View source for Team:Tokyo Tech/Experiment/FimE dependent fim switch state assay

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      <h1>FimE dependent <i>fim</i> switch state assay</h1>
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     <h2 class="content2">contents</h2>
      <h3 class="link"><a href="#Introduction">1. Introduction</a></h3>
      <h3 class="link"><a href="#Summary">2. Summary of the Experiment</a></h3>
      <h3 class="link"><a href="#Results">3. Results</a></h3>
       <h3 class="link2"><a href="#Result1">3.1. Arabinose-dependent FimE (wild-type) expression</a></h3>
       <h3 class="link2"><a href="#Result2">3.2. FLA analysis</a></h3>
      <h3 class="link"><a href="#Discussion">4. Discussion</a></h3>               
      <h3 class="link"><a href="#Materials">5. Materials and Methods</a></h3>
       <h3 class="link2"><a href="#Const">5.1.  Construction</a></h3>
       <h3 class="link2"><a href="#Protocol">5.2. Assay Protocol</a></h3>
        <h3 class="link3"><a href="#Protol1">5.2.1 Arabinose dependent FimE expression</a></h3>
        <h3 class="link3"><a href="#Protol2">5.2.2. FLA analysis</a></h3>
      <h3 class="link"><a href="#Reference">6. Reference</a></h3>
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          <h2 id="Introduction" class="smalltitle">1. Introduction</h2>
　　　　　　<p class="text">To confirm the function of <i>fim</i> switch in the presence of FimE(wild-type), we constructed two Biobrick parts, <a href="http://parts.igem.org/Part:BBa_K1632013">BBa_K1632013</a>　(Fig. 3-5-1-1). <a href="http://parts.igem.org/Part:BBa_K1632013">BBa_K1632013</a> enables arabinose-inducible expression of the FimE (wild-type).  In <a href="http://parts.igem.org/Part:BBa_K1632007">BBa_K1632007</a> and <a href="http://parts.igem.org/Part:BBa_K1632008">BBa_K1632008</a>, either the <i>fim</i> switch [default ON] or the <i>fim</i> switch [default OFF] is placed upstream of the GFP coding sequence. </p> 
　　　　　　    <table width="940 px" border="0px">
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      <td width="940px"><div align="center"><img src="https://static.igem.org/mediawiki/2015/8/8a/Tokyo_Tech_arabinosefimEsummary.png" width="450px"/>
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      <h4 align="center" class="fig"><strong>Fig.3-5-1-1.</strong>&nbsp;New plasmids we constructed to confirm the function of fim switch</h4>
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          <h2 id="Summary" class="smalltitle">2. Summary of the Experiment</h2>
　　　　　　<p class="text"> Our purpose is to confirm that FimE (wild-type) inverts the <i>fim</i> switch (wild-type) from ON to OFF and from OFF to ON (Fig.3-5-2-1). We prepared six plasmids below (Fig.3-5-2-2). We measured the fluorescence intensity from the GFP expression in the presence of arabinose. From the results, we confirmed that our <i>fim</i> switch (wild-type) is inverted from ON to OFF and OFF to ON. From the results we also confirmed our <i>fim</i> switch (wild-type) is not inverted by the endogenous FimB and FimE and that FimE(wild-type) expression doesn’t have effect on the GFP expression.In order to confirm inversion more precisely, we also show the inversion ration of ON and inversion in the level of DNA sequencing </p>
<p class="text4">
(1) P<sub>BAD/<i>araC</i></sub>_<i>fimE</i>(wild-type) (pSB6A1) + <i> fim</i> switch[default ON](wild-type)_<i>gfp</i> (pSB3K3)<br>
(2) P<sub>BAD/<i>araC</i></sub>_<i>fimE</i>(wild-type) (pSB6A1) + <i> fim</i> switch[default OFF](wild-type) _<i>gfp</i> (pSB3K3)<br>
(3) Positive control 1 : (pSB6A1) + <i>fim</i> switch[default ON](wild-type) _<i>gfp</i> (pSB3K3)<br>
(4) Negative control 1 : (pSB6A1) + <i>fim</i> switch[default OFF](wild-type) _<i>gfp</i> (pSB3K3)<br>	
(5) Positive control 2 : P<sub>BAD/<i>araC</i></sub>_<i>fimE</i>(wild-type) (pSB6A1) + Pcon_<i>gfp</i> (pSB3K3)<br>
(6) Negative control 2 : P<sub>BAD/<i>araC</i></sub>_<i>fimE</i>(wild-type) (pSB6A1) + promoter less_<i>gfp</i> (pSB3K3)<br>

