAD/araC_fimB(wild-type) (pSB6A1) + ''fim'' switch[default OFF](wild-type)_gfp (pSB3K3) <br>
+
(2) PBAD/''araC''_''fimB''(wild-type) (pSB6A1) + ''fim'' switch[default OFF](wild-type)_''gfp'' (pSB3K3) <br>
(3) pSB6A1 + ''fim'' switch[default ON](wild-type)_gfp (pSB3K3) …positive control 1<br>
+
(3) pSB6A1 + ''fim'' switch[default ON](wild-type)_''gfp'' (pSB3K3) …positive control 1<br>
(4) pSB6A1 + ''fim'' switch[default OFF](wild-type)_gfp (pSB3K3) …negative control 1<br>
+
(4) pSB6A1 + ''fim'' switch[default OFF](wild-type)_''gfp'' (pSB3K3) …negative control 1<br>
(5) PBAD/araC_fimB(wild-type) (pSB6A1) + J23119_gfp(pSB3K3) …positive control 2 <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>
+
(6) PBAD/''araC''_''fim''B(wild-type) (pSB6A1) + rbs_''gfp''(pSB3K3) …negative control 2 <br>
  
 
[[Image:Tokyo_Tech_FimB_assay.png |thumb|center|900px|<b>Fig. 1. </b>Plasmids]]<br>
 
[[Image:Tokyo_Tech_FimB_assay.png |thumb|center|900px|<b>Fig. 1. </b>Plasmids]]<br>
  
=====2. Assay protocol=====
+
=====Flow cytometer=====
 +
======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>
+
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>
 
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. 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>
 
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>
 
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>
 
9. Remove the supernatant.<br>
10. Add 1 mL of LB containing Amp and Kan, and suspend.<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>
 
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;">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 2 mM arabinose (final concentration of arabinose is 20 microM)<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;">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>
 
<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>
<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 ℃, shaking at 180 rpm for 6.5 hours. (Measure OD590 of all the samples every hour.)<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>
 
13. After the incubation, take the samples, and centrifuge at 9000x g, 1min, 4℃.<br>
Line 50: Line 51:
 
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>
 +
 +
======3.Results======
 +
[[Image:Tokyo_Tech_FimB_assay_Results.png |thumb|center|700px|<b>Fig. 2. </b>The histograms of the samples measured by flow cytometer]]<br>
 +
 +
=====Supplemental experiments=====
 +
 +
======2.Assay protocol======
 +
1. After the assay of “Arabinose dependent FimE expression”, miniprep cell culture ((1)-①, (1)-③, (2)-① and (2)-③) of leftover as here.(https://parts.igem.org/Help:Protocols/Miniprep)<br>
 +
2. Turn on water bath to 42℃.<br>
 +
3. Take competent DH5alpha strain from -80℃ freezer and leave at rest on ice.<br>
 +
4. Add 3 µl of each plasmids in a 1.5 ml tube.<br>
 +
5. Put 25 µl competent cell into each 1.5 ml tubes with plasmid.<br>
 +
6. Incubate on ice for 15 min.<br>
 +
7. Put tubes with DNA and competent cells into water bath at 42℃ for 30 seconds.<br>
 +
8. Put tubes back on ice for 2 minutes.<br>
 +
9. Add 125 µl of SOC medium. Incubate tubes for 30 minutes at 37℃.<br>
 +
10. Make a 1:5 dilution in 150 µl of fresh SOC medium.<br>
 +
11. Spread about 100 µl of the resulting culture of LB plate containing kanamycin. <br>
 +
12. Incubate LB plate for 14-15 hours at 37℃.<br>
 +
13. Set the plate reader to measure GFP.<br>
 +
14. Scan the each plates with the plate reader. (We used FujiFilm FLA-5100 Fluorescent Image Analyzer from FUJIFilm Life Science.)<br>
 +
15. Analyze the scanning data by changing the scale type (Bezier) and adjusting the range. (We analyzed by using the software, Multi Gauge ver. 2.0 from FUJIFilm Life Science.)<br>
 +
16. Counted out the all colonies and those with fluorescence.<br>
 +
17. Prepare three overnight cultures for each sample in 3 mL of LB medium containing kanamycin (30 microg / mL) shaking at 180 rpm for 12h.<br>
 +
18. Miniprep each samples and ask DNA sequencing of each samples for Biomaterial Analysis Center, Technical Department.<br>
 +
 +
=====3.Results=====
 +
[[Image:Tokyo_Tech_FLA_colony_FimB.png |thumb|center|600px|<b>Fig. 3. </b> Determination of percemtage of [ON] state and colony formation using plasmid mixture extracted cell expressing FimB.]]<br>
 +
[[Image:Tokyo_Tech_FImB_sequence.png |thumb|center|600px|<b>Fig. 4. </b> DNA sequencing results of <i>fim</i> switch(wild-type)]]<br>
  
 
====Invertion assay with FimE====
 
====Invertion assay with FimE====
Line 55: Line 85:
 
