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| <div class="9u 12u(narrower)"> | | <div class="9u 12u(narrower)"> |
− | <p>The reporter gene (i.e. <span class="i_enph">GFPmut3b</span> from part <a class="i_linker" href="http://parts.igem.org/Part:BBa_I13504">BBa_I13504</a>) was amplified using the Phusion polymerase from New England Biolabs and primers matching the prefix and suffix.</p> | + | <p>The reporter gene (i.e. <span class="i_enph italic">GFPmut3b</span> from part <a class="i_linker" href="http://parts.igem.org/Part:BBa_I13504">BBa_I13504</a>) was amplified using the Phusion polymerase from New England Biolabs and primers matching the prefix and suffix.</p> |
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| <p>We used <span class="i_enph quantity">50 ng</span> of template with an annealing temperature of <span class="i_enph quantity">59 °C</span> and an extension time of <span class="i_enph quantity">90 seconds</span>. The PCR was confirmed by electrophoresis and subsequently purified with NucleoSpin Gel and PCR Clean-Up Kit from Macherey-Nigel. <span class="i_enph quantity">125 ng</span> of purified PCR were digested with <span class="i_enph quantity">1 μl</span> of <span class="i_enph">XbaI</span> and <span class="i_enph quantity">1 μl</span> of <span class="i_enph">PstI</span> overnight at <span class="i_enph quantity">37°C</span>. The following morning the sample was treated with <span class="i_enph quantity">1 μl</span> of <span class="i_enph">DpnI</span> for 2 hour at <span class="i_enph quantity">37°C</span> and the enzymes were deactivated for <span class="i_enph quantity">20 min</span> at <span class="i_enph quantity">80 °C</span>.</p> | | <p>We used <span class="i_enph quantity">50 ng</span> of template with an annealing temperature of <span class="i_enph quantity">59 °C</span> and an extension time of <span class="i_enph quantity">90 seconds</span>. The PCR was confirmed by electrophoresis and subsequently purified with NucleoSpin Gel and PCR Clean-Up Kit from Macherey-Nigel. <span class="i_enph quantity">125 ng</span> of purified PCR were digested with <span class="i_enph quantity">1 μl</span> of <span class="i_enph">XbaI</span> and <span class="i_enph quantity">1 μl</span> of <span class="i_enph">PstI</span> overnight at <span class="i_enph quantity">37°C</span>. The following morning the sample was treated with <span class="i_enph quantity">1 μl</span> of <span class="i_enph">DpnI</span> for 2 hour at <span class="i_enph quantity">37°C</span> and the enzymes were deactivated for <span class="i_enph quantity">20 min</span> at <span class="i_enph quantity">80 °C</span>.</p> |
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| <div style="display:none;"> | | <div style="display:none;"> |
− | <p>Each promoter containing plasmid was digested with <span class="i_enph quantity">1 μl</span> of <span class="i_enph">SpeI</span> and <span class="i_enph quantity">1 μl</span> of <span class="i_enph">PstI</span> at <span class="i_enph quantity">37°C</span> overnight. The day after add <span class="i_enph quantity">1 μl</span> of phosphatase (CIP from New England Biolabs) for <span class="i_enph quantity">2 hours</span> at <span class="i_enph quantity">37°C</span>. The enzymes were then heat deactivated.</p> | + | <p>Each promoter containing plasmid was digested with <span class="i_enph quantity">1 μl</span> of <span class="i_enph">SpeI</span> and <span class="i_enph quantity">1 μl</span> of <span class="i_enph">PstI</span> at <span class="i_enph quantity">37°C</span> overnight. The day after <span class="i_enph quantity">1 μl</span> of phosphatase (CIP from New England Biolabs) was added for <span class="i_enph quantity">2 hours</span> at <span class="i_enph quantity">37°C</span>. The enzymes were then heat deactivated.</p> |
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| <p>The digestion reactions were assembled in this way:</p> | | <p>The digestion reactions were assembled in this way:</p> |
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| <i class="liticon wow bounceInLeft delay02 flaticon-bacteria"></i> <h4 class="header4 displayControl">Glycerol stocks preparation and Sample Growth </h4> | | <i class="liticon wow bounceInLeft delay02 flaticon-bacteria"></i> <h4 class="header4 displayControl">Glycerol stocks preparation and Sample Growth </h4> |
| <div style="display:none;"> | | <div style="display:none;"> |