    <table width="940 px" border="0px">
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      <td width="940px"><div align="center"><img src="https://static.igem.org/mediawiki/2015/0/06/Tokyo_Tech_arabinosefimE.png" width="700px" />
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      <h4 align="center" class="fig"><strong>Fig.3-5-2-1.</strong>&nbsp;Plasmids for the experiment of FimE dependent <i>fim</i> switch state assay</h4>
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          <h2 id="Results" class="smalltitle">3. Results</h2>
              <h3 id="Result1" class="sub5">3.1. Arabinose dependent FimE(wild-type) expression</h3>
　　　　　　    <p class="text2">We tried to confirm that <i>fim</i> switch is unidirectionally inverted in the presence of FimE (wild-type) by using GFP as a reporter, under 4 different concentrations of arabinose. In the medium with 0 M arabinose, we supplemented the medium with 1.0 % glucose in order to repress the leakage in the P<sub>BAD/<i>araC</i></sub> promoter. Fig. 3-5-3-1 shows the histograms of the samples measured by the flow cytometer. In the results of the reporter cell (1), when the Induction of FimE(wild-type) expression increases, the fluorescence intensity decreases. From this fact, we confirmed that the <i>fim</i> switch (wild-type) is inverted from ON to OFF by FimE (wild-type). From the result of the reporter cell (2), even when the expression amount of FimE (wild-type) increases, the expression amount of GFP in the reporter cell (2) does not change. From this fact, we confirmed that the <i>fim</i> switch (wild-type) is inverted only from ON to OFF by FimE (wild-type). From the results of the two reporter cells (1) and (2), we successfully confirmed that FimE (wild-type) inverts the <i>fim</i> switch only from ON to OFF.<br>&nbsp;&nbsp; 
　The results of positive control 1 and negative control 1 confirmed that the endogenous FimB and FimE did not invert our fim switch (wild-type). Also, the result of positive control 2 and negative control 2, indicates that the expression of FimE (wild-type) do not have effects on the GFP expression.
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                 <table width="940 px" border="0px">
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                   <td width="940px"><div align="center"><img src="https://static.igem.org/mediawiki/2015/c/cc/Tokyo_Tech_arabinose_fimE_result1.png" width="800px"/>
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      <h4 align="center" class="fig"><strong>Fig. 3-5-3-1.</strong> The histogram of the samples measured by flow cytometer</h4>
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              <h3 id="Result2" class="sub5">3.2. FLA analysis</h3>
　　　　　　    <p class="text2">写真とシークエンスデータ</p>
          <h2 id="Discussion" class="smalltitle">4. Discussion</h2>





























































          <h2 id="Materials" class="smalltitle">5. Materials and Methods</h2>
              <h3 id="Const" class="sub5">5.1.  Construction</h3>
               <h3 class="sub5">-Strain</h3>

