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- | <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true"
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- | Name="Table Theme"/>
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| |
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| |
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| |
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| |
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| |
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| |
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| |
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| |
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| |
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| |
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| |
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| |
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| |
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| |
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| |
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| |
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| |
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| |
- |
| |
- | <p class=MsoNormal><b><u><span lang=EN-US style='color:red;mso-ansi-language:
| |
- | EN-US'>Spectrophotometric Absorption Measurements: </span></u></b><span
| |
- | lang=EN-US style='mso-ansi-language:EN-US'> We used </span><b>Thermo Sc</b><b><span
| |
- | lang=EN-US style='mso-ansi-language:EN-US'>ientific Varioskan Flash Multimode
| |
- | Reader for our absorption measurements.</span></b></p>
| |
- |
| |
- | <p class=MsoNormal><span lang=EN-US style='mso-ansi-language:EN-US'>1)
| |
- | Measurements of Dyes:</span></p>
| |
- |
| |
- | <p class=MsoNormal><span style='mso-no-proof:yes'><img width=604 height=440
| |
- | id="_x0000_i1031" src="https://static.igem.org/mediawiki/2014hs/8/88/Fimage001.jpg"
| |
- | alt="https://static.igem.org/mediawiki/2014hs/8/88/Fimage001.jpg"></span></p>
| |
- |
| |
- | <p class=MsoNormal><span lang=EN-US style='mso-ansi-language:EN-US'>Graphic 1:
| |
- | This graphic shows our absorption rate of our dye (Methylene Blue) in different
| |
- | concentrations. ( 1X=8 uM, 10X=80uM, 100X=800uM, 1000X=uM) </span></p>
| |
- |
| |
- | <p class=MsoNormal><span lang=EN-US style='mso-ansi-language:EN-US'>In the
| |
- | Graphic 1, we have got the correct absorption rates for every concentration of
| |
- | Methylene Blue dyes that correlates with the same results in the literature.</span></p>
| |
- |
| |
- | <p class=MsoNormal><span lang=EN-US style='mso-ansi-language:EN-US'>2)
| |
- | Preparations of Lysates : <o:p></o:p></span></p>
| |
- |
| |
- | <p class=MsoNormal><span style='font-size:11.5pt;line-height:115%;font-family:
| |
- | "Arial","sans-serif";color:black'>The verified plasmids were then cultivated in
| |
- | liquid culture for isolation. The 50 mL’s of bacteria containing the parts
| |
- | BBa_K1291071 and BBa_K1291299 were centrifuged at 4100 rpm for 5 minutes, the
| |
- | supernatant was discarded and then lysis solution was added. After 10 minutes
| |
- | of incubation in ice, the lysates were sonicated in the minimum activity level.
| |
- | After a spin of 15000 rpm for 10 minutes, the supernatant was transferred to
| |
- | eppendorphs and dye was added; the solution was boiled at 95</span></span><span
| |
- | style='font-size:11.5pt;line-height:115%;font-family:"Arial","sans-serif";
