Latest revision as of 22:47, 21 October 2019

Project

Contribution

We added quantitative experimental characterization to the following biobricks:

These parts are a family of constitutive promoters. They were present in plasmid BBa_J61002). We analyzed the influence of the mentioned promoters on the expression of RFP, both on protein level (fluorescence spectroscopy) and mRNA level (RT-qPCR).

Materials and Methods

Forward and reverse primers were designed for RFP mRNA amplification:

forward primer: atggcttcctccgaagacg
reverse primer: actcaccgtcttgcaggg

The expected amplification product has a length of 352 bp.

Lyophilized biobricks were resuspended in 10 µL ddH₂O according to the provided protocol. A vial of competent cells was retrieved from storage at -70 °C and thawed on ice. Then, 1 µL plasmid DNA was added to 80 µL electrocompetent E. coli MG1655 and gently mixed.
The cell-DNA suspension was transferred to a chilled electroporation cuvette. Electroporation was performed at 2500 V. Cells were immediately recovered in 1 mL pre-warmed 2YT medium and incubated at 37 °C for 1 h. Aliquots of the recovery medium were plated out on agar plates containing 100 µg/mL ampicillin.
The plates were incubated overnight at 37 °C. Single colonies were picked from the plates and grown overnight in 5 mL 2YT medium supplemented with 100 µg/mL ampicillin in glass test tubes at 37 °C and 130 rpm.
The optical density (OD₆₀₀) of the overnight cultures was determined and a 5 mL main culture in 2YT medium supplemented with 100 µg/mL ampicillin in glass test tubes was inoculated with a starting OD₆₀₀ of 0.2. After 3 h of growth fluorescence of 50 µL of the cell suspension was measured (excitation: 540 nm and emission: 590 nm).
Also, 500 µL of culture was centrifuged and the pellet was used for RNA extraction. This was done using the Zymo QuickRNA Fungal/Bacterial Mini Prep Kit following the user manual. In addition to that, E. coli MG1655 was cultured and harvested for RNA isolation without the plasmid BBa_J61002 to ensure, that our primers specifically bind to RFP mRNA.

Subsequently RT-qPCR was performed. For the reverse transcription 0.5 µL random hexamers (stock = 50 µM), 0.5 µL dNTPs (stock = 10 mM each), 0.5 µL RNA template and 5.5 µL RNAse-free water where mixed and incubated at 65 °C for 5 min, followed by 5 min on ice.
The reverse transcription mix was prepared and added to each reaction. It composed of 2 µL 5x SSIV buffer, 0.5 µL DTT and 0.5 µL SSIV per reaction. The mixture was incubated at RT for 10 min, followed by incubation at 50 °C for another 10 min. The reaction was inactivated by incubation at 80 °C for 10 min. QPCR was performed in triplicates using KAPA SYBR FAST qPCR Mix (2x). 10 µL 2x KAPA Mix, 0.4 µL each forward and reverse primer (stock = 10 pmol/µL), 1 µL cDNA and 8.2 µL water were mixed. Initial denaturation was performed at 95 °C for 3 min, followed by 44 cycles of denaturation (95 °C, 15 sec), annealing (58 °C, 30 sec) and elongation (68 °C, 45 sec).

SYBR, included in the KAPA mix, binds to double stranded DNA absorbing blue light and emitting green light. Therefore, the fluorescence signal increases with the increasing amount of double stranded DNA. The higher the concentration of the template in the sample, the faster the fluorescence exceeds the threshold value. The number of cycles at which this happens is called threshold cycle (C_t). The amount of RNA present in the sample can be calculated as follows: if sample A showed a C_t of 8 and sample B showed a C_t of 11, sample A contained 2³ = 8 times more template.

A melting curve was run afterwards in a temperature range between 60 °C and 93.9 °C (in 1 °C steps), to verify, that one specific amplification product was generated.

To validate, that the expected amplification product was generated, 5 µL of the amplification mix was added to 1 µL of 6 x loading dye and loaded onto a 2 % agarose gel. A marker was also loaded (ThermoFisher 1 kb Plus DNA ladder). In addition to that, the gel contained the nucleic acid stain GelRed to visualize the nucleic acid when exposed under UV light. The gel was run at 80 V (1V/cm) for 1 h.

Results

Results of fluorescence spectroscopy

Investigating the grown cell cultures by fluorescence spectroscopy clearly showed emission at 590nm (excitation at 540 nm) indicating a successful expression of RFP.

Promoter	Emission at 590 nm
BBa_J23100	192 +- 13
BBa_J23112	30 +- 1
BBa_J23118	345 +- 4

Results of RT-qPCR

Figure1 - Fluorescence signal over cycle number for amplification of the target cDNA of E. coli cells containing the plasmid BBa_J61002 with different promoters (100, 112, 118). Means and standard deviation of technical triplicates are shown.

Figure2 - Fluorescence signal over cycle number for amplification of the target cDNA of E. coli cells without the plasmid BBa_J61002. Means and standard deviation of technical triplicates are shown.

Figure 1 shows the fluorescence signal over cycle number for amplification of the target cDNA of E. coli cells containing the plasmid BBa_J61002 with different promoters (100, 112, 118). As expected, the E. coli without the plasmid (-) showed amplification of unspecific targets (figure 2), which can be derived from high C_t values. This indicates, that the primers we designed bind to RFP cDNA specifically.