=====1. Construction=====
 
=====1. Construction=====
  
All the samples were DH5α strain with antibiotic resistance to ampicillin and kanamycin.<br>
+
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](wild-type)_gfp (pSB3K3) <br>
+
(1) PBAD/''araC''_''fimE''(wild-type) (pSB6A1) + ''fim'' switch[default ON](wild-type)_''gfp'' (pSB3K3) <br>
(2) PBAD/''araC''_fimE(wild-type) (pSB6A1) + ''fim'' switch[default OFF](wild-type)_gfp (pSB3K3) <br>
+
(2) PBAD/''araC''_''fimE''(wild-type) (pSB6A1) + ''fim'' switch[default OFF](wild-type)_''gfp'' (pSB3K3) <br>
(3) pSB6A1 + ''fim'' switch[default ON](wild-type)_gfp (pSB3K3) …positive control 1<br>
+
(3) pSB6A1 + ''fim'' switch[default ON](wild-type)_''gfp'' (pSB3K3) …positive control 1<br>
(4) pSB6A1 + ''fim'' switch[default OFF](wild-type)_gfp (pSB3K3) …negative control 1<br>
+
(4) pSB6A1 + ''fim'' switch[default OFF](wild-type)_''gfp'' (pSB3K3) …negative control 1<br>
(5) PBAD/''araC''_fimE(wild-type) (pSB6A1) + J23119_gfp (pSB3K3) …positive control 2 <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>
+
(6) PBAD/''araC''_''fimE''(wild-type) (pSB6A1) + rbs_''gfp'' (pSB3K3) …negative control 2 <br>
  
[[Image:Tokyo_Tech_FimE_assay.png|thumb|center|900px|<b>Fig. 2. </b>Plasmids]]<br>
+
[[Image:Tokyo_Tech_FimE_assay.png|thumb|center|900px|<b>Fig. 5. </b>Plasmids]]<br>
 +
=====Flow cytometer=====
 +
======2. Assay protocol======
  
=====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.<br>
 
+
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 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 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. 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>
 
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. 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>
 
7. Remove the supernatant.<br>
8. Take the samples, and centrifuge at 5000x g, 1 min, 25 ℃ <br>
+
8. Suspend the pellet in 1 mL of LB containing Amp and Ka, 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>
 
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, glucose (final concentration is 1.0 %) and 30 microL 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 90: Line 120:
 
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>
 +
======3.Results======
 +
[[Image:Tokyo_Tech_FimE_assay_Results.png |thumb|center|700px|<b>Fig. 6. </b>The histograms of the samples measured by flow cytometer]]<br>
 +
 +
=====Supplemental experiments=====
 +
 +
======2.Assay protocol======
 +
1. After the assay of “Arabinose dependent FimE expression”, miniprep cell culture ((1)-①, (1)-③, (2)-① and (2)-③) of leftover as here.<br>(https://parts.igem.org/Help:Protocols/Miniprep)
 +
2. Turn on water bath to 42℃.<br>
 +
3. Take competent DH5alpha strain from -80℃ freezer and leave at rest on ice.<br>
 +
4. Add 3 µl of each plasmids in a 1.5 ml tube.<br>
 +
5. Put 25 µl competent cell into each 1.5 ml tubes with plasmid.<br>
 +
6. Incubate on ice for 15 min.<br>
 +
7. Put tubes with DNA and competent cells into water bath at 42℃ for 30 seconds.<br>
 +
8. Put tubes back on ice for 2 minutes.<br>
 +
9. Add 125 µl of SOC medium. Incubate tubes for 30 minutes at 37℃.<br>
 +
10. Make a 1:5 dilution in 150 µl of fresh SOC medium.<br>
 +
11. Spread about 100 µl of the resulting culture of LB plate containing kanamycin. <br>
 +
12. Incubate LB plate for 14-15 hours at 37℃.<br>
 +
13. Set the plate reader to measure GFP.<br>
 +
14. Scan the each plates with the plate reader. (We used FujiFilm FLA-5100 Fluorescent Image Analyzer from FUJIFilm Life Science.)<br>
 +
15. Analyze the scanning data by changing the scale type (Bezier) and adjusting the range. (We analyzed by using the software, Multi Gauge ver. 2.0 from FUJIFilm Life Science.)<br>
 +
16. Counted out the all colonies and those with fluorescence.<br>
 +
17. Prepare three overnight cultures for each sample in 3 mL of LB medium containing kanamycin (30 microg / mL) shaking at 180 rpm for 12h.<br>
 +
18. Miniprep each samples and ask DNA sequencing of each samples for Biomaterial Analysis Center, Technical Department.<br>
 +
 +
======3.Results======
 +
[[Image:Tokyo_Tech_FLA_FImE_.png |thumb|center|700px|<b>Fig. 7. </b>Determination of percemtage of [ON] state and colony formation using plasmid mixture extracted cell expressing FimE]]<br>
 +
[[Image:Tokyo_Tech_sequence_FimE.png |thumb|center|700px|<b>Fig. 8. </b>DNA sequencing results of <i>fim</i> switch(wild-type)]]<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===
  