| |
- | color:#545454; '><span style='orphans: auto;text-align:start;
| |
- | widows: auto;-webkit-text-stroke-width: 0px;white-space:pre-wrap;word-spacing:
| |
- | 0px'> °C for 5 minutes. The lysate were stored at -20 °C.</span></span></p>
| |
- |
| |
- | <p class=MsoNormal><span lang=EN-US style='mso-ansi-language:EN-US'>3)
| |
- | Decolorization Assay:</span></p>
| |
- |
| |
- | <p class=MsoNormal><span lang=EN-US style='mso-ansi-language:EN-US'>In this
| |
- | assay we mixed our liquid cell culture and also our lysates with dyes. All the
| |
- | concentrations of the solutions are adjusted according to the literature. We
| |
- | measured the first absorptions of the samples and compared it with the 2<sup>nd</sup>
| |
- | measurement in the end of 1<sup>st</sup> hour.</span></p>
| |
- |
| |
- | <p class=MsoNormal><span style='mso-no-proof:yes'><img width=604 height=300
| |
- | id="_x0000_i1030" src="https://static.igem.org/mediawiki/2014hs/f/fb/Fimage002.jpg"
| |
- | alt="https://static.igem.org/mediawiki/2014hs/f/fb/Fimage002.jpg"></span> </p>
| |
- |
| |
- | <p class=MsoNormal><span lang=EN-US style='mso-ansi-language:EN-US'>Graphic 2:
| |
- | This graphic shows our absorption rate of our mixed sample that includes
| |
- | Lysates of our BL-21 Bacteria that have LiP gene and Methylene Blue
| |
- | (80uM) dyes and for the activate the LiP we also added Hydrogen Peroxide
| |
- | (800uM) measured in the 1 hour.</span></p>
| |
- |
| |
- | <p class=MsoNormal><span lang=EN-US style='mso-ansi-language:EN-US'>The result
| |
- | of Graphic-2 is that our Lysates of BL-21 Bacteria that have LiP gene degraded
| |
- | the Methylene Blue (80uM) dye with approximately %83,3 efficiency.</span></p>
| |
- |
| |
- | <p class=MsoNormal><span lang=EN-US style='mso-ansi-language:EN-US'> </span></p>
| |
- |
| |
- | <p class=MsoNormal><span style='mso-no-proof:yes'><img width=605 height=516
| |
- | id="_x0000_i1029" src="https://static.igem.org/mediawiki/2014hs/7/7d/Fimage003.jpg"
| |
- | alt="https://static.igem.org/mediawiki/2014hs/7/7d/Fimage003.jpg"></span><span
| |
- | lang=EN-US style='mso-ansi-language:EN-US'>Graphic 3: This graphic shows our
| |
- | absorption rate of our mixed samples that includes Lysates of our BL-21
| |
- | Bacteria that have HRP gene and other Bacteria that have LiP gene and Methyl
| |
- | Green (150uM) dyes and for the activate the HRP and LiP we also added Hydrogen
| |
- | Peroxide (8.8 uM-HRP), (0,176 uM-LiP) concentrations that measured in the
| |
- | different time periods. </span></p>
| |
- |
| |
- | <p class=MsoNormal><u><span lang=EN-US style='color:red;mso-ansi-language:EN-US'>The
| |
- | result of Graphic-3 is that our Lysates of BL-21 Bacteria that have HRP gene
| |
- | and other Bacteria that have LiP gene degraded the Methyl Green (8.8 uM) dye
| |
- | with approximately %71,4 efficiency in the end of the 7 hours.</span></u></p>
| |
- |
| |
- | <p class=MsoNormal><span lang=EN-US style='mso-ansi-language:EN-US'> </span></p>
| |
- |
| |
- | <p class=MsoNormal><span lang=EN-US style='mso-ansi-language:EN-US'> </span></p>
| |
- |
| |
- | <p class=MsoNormal><span style='mso-no-proof:yes'><img width=475 height=304
| |
- | id="_x0000_i1028" src="https://static.igem.org/mediawiki/2014hs/e/ed/Fimage004.png"
| |
- | alt="https://static.igem.org/mediawiki/2014hs/e/ed/Fimage004.png"></span><span
| |
- | lang=EN-US style='mso-ansi-language:EN-US'>.</span></p>
| |
- |
| |