− | <p>A single colony was inoculated with a sterile pipette tip in a test tube with <span class="i_enph quantity">10 ml</span> of LB and antibiotic (1000:1 LB to antibiotic ratio) and placed in the thermoshaker (<span class="i_enph quantity">190 rpm</span>, <span class="i_enph quantity">37°C</span>. When the culture got cloudy</span>, <span class="i_enph quantity">40 ml</span> of LB+antibiotic were added to reach a final volume of <span class="i_enph quantity">50 ml</span>. The cells were grown until an OD<sub>600</sub> of <span class="i_enph quantity">0.5</span> and then centrifuged at <span class="i_enph quantity">4100 rpm</span> for <span class="i_enph quantity">10 minutes</span> at <span class="i_enph quantity">4 °C</span>. The supernatant was discarded and the cells were resuspend in <span class="i_enph quantity">5 ml</span> of LB + antibiotic + glycerol (<span class="i_enph quantity">20% v/v</span>). The cells were kept on ice and were promptly aliquoted into <span class="i_enph quantity">200 μl</span> tubes and frozen at <span class="i_enph quantity">-80°C</span> immediately. From this protocol we obtained a <span class="i_enph quantity">10X</span> concentrated glycerol stock for each sample.</p> | + | <p>A single colony was inoculated with a sterile pipette tip in a test tube with <span class="i_enph quantity">10 ml</span> of LB and antibiotic (1000:1 LB to antibiotic ratio) and placed in the thermoshaker (<span class="i_enph quantity">190 rpm</span>, <span class="i_enph quantity">37°C</span>). When the culture got cloudy</span>, <span class="i_enph quantity">40 ml</span> of LB+antibiotic were added to reach a final volume of <span class="i_enph quantity">50 ml</span>. The cells were grown until an OD<sub>600</sub> of <span class="i_enph quantity">0.5</span> and then centrifuged at <span class="i_enph quantity">4100 rpm</span> for <span class="i_enph quantity">10 minutes</span> at <span class="i_enph quantity">4 °C</span>. The supernatant was discarded and the cells were resuspend in <span class="i_enph quantity">5 ml</span> of LB + antibiotic + glycerol (<span class="i_enph quantity">20% v/v</span>). The cells were kept on ice and were promptly aliquoted into <span class="i_enph quantity">200 μl</span> tubes and frozen at <span class="i_enph quantity">-80°C</span> immediately. From this protocol we obtained a <span class="i_enph quantity">10X</span> concentrated glycerol stock for each sample.</p> |
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| <p>The glycerol stock was thaw and added into <span class="i_enph quantity">10 ml</span> of LB with antibiotic, giving a starting culture with an OD<sub>600</sub> of <span class="i_enph quantity">0.1</span>. The sample were grown in a <span class="i_enph quantity">50 mL</span> conical plastic tube in the termoshaker at <span class="i_enph quantity">37°C</span> and were grown until an OD<sub>600</sub> <span class="i_enph quantity">0.7</span>. At this point <span class="i_enph quantity">3 ml</span> of the culture were transferred in a new tube, centrifuged it, and stored at <span class="i_enph quantity">-20°C</span>, except if otherwise indicated.</p> | | <p>The glycerol stock was thaw and added into <span class="i_enph quantity">10 ml</span> of LB with antibiotic, giving a starting culture with an OD<sub>600</sub> of <span class="i_enph quantity">0.1</span>. The sample were grown in a <span class="i_enph quantity">50 mL</span> conical plastic tube in the termoshaker at <span class="i_enph quantity">37°C</span> and were grown until an OD<sub>600</sub> <span class="i_enph quantity">0.7</span>. At this point <span class="i_enph quantity">3 ml</span> of the culture were transferred in a new tube, centrifuged it, and stored at <span class="i_enph quantity">-20°C</span>, except if otherwise indicated.</p> |
| </div> | | </div> |
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− | <i class="liticon wow bounceInLeft delay03 flaticon-atom27"></i> <h4 class="header4 displayControl">Fluorescence readings: Tecan INFINITE ® 200 PRO Plate Reader </h4> | + | <i class="liticon wow bounceInLeft delay03 flaticon-atom27"></i> <h4 class="header4 displayControl">Fluorescence readings: Tecan INFINITE 200 PRO Plate Reader </h4> |
| <div style="display:none;" class="row"> | | <div style="display:none;" class="row"> |
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| </div> | | </div> |
| <div class="7u 12u(narrower)"> | | <div class="7u 12u(narrower)"> |
