Difference between revisions of "Team:Tokyo Tech/Project/FimB dependent fim switch state assay"

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      <h1>FimB dependent <i>fim</i> switch state_assay</h1>
 
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    <h4 class="subtitle"><strong>We have characterized previous parts.</strong></h4>
 
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    <h2 class="content2">contents</h2>
 
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      <h3 class="link"><a href="#Introduction">1. Introduction</a></h3>
 
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      <h3 class="link"><a href="#Summary">2. Summary of the Experiment</a></h3>
 
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      <h3 class="link"><a href="#Results">3. Results</a></h3>
 
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      <h3 class="link2"><a href="#Result1">3.1. Arabinose dependent FimE expression</a></h3>
 
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      <h3 class="link2"><a href="#Result2">3.2. FLA analysis</a></h3>               
 
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      <h3 class="link"><a href="#Materials">4. Materials and Methods</a></h3>
 
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      <h3 class="link2"><a href="#Const">4.1.  Construction</a></h3>
 
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      <h3 class="link2"><a href="#Protocol">4.2. Assay Protocol</a></h3>
 
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        <h3 class="link3"><a href="#Protol1">4.2.1 Arabinose dependent FimE expression</a></h3>
 
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        <h3 class="link3"><a href="#Protol2">4.2.2. FLA analysis</a></h3>
 
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      <h3 class="link"><a href="#Reference">6. Reference</a></h3>
 
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          <h2 id="Introduction" class="smalltitle">1. Introduction</h2>
 
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　　　　　　<p class="text">To confirm the function of fim switch in the presence of FimE, we constructed two new genetic circuit parts, BBa_K1632013 and BBa_K1632002 (Fig. 3-5-1). BBa_K1632013 enables arabinose-inducible expression of wild type FimE. In BBa_K1632013 and BBa_K1632002, either [ON] or [OFF] fim switch is placed upstream of GFP coding sequence. </p>
 
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      <td width="940px"><div align="center"><img src="https://static.igem.org/mediawiki/2015/8/8a/Tokyo_Tech_arabinosefimEsummary.png" />
 
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      <h4 align="center" class="fig"><strong>Fig.3-7-2-1.</strong>&nbsp; New plasmids we constructed to confirm the function of <i>fim</i> switch</h4>
 
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          <h2 id="Summary" class="smalltitle">2. Summary of the Experiment</h2>
 
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　　　　　　<p class="text">Our purpose is to confirm that FimE inverts fimswitch only from ON to OFF. Taking endogenous FimB and FimE into account, we prepared six plasmids sets shown below. We measured the fluorescence intensity by GFP expression when we added arabinose.　また、我々はFimSが本当に反転しているかどうかを確認するために、FLAを使った解析とシークエンスデータの解析を行いました。</p>
 
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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="800px" />
 
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      <h4 align="center" class="fig"><strong>Fig.3-7-2-1.</strong>&nbsp;Plasmids for the experiment of FimE dependent fim switch state assay</h4>
 
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          <h2 id="Results" class="smalltitle">3. Results</h2>
 
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              <h3 id="Result1" class="sub5">3.1. Arabinose dependent FimE expression</h3>
 
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　　　　　　    <p class="text2">
 
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Fig.3-5-3-1 はフローサイトメーターで測定した各サンプルのヒストグラムである。レポーターセルCとDの結果から、内在性のFImBとFimEは無視できることがわかる。また、レポーターセルE,Fの結果から、FImEの発現はヒストグラムの波形にほとんど影響を与えないことがわかる。
 
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　レポーターセルAの結果は、FimEの発現量が増加すると、蛍光強度が減少することを示している。このことより、我々はFimSがFimEによってOn状態からOff状態に切り替わっていることを確信した。また、レポーターセルBの結果はFimEの発現量が増加してもレポーターセルBのGFP発現量は変化しないことを示している。これより、fimSはFimEによってOff状態からOn状態に切り替わらないことを確信した。以上二つのレポーターセルA,Bの結果より、FimEはFimSをinverts fimswitch only from ON to OFF していることを確信した。
 
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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="900px"/>
 
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      <h4 align="center" class="fig"><strong>Fig. 3-5-3-1.</strong></h4>
 
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              <h3 id="Result2" class="sub5">3.2. FLA analysis</h3>
 
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　　　　　　    <p class="text2">写真とシークエンスデータ</p>
 
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          <h2 id="Discussion" class="smalltitle">4. Discussion</h2>
 
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　　　　　　    <p class="text">When FimB concentration increased by increasing arabinose concentration, we confirmed that Fluorescence intensity was decreased in both of ON to OFF and OFF to ON.<br>
 
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&nbsp;&nbsp;According to [1], increasing switching frequency by increasing FimB expression decrease mean expression because it is enough time for FimB to bind to the inversion sequences and disrupt transcription initiation or elongation.<br>
 
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&nbsp;&nbsp;Similar increase dependent on FimB expression was found in control samples(図).  Because FimE expression decreases cell growth rate, decreased dilution rate of proteins including GFP from leaky expression in the cells could slightly increase of fluorescence in a cell.
 