- | <p class=MsoNormal><span lang=EN-US style='mso-ansi-language:EN-US'>Graphic 4:
| |
- | This graphic shows our absorption rate of our mixed samples that includes
| |
- | Lysates of our BL-21 Bacteria that have HRP gene and other Bacteria that have
| |
- | LiP gene and Methyl Green (150uM) dyes and for the activate the HRP and LiP we
| |
- | also added Hydrogen Peroxide (8.8 uM-HRP), (0,176 uM-LiP) concentrations that
| |
- | measured in the beginning of the experiment and in the 1<sup>st</sup> hour. </span></p>
| |
- |
| |
- | <p class=MsoNormal><span lang=EN-US style='mso-ansi-language:EN-US'>The result
| |
- | of Graphic-4 is that our Lysates of BL-21 Bacteria that have HRP gene and other
| |
- | Bacteria that have LiP gene degraded the Methyl Green (8.8 uM) dye with
| |
- | approximately %71,4 efficiency. <u><span style='color:red'>Also in this graphic
| |
- | the most important point that our control group BL-21 bacteria that don’t
| |
- | include our genes of enzymes(HRP or LiP) couldn’t degrade the Methyl Green (8.8
| |
- | uM) dye.</span></u></span></p>
| |
- |
| |
- | <p class=MsoNormal><span lang=EN-US style='mso-ansi-language:EN-US'> </span></p>
| |
- |
| |
- | <p class=MsoNormal><span style='mso-no-proof:yes'><img width=610 height=320
| |
- | id="_x0000_i1027" src="https://static.igem.org/mediawiki/2014hs/a/a9/Fimage005.jpg"
| |
- | alt="https://static.igem.org/mediawiki/2014hs/a/a9/Fimage005.jpg"></span></p>
| |
- |
| |
- | <p class=MsoNormal><span lang=EN-US style='mso-ansi-language:EN-US'>Graphic 5:
| |
- | This graphic shows our absorption rate of our mixed sample that includes liquid
| |
- | culture of our BL-21 Bacteria that have LiP gene and Methylene Blue
| |
- | (80uM) dyes and for the activate the LiP we also added Hydrogen Peroxide
| |
- | (800uM) measured in the 1 hour.</span></p>
| |
- |
| |
- | <p class=MsoNormal><span lang=EN-US style='mso-ansi-language:EN-US'>The result
| |
- | of Graphic-5 is that our Liquid cultures of BL-21 Bacteria that have LiP gene
| |
- | also degraded the Methylene Blue (80uM) dye with approximately %70 efficiency. </span></p>
| |
- |
| |
- | <p class=MsoNormal><span lang=EN-US style='mso-ansi-language:EN-US'> </span></p>
| |
- |
| |
- | <p class=MsoNormal><span lang=EN-US style='mso-ansi-language:EN-US'> </span></p>
| |
- |
| |
- | <p class=MsoNormal><span lang=EN-US style='mso-ansi-language:EN-US'> </span></p>
| |
- |
| |
- | <p class=MsoNormal><span lang=EN-US style='mso-ansi-language:EN-US'> </span></p>
| |
- |
| |
- | <p class=MsoNormal><span lang=EN-US style='mso-ansi-language:EN-US'> </span></p>
| |
- |
| |
- | <p class=MsoNormal><span lang=EN-US style='mso-ansi-language:EN-US'> </span></p>
| |
- |
| |
- | <p class=MsoNormal><span style='mso-no-proof:yes'><img width=603 height=359
| |
- | id="_x0000_i1026" src="https://static.igem.org/mediawiki/2014hs/e/eb/Fimage006.jpg"
| |
- | alt="https://static.igem.org/mediawiki/2014hs/e/eb/Fimage006.jpg"></span></p>
| |
- |
| |
- | <p class=MsoNormal><span lang=EN-US style='mso-ansi-language:EN-US'>Graphic 6:
| |
- | This graphic shows our absorption rate of our mixed sample that includes liquid
| |
- | culture of our BL-21 Bacteria that have HRP gene and Methylene Blue
| |
- | (80uM) dyes and for the activate the HRP we also added Hydrogen Peroxide
| |
- | (800uM) measured in the 1 hour.</span></p>
| |
- |
| |
- | <p class=MsoNormal><span lang=EN-US style='mso-ansi-language:EN-US'>The result
| |
- | of Graphic-6 is that our Liquid cultures of BL-21 Bacteria that have LiP gene