− | <p>The cells were thawed and resuspended in <span class="i_enph quantity">3 ml</span> of PBS. An aliquot of <span class="i_enph quantity">150 μl</span> of each sample was placed into a <b>White, Flat-bottomed, 96-well Costar Plate</b> (code: 3917) and fluorescence intensities were taken with a <b>Tecan Infinite® 200 Pro Plate Reader</b> (made in Switzerland). Excitation wavelength and emission wavelength were <span class="i_enph quantity">395 nm</span> and <span class="i_enph quantity">509 nm</span>, respectively. The gain was optimized at <span class="i_enph quantity">70 V</span> and kept constant for each sample. PBS was used as blank. To obtain technical replicates, fluorescence intensities were acquired for three aliquots of the same biological sample, keeping the same instrumental conditions. The raw data were adjusted for the blank value and the MEANS across the replicates with their relative standard deviation were plotted.</p> | + | <p>The cells were thawed and resuspended in <span class="i_enph quantity">3 ml</span> of PBS. An aliquot of <span class="i_enph quantity">150 μl</span> of each sample was placed into a <b>White, Flat-bottomed, 96-well Costar Plate</b> (code: 3917) and fluorescence intensities were taken with a <b>Tecan Infinite 200 Pro Plate Reader</b> (made in Switzerland). Excitation wavelength and emission wavelength were <span class="i_enph quantity">395 nm</span> and <span class="i_enph quantity">509 nm</span>, respectively. The gain was optimized at <span class="i_enph quantity">70 V</span> and kept constant for each sample. PBS was used as blank. To obtain technical replicates, fluorescence intensities were acquired for three aliquots of the same biological sample, keeping the same instrumental conditions. The raw data were adjusted for the blank value and the means across the replicates with their relative standard deviation were plotted.</p> |
| </div> | | </div> |
| </div> | | </div> |
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| </div> | | </div> |
| </div> | | </div> |
− | <p>Primers were designed for the reporter gene <span class="i_enph italic">GFP_mut3b</span> and for the housekeeping gene <span class="i_enph italic">idnT</span> <span class="lesser">(D-gluconate transporter)</span> as indicated above.</p> | + | <p>Primers were designed for the reporter gene <span class="i_enph italic">GFPmut3b</span> and for the housekeeping gene <span class="i_enph italic">idnT</span> <span class="lesser">(D-gluconate transporter)</span> as indicated above.</p> |
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| <p> We then analyzed the raw data, calculating the relative fold expression of each GPF device compared to the housekeeping (ΔCt) and the related standard deviation:</p> | | <p> We then analyzed the raw data, calculating the relative fold expression of each GPF device compared to the housekeeping (ΔCt) and the related standard deviation:</p> |
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| <i class="liticon wow bounceInLeft delay03 flaticon-test17"></i> <h4 class="header4 displayControl">Fluorescence readings: E. coli S30 Extract System for DNA Circular </h4> | | <i class="liticon wow bounceInLeft delay03 flaticon-test17"></i> <h4 class="header4 displayControl">Fluorescence readings: E. coli S30 Extract System for DNA Circular </h4> |
| <div style="display:none;"> | | <div style="display:none;"> |
− | <p>Miniprepped DNA was purified and extracted through phenol/chloroform extraction followed by ethanol precipitation. Reactions were set following <b>Promega <i>E. coli</i> S30 Extract System Technical Bulletin</b> and were performed using <b>Qiagen Rotor-Gene Q</b>(made in USA). Parameters for this specific experiment were set as follows:</p> | + | <p>Miniprepped DNA was purified and extracted through phenol/chloroform extraction followed by ethanol precipitation. Reactions were set following <b>Promega <i>E. coli</i> S30 Extract System Technical Bulletin</b> and were performed using <b>Qiagen Rotor-Gene Q</b> (made in USA). Parameters for this specific experiment were set as follows:</p> |
| <ul class="customlist arrowed"> | | <ul class="customlist arrowed"> |
| <li>Green channel gain: <span class="i_enph quantity">0.67</span></li> | | <li>Green channel gain: <span class="i_enph quantity">0.67</span></li> |
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| <h4 class="header4-resume wow fadeInDown">Ratios across promoters are kept the same</h4> | | <h4 class="header4-resume wow fadeInDown">Ratios across promoters are kept the same</h4> |
− | <p><span class="i_enph">J23101/J23106</span> fluorescence ratios ranged from <span class="i_enph quantity">2.0</span> to <span class="i_enph quantity">4.5</span>, depending on the strain and the technique. Differently from the other two promoters, <span class="i_enph">J23117</span> showed a very low GFP production, as it was not detectable by eye or using the trans-illuminator and showed little fluorescence with the three techniques used.</p> | + | <p><span class="i_enph">J23101/J23106</span> fluorescence ratios ranged from <span class="i_enph quantity">2.0</span> to <span class="i_enph quantity">4.5</span>, depending on the strain and the technique. Differently from the other two promoters, <span class="i_enph">J23117</span> showed a very low GFP production, as it was <span class="i_enph">not detectable by eye</span> or using the trans-illuminator and showed little fluorescence with the three techniques used.</p> |