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          <h2 id="Materials" class="smalltitle">5. Materials and Methods</h2>
 
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              <h3 id="Const" class="sub5">5.1.  Construction</h3>
 
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              <h3 class="sub5">-Strain</h3>
 
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　　　　　　    <p class="text2">All the samples were DH5alpha strain.</p>
 
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              <h3 class="sub5">-Plasmids</h3>
 
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　　　　　　    <p class="text2">A. Pbad/araC_<i>fimB</i>(wild-type) (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/e/e1/Tokyo_Tech_PBAD_fimB_fimswitchon_gfp.png"/>
 
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      <h4 align="center" class="fig"><strong>Fig. 3-4-5-1.</strong></h4>
 
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　　　　　　    <p class="text2">B. Pbad/araC_<i>fimB</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/2015/4/47/Tokyo_Tech_PBAD_fimB_fimswitchoff_gfp.png"/>
 
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      <h4 align="center" class="fig"><strong>Fig. 3-4-5-2.</strong></h4>
 
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　　　　　　    <p class="text2">C. promoter less M256ICysE(pSB6A1)+ <i>fim</i> switch[default ON](wild-type)_<i>gfp</i>(pSB3K3)…Positive control 1</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. 3-4-5-3.</strong></h4>
 
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　　　　　　    <p class="text2">D. promoter less M256ICysE(pSB6A1)+ <i>fim</i> switch[default OFF](wild-type)_<i>gfp</i>(pSB3K3)…Negative control 1</p>
 
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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. 3-4-5-4.</strong></h4>
 
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　　　　　　    <p class="text2">E. Pbad/araC-<i>fimB</i> (pSB6A1) +J23119 promoter_<i>gfp</i> (pSB3K3)…Positive control2</p>
 
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                  <td width="980px"><div align="center"><img src="https://static.igem.org/mediawiki/2015/7/79/Tokyo_Tech_PBAD_fimB_J23119_gfp.png"/>
 
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      <h4 align="center" class="fig"><strong>Fig. 3-4-5-5.</strong></h4>
 
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　　　　　　    <p class="text2">F. Pbad/araC-<i>fimB</i> (pSB6A1) +promoter less <i>gfp</i> (pSB3K3)…Negative control2</p>
 
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                  <td width="980px"><div align="center"><img src="https://static.igem.org/mediawiki/2015/4/42/Tokyo_Tech_PBAD_fimB_gfp.png"/>
 
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      <h4 align="center" class="fig"><strong>Fig. 3-4-5-6.</strong></h4>
 
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              <h3 id="Protocol" class="sub5">5.2. Assay Protocol</h3>
 
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              <h3 id="Protol1" class="sub6">5.2.1. Arabinose dependent FimE expression</h3>
 
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                <p class="text2"></p>
 
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                    <p class="text4">
 
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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 0.5 percent) at 37 ℃ for 12h.<br>
 
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2. Make a 1:100 dilution in 3 mL of fresh LB containing Amp, Kan and glucose (final concentration of mass of glucose is 0.5 percent).<br>
 
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3. Grow the cells at 37 ℃ until the observed OD590 reaches 0.4 (Fresh Culture)<br>
 
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4. After incubation, take 1 mL of the samples, and centrifuge at 5000x g, 1 min, 25 ℃.<br>
 
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5. Remove the supernatant by using P1000 pipette.<br>
 
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6. Suspend the pellet in 1 mL of LB containing Amp and Kan, and centrifuge at 5000x g, 1 min, 25 ℃ <br>
 
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7. Remove the supernatant by using P1000 pipette.<br>
 
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8. Take the samples, and centrifuge at 5000x g, 1 min, 25 ℃.<br>
 
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9. Remove the supernatant by using P1000 pipette.<br>
 
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10. Add 1 mL of LB containing Amp and Kan, and suspend.<br>
 
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11. Add 30 microL of suspension in the following medium.<br>
 
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&nbsp;&nbsp;&nbsp;① 3 mL of LB containing Amp, Kan, glucose (final concentration of mass of glucose is 0.5 percent) and 30 microL of sterile water.<br>
 
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&nbsp;&nbsp;&nbsp;② 3 mL of LB containing Amp, Kan and 30 microL of 2 mM　arabinose (final concentration of arabinose is 20 microM)<br>
 
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&nbsp;&nbsp;&nbsp;③ 3 mL of LB containing Amp, Kan and 30 microL of 20 mM arabinose (final concentration of arabinose is 200 microM)<br>
 
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&nbsp;&nbsp;&nbsp;④ 3 mL of LB containing Amp, Kan and 30 microL of 500 mM arabinose (final concentration of arabinose is 5 mM)<br>
 
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&nbsp;&nbsp;&nbsp;※ As for C and D, the suspension were added only in medium ① and ④. <br>
 
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12. Grow the samples at 37 ℃ for 6.5 hours.<br>
 
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13. Measure OD590 of all the samples every hour.<br>
 
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14. Start preparing the flow cytometer 1 h before the end of incubation.<br>
 
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15. After the incubation, take the samples, and centrifuge at 9000x g, 1min, 4℃.<br>
 
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16. Remove the supernatant by using P1000 pipette.<br>
 
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17. Add 1 mL of filtered PBS (phosphate-buffered saline) and suspend. (The ideal of OD is 0.3)<br>
 
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18. Dispense all of each suspension into a disposable tube through a cell strainer.<br>
 
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19. Use flow cytometer to measure the fluorescence of GFP. (We used BD FACSCaliburTM Flow Cytometer of Becton, Dickenson and Company.)<br>
 
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              <h3 id="Protol2" class="sub6">5.2.2. FLA analysis</h3>
 
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                <p class="text2"></p>
 
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1. コピペ。<br>
 
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2. コピペ。<br>
 
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3. コピペ。<br>
 
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4. コピペ。<br>
 
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5. コピペ。<br>
 
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6. コピペ。<br>
 
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7. コピペ。<br>
 
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8. コピペ。<br>
 
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9. コピペ。<br></p>
 
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          <h2 id="Reference" class="smalltitle">6. Reference</h2>
 
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　　　　　　<p class="text">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</p>
 
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　　　　　　<p class="text">2. 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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Latest revision as of 17:39, 16 September 2015