　　　　　　    <p class="text2">All the samples were DH5alpha strain.</p>
              <h3 class="sub5">-Plasmids</h3>
　　　　　　    <p class="text2">(1) P<sub>BAD/<i>araC</i></sub>_<i>fimE</i>(wild-type) (pSB6A1)+ <i>fim</i> switch[default ON](wild-type)_<i>gfp</i> (pSB3K3)</p>
                 <table width="980 px" border="0px">
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                   <td width="980px"><div align="center"><img src="https://static.igem.org/mediawiki/2015/0/0f/Tokyo_Tech_PBAD_fimE_fimswitchon_gfp.png"/>
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      <h4 align="center" class="fig"><strong>Fig. 3-5-4-1.</strong></h4>
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　　　　　　    <p class="text2">(2) P<sub>BAD/<i>araC</i></sub>_<i>fimE</i>(wild-type) (pSB6A1)+ <i>fim</i> switch[default OFF](wild-type)_<i>gfp</i> (pSB3K3)
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                   <td width="980px"><div align="center"><img src="https://static.igem.org/mediawiki/2015/a/a2/Tokyo_Tech_PBAD_fimE_fimswitchoff_gfp.png"/>
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      <h4 align="center" class="fig"><strong>Fig. 3-5-4-2.</strong></h4>
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　　　　　　    <p class="text2">(3) Positive control 1 : (pSB6A1) + <i>fim</i> switch[default ON](wild-type) _<i>gfp</i> (pSB3K3)</p>
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                   <td width="980px"><div align="center"><img src="https://static.igem.org/mediawiki/2015/0/07/Tokyo_Tech_cysE_fimswitchon.png"/>
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      <h4 align="center" class="fig"><strong>Fig. 3-5-4-3.</strong></h4>
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　　　　　　    <p class="text2">(4) Negative control 1 : (pSB6A1) + <i>fim</i> switch[default OFF](wild-type) _<i>gfp</i> (pSB3K3)</p>
                 <table width="980 px" border="0px">
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                   <td width="980px"><div align="center"><img src="https://static.igem.org/mediawiki/2015/8/89/Tokyo_Tech_cysE_fimswitchoff.png"/>
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      <h4 align="center" class="fig"><strong>Fig. 3-5-4-4.</strong></h4>
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　　　　　　    <p class="text2">(5) Positive control 2 : P<sub>BAD/<i>araC</i></sub>_<i>fimE</i> (wild-type) (pSB6A1) + Pcon_<i>gfp</i> (pSB3K3)</p>
                 <table width="980 px" border="0px">
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                   <td width="980px"><div align="center"><img src="https://static.igem.org/mediawiki/2015/c/c5/Tokyo_Tech_PBAD_fimE_J23119_gfp.png"/>
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      <h4 align="center" class="fig"><strong>Fig. 3-5-4-5.</strong></h4>
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　　　　　　    <p class="text2">(6) Negative control 2 : P<sub>BAD/<i>araC</i></sub>_<i>fimE</i> (wild-type) (pSB6A1) + promoter less_<i>gfp</i> (pSB3K3)</p>
                 <table width="980 px" border="0px">
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                   <td width="980px"><div align="center"><img src="https://static.igem.org/mediawiki/2015/a/a4/Tokyo_Tech_PBAD_fimE_gfp.png"/>
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      <h4 align="center" class="fig"><strong>Fig. 3-5-4-6.</strong></h4>
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              <h3 id="Protocol" class="sub5">4.2. Assay Protocol</h3>
               <h3 id="Protol1" class="sub6">4.2.1. Arabinose dependent FimE expression</h3>
                 <p class="text2"></p>
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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 is 1.0 percent) 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>
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>
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>
&nbsp;&nbsp;&nbsp;① 3 mL of LB containing Amp, Kan, glucose (final concentration of glucose is 1.0 %) and 30 microL sterile water<br>
&nbsp;&nbsp;&nbsp;② 3 mL of LB containing Amp, Kan and 30 microL of  500μM arabinose (final concentration of arabinose is 5 microM)<br>
&nbsp;&nbsp;&nbsp;③ 3 mL of LB containing Amp, Kan and 30 microL of 1 mM arabinose (final concentration of arabinose is 10 microM)<br>
&nbsp;&nbsp;&nbsp;④3 mL of LB containing Amp, Kan and 30 microL of 2 mM arabinose (final concentration of arabinose is 20 microM)<br>
&nbsp;&nbsp;&nbsp;※ As for C and D, 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.)<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>

               <h3 id="Protol2" class="sub6">4.2.2. FLA analysis</h3>
                 <p class="text2"></p>
                   <p class="text3"></p>
                    <p class="text4">
1. After the assay of “Arabinose dependent FimB expression”, miniprep cell culture (A,B,
,C and D) of leftover as here.(http://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 150µl 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></p>
                   
          <h2 id="Reference" class="smalltitle">5. Reference</h2>
　　　　　　<p class="text">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<p><br><br><br>
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