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| <h4 class="header4-resume wow fadeInDown">Different techniques lead to the same results, with different sensitivities</h4> | | <h4 class="header4-resume wow fadeInDown">Different techniques lead to the same results, with different sensitivities</h4> |
− | <p>The best way to perform a characterization is to use various techniques. Throughout our experiments we saw that each instrument has a specific sensitivity, which alters the output data. The FACS happened to be the most accurate among all, due to its extremely high intrinsic sentivity. The plate reader also showed a good accuracy while the fluorimeter was not able to detect the weakest promoter from the background noise, due to its low intrinsic sensitivity. The qPCR and the Cell-Free Extract also gave positive results, in line with our expectations.</p> | + | <p>The best way to perform a characterization is to <span class="i_enph">use various techniques</span>. Throughout our experiments we saw that each instrument has a specific sensitivity, which alters the output data. The <span class="i_enph">FACS happened to be the most accurate among all</span>, due to its extremely high intrinsic sentivity. The plate reader also showed a good accuracy while the <span class="i_enph">fluorimeter was not able to detect the weakest promoter</span> from the background noise, due to its low intrinsic sensitivity. The qPCR and the Cell-Free Extract also gave positive results, in line with our expectations.</p> |
| | | |
| <h4 class="header4-resume wow fadeInDown">Bacterial strain does matter</h4> | | <h4 class="header4-resume wow fadeInDown">Bacterial strain does matter</h4> |
− | <p>The three promoters behaved differently in the different bacterial strains used. The bacterial strain which gave the highest fluorescence was NEB10Beta cells in all cases showed a significant increased expression of the protein, compared to JM109 and NEB Express. We hypothesized this discordance among strains is due to their different genotypes. A different bacterial proteome (i.e. presence/lack of specific proteases and/or chaperonins, polymerases efficiency) may alter protein production, processing and folding, thus fluorescence emission.</p> | + | <p>The three promoters behaved differently in the different bacterial strains used. The bacterial strain which gave the highest fluorescence was <span class="i_enph">NEB10β cells in all cases showed a significant increased expression of the protein</span>, compared to JM109 and NEB Express. We hypothesized this discordance among strains is due to their <span class="i_enph">different genotypes</span>. A different bacterial proteome (i.e. presence/lack of specific proteases and/or chaperonins, polymerases efficiency) may alter protein production, processing and folding, thus fluorescence emission.</p> |
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− | <h4 class="header4-resume wow fadeInDown">Looking at promoters in a different angle</h4> | + | <h4 class="header4-resume wow fadeInDown">Looking at promoters from a different angle</h4> |
− | <p>Characterization in vitro using the qPCR allows to quantify the promoter strength by measuring transcript level, rather than just looking at the protein production. This approach gives a better understanding on the promoter`s nature, since it`s well known that the central dogma in biology is not always respected. </p> | + | <p>Characterization in vitro using the qPCR allows to <span class="i_enph">quantify the promoter strength by measuring transcript level</span>, rather than just looking at the protein production. This approach gives a <span class="i_enph">better understanding on the promoter`s nature</span>, since it`s well known that the central dogma in biology is not always respected. </p> |
| | | |
| <h4 class="header4-resume wow fadeInDown"><i>In vitro</i> conditions mimic the <i>in vivo</i> reality</h4> | | <h4 class="header4-resume wow fadeInDown"><i>In vitro</i> conditions mimic the <i>in vivo</i> reality</h4> |
− | <p>Comparing the results obtained from the cell-free extract to the others, we discovered that the promoters behave the same when working <i>in vitro</i> or in living bacteria.</p> | + | <p>Comparing the results obtained from the cell-free extract to the others, we discovered that the <span class="i_enph">promoters behave the same when working <i>in vitro</i> or in living bacteria</span>.</p> |
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| </section> | | </section> |