PCR after the invertion experiment of BBa_K1632004
+
Composite of BBa_K1632004, BBa_I13500
  
 
===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
+
[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 <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
+
[3]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
+
[4]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 03:56, 19 September 2015

fim switch[default OFF](wild-type)


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 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](wild-type)_gfp (pSB3K3)
(2) PBAD/araC_fimB(wild-type) (pSB6A1) + fim switch[default OFF](wild-type)_gfp (pSB3K3)
(3) pSB6A1 + fim switch[default ON](wild-type)_gfp (pSB3K3) …positive control 1
(4) pSB6A1 + fim switch[default OFF](wild-type)_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

Fig. 1. Plasmids

Flow cytometer
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 concentration 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 (3) and (4), 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.)

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

Supplemental experiments
2.Assay protocol

1. After the assay of “Arabinose dependent FimE expression”, miniprep cell culture ((1)-①, (1)-③, (2)-① and (2)-③) of leftover as here.(https://parts.igem.org/Help:Protocols/Miniprep)
2. Turn on water bath to 42℃.
3. Take competent DH5alpha strain from -80℃ freezer and leave at rest on ice.
4. Add 3 µl of each plasmids in a 1.5 ml tube.
5. Put 25 µl competent cell into each 1.5 ml tubes with plasmid.
6. Incubate on ice for 15 min.
7. Put tubes with DNA and competent cells into water bath at 42℃ for 30 seconds.
8. Put tubes back on ice for 2 minutes.
9. Add 125 µl of SOC medium. Incubate tubes for 30 minutes at 37℃.
10. Make a 1:5 dilution in 150 µl of fresh SOC medium.
11. Spread about 100 µl of the resulting culture of LB plate containing kanamycin.
12. Incubate LB plate for 14-15 hours at 37℃.
13. Set the plate reader to measure GFP.
14. Scan the each plates with the plate reader. (We used FujiFilm FLA-5100 Fluorescent Image Analyzer from FUJIFilm Life Science.)
15. Analyze the scanning data by changing the scale type (Bezier) and adjusting the range. (We analyzed by using the software, Multi Gauge ver. 2.0 from FUJIFilm Life Science.)
16. Counted out the all colonies and those with fluorescence.
17. Prepare three overnight cultures for each sample in 3 mL of LB medium containing kanamycin (30 microg / mL) shaking at 180 rpm for 12h.
18. Miniprep each samples and ask DNA sequencing of each samples for Biomaterial Analysis Center, Technical Department.

3.Results
Fig. 3. Determination of percemtage of [ON] state and colony formation using plasmid mixture extracted cell expressing FimB.

Fig. 4. DNA sequencing results of fim switch(wild-type)

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

Fig. 5. Plasmids

Flow cytometer
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. 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 Ka, 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.)

3.Results
Fig. 6. The histograms of the samples measured by flow cytometer

Supplemental experiments
2.Assay protocol

1. After the assay of “Arabinose dependent FimE expression”, miniprep cell culture ((1)-①, (1)-③, (2)-① and (2)-③) of leftover as here.
(https://parts.igem.org/Help:Protocols/Miniprep) 2. Turn on water bath to 42℃.
3. Take competent DH5alpha strain from -80℃ freezer and leave at rest on ice.
4. Add 3 µl of each plasmids in a 1.5 ml tube.
5. Put 25 µl competent cell into each 1.5 ml tubes with plasmid.
6. Incubate on ice for 15 min.
7. Put tubes with DNA and competent cells into water bath at 42℃ for 30 seconds.
8. Put tubes back on ice for 2 minutes.
9. Add 125 µl of SOC medium. Incubate tubes for 30 minutes at 37℃.
10. Make a 1:5 dilution in 150 µl of fresh SOC medium.
11. Spread about 100 µl of the resulting culture of LB plate containing kanamycin.
12. Incubate LB plate for 14-15 hours at 37℃.
13. Set the plate reader to measure GFP.
14. Scan the each plates with the plate reader. (We used FujiFilm FLA-5100 Fluorescent Image Analyzer from FUJIFilm Life Science.)
15. Analyze the scanning data by changing the scale type (Bezier) and adjusting the range. (We analyzed by using the software, Multi Gauge ver. 2.0 from FUJIFilm Life Science.)
16. Counted out the all colonies and those with fluorescence.
17. Prepare three overnight cultures for each sample in 3 mL of LB medium containing kanamycin (30 microg / mL) shaking at 180 rpm for 12h.
18. Miniprep each samples and ask DNA sequencing of each samples for Biomaterial Analysis Center, Technical Department.

3.Results
Fig. 7. Determination of percemtage of [ON] state and colony formation using plasmid mixture extracted cell expressing FimE

Fig. 8. DNA sequencing results of fim switch(wild-type)

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

Composite of BBa_K1632004, 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