| |
- | also degraded the Methylene Blue (80uM) dye with approximately %65 efficiency.</span></p>
| |
- |
| |
- | <p class=MsoNormal><span lang=EN-US style='mso-ansi-language:EN-US'> </span></p>
| |
- |
| |
- | <p class=MsoNormal><span lang=EN-US style='mso-ansi-language:EN-US'> </span></p>
| |
- |
| |
- | <p class=MsoNormal><span lang=EN-US style='mso-ansi-language:EN-US'>The Results
| |
- | of Graphic 5 and 6 show that our liquid culture of our BL-21 Bacteria that have
| |
- | HRP gene and other Bacteria that have LiP gene could degraded the Methylene
| |
- | Blue (80uM) dye but our Lysates of our BL-21 Bacteria that have HRP gene and
| |
- | other Bacteria that have LiP gene degraded the Methylene Blue (80uM) dye more
| |
- | efficiently. This results gave us an opinion that when our enzymes have further
| |
- | interaction with dyes they could degrade much more.</span></p>
| |
- |
| |
- | <p class=MsoNormal><span lang=EN-US style='mso-ansi-language:EN-US'> </span></p>
| |
- |
| |
- | <p class=MsoNormal><span lang=EN-US style='mso-ansi-language:EN-US'> </span></p>
| |
- |
| |
- | <p class=MsoNormal><span lang=EN-US style='mso-ansi-language:EN-US'> </span></p>
| |
- |
| |
- | <p class=MsoNormal><span lang=EN-US style='mso-ansi-language:EN-US'> </span></p>
| |
- |
| |
- | <p class=MsoNormal><span lang=EN-US style='mso-ansi-language:EN-US'> </span></p>
| |
- |
| |
- | <p class=MsoNormal><span lang=EN-US style='mso-ansi-language:EN-US'> </span></p>
| |
- |
| |
- | <p class=MsoNormal><span lang=EN-US style='mso-ansi-language:EN-US'> </span></p>
| |
- |
| |
- | <p class=MsoNormal><span style='mso-no-proof:yes'><img width=604 height=283
| |
- | id="_x0000_i1025" src="https://static.igem.org/mediawiki/2014hs/6/60/Fimage007.jpg"
| |
- | alt="https://static.igem.org/mediawiki/2014hs/6/60/Fimage007.jpg"></span></p>
| |
- |
| |
- | <p class=MsoNormal><span lang=EN-US style='mso-ansi-language:EN-US'> </span></p>
| |
- |
| |
- | <p class=MsoNormal><span lang=EN-US style='mso-ansi-language:EN-US'>Graphic 7:
| |
- | This graphic shows our absorption rate of our mixed sample that includes liquid
| |
- | culture of our Control BL-21 bacteria that don’t have any HRP or LiP gene
| |
- | and Methylene Blue (80uM) dyes and for the activate the any peroxidases,
| |
- | Control BL-21 bacteria have, we also added Hydrogen Peroxide (800uM) measured
| |
- | in the 1 hour.</span></p>
| |
- |
| |
- | <p class=MsoNormal><span lang=EN-US style='mso-ansi-language:EN-US'>The result
| |
- | of Graphic-7 is that our Liquid culture of BL-21 Bacteria that don’t have any
| |
- | HRP or LiP gene could degrade the Methylene Blue (80uM) dye with approximately
| |
- | % 18 efficiency. This result show that Control bacteria can degrade the the
| |
- | Methylene Blue (80uM) dye but at the very low percentage when compared with our
| |
- | liquid culture BL-21 Bacteria that have HRP gene and other Bacteria that have
| |
- | LiP gene so we thought that some peroxidases also can degrade the dyes but our
| |
- | enzymes HRP and LiP have very high catalytic ability for the our
| |
- | dyes(Methlylene Blue and Methyl Green). These results and graphics show that
| |
- | our enzymes are functional and working properly.</span></p>
| |
- |
| |
- | </div>
| |
- |
| |
- | </body>
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- | </head>
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- | </html>
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