To quantify the expression levels of RFP under different promoters, the cDNA of a house-keeping gene was amplified as well. After we spoke to Dr. Trachtmann, who is an expert in performing and evaluating RT-qPCR experiments, we chose a GTPase-activation protein (GAP) as a housekeeping gene. The specific primers to amplify the desired house-keeping gene were kindly provided by Dr. Trachtmann.

Figure 3 - Fluorescence signal over cycle number for amplification of the house keeping gene GAP cDNA of E. coli cells containing the plasmid BBa_J61002 with different promoters (100, 112, 118). Means and standard deviation of technical triplicates are shown.

Figure 4 - Fluorescence signal over cycle number for amplification of the house keeping gene GAP cDNA of E. coli cells without the plasmid BBa_J61002. Means and standard deviation of technical triplicates are shown

Figure 3 and 4 showed that GAP is present in all samples. Under the assumption, that GAP is expressed under constant rates, one is able to quantify the expression of the target gene relatively to the expression of the house-keeping gene. Analytik Jena provided a qPCR device, which came with a software to interpret our results using the ddCt-method. Using this method, revealed the results shown in figure 5.

Figure 5 - Relative quantification of RFP expression compared to the expression of the house-keeping gene GAP in E. coli MG1655. Cells contained the plasmid BBa_J61002 with different promoters: BBa_J23100, BBa_J23112 and BBa_J23118. As a control E. coli MG1655 were cultivated without the plasmid.

RFP expression was about 7-fold higher than the expression of GAP in cells containing promoter BBa_J23100. Compared to that, the expression of RFP in cells containing promoter BBa_J23112 was about 55-fold higher than expression of the house-keeping gene. The highest expression of RFP compared to GAP was observed in cells containing promoter BBa_J23118. Here, RFP expression was 74-fold compared to GAP expression.

Figure 6: Melting curve of the amplification mix of the target cDNA (RFP) of E. coli cells containing the plasmid BBa_J61002 with different promoters (100, 112, 118). Means and standard deviation of technical triplicates are shown.

Figure 7 - Melting curve of the amplification mix of the house keeping gene GAP cDNA of E. coli cells containing the plasmid BBa_J61002 with different promoters (100, 112, 118). Means and standard deviation of technical triplicates are shown.

Figure 8 - Melting curve of the amplification mix of the house keeping gene GAP cDNA of E. coli cells without the plasmid BBa_J61002. Means and standard deviation of technical triplicates are shown.

Figure 9: Melting curve of the amplification mix of the target cDNA (RFP) of E. coli cells without the plasmid BBa_J61002. Means and standard deviation of technical triplicates are shown.

The melting curves showed that specific amplification products were generated (figure 6, figure 7, figure 8). As E. Coli MG1655 does not contain any target sequences for RFP primers when no plasmid is present, the melting curves show unspecific product generation (figure 9).

Results of agarose gel electrophoresis

Exposing the gel under UV light, reveals that the expected amplification product was generated (about 350 bp).

Figure 10: Amplification mix loaded onto a 2 % agarose gel reveals production of one amplification product for cells containing the plasmid with different promoters.

Conclusion

We were surprised by the results of the characterization of the Anderson promoters as they do not meet our expectations. Actually, we expected that the use of different promoter strengths would result in different levels of mRNA in a way that weak promoters would result in weak fluorescence and low corresponding mRNA levels and strong promoters would lead to high corresponding mRNA levels. More precisely, we expected cells with the promoter BBa_J23100 to exhibit the highest mRNA level, as it was supposed to be the strongest promoter of the ones we characterized.
When measuring fluorescence, we obtained the expected results: it was revealed, that cells with the promoter BBa_J23100 (strongest promoter) produced more RFP than cells with the promoter BBa_J23118. Cells with the weakest promoter (BBa_J23112) produced fewest amounts of RFP.
Possible reasons for the discrepancy between promoter strength, RFP amount and mRNA concentration could lie in the promoter library itself: Changes in the promoter sequence to generate the library could lead to impaired secondary structure of the mRNA. This in turn may influence the binding of ribosomes and the translation initiation efficiency significantly.
It may be possible, that the differences in fluorescence are mainly caused by changes on the translational level and not on transcriptional level, as to date promotor strength for these biobricks was only determined by fluorescence measurements.

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+    <div class="container has-text-centered">
-        <h2 class="subtitle">
+      <h2 class="subtitle">
-          Project
+        Project
-        </h2>
+      </h2>
-        <h1 class="title">
+      <h1 class="title">
-          Contribution
+        Contribution
-        </h1>
+      </h1>
-      </div>
      </div>
-   </section>
+   </div>
-  <div style="padding: 1rem 0;" class="container">
+</section>
-    <div class="columns is-centered">
+<div style="padding: 1rem 0;" class="container">
-      <div class="column is-three-quarters">
+  <div class="columns is-centered">
-        We added quantitative experimental characterization to the following biobricks:
+    <div class="column is-three-quarters">
+      We added quantitative experimental characterization to the following biobricks:
+      <ul>
+        <li><a href="http://parts.igem.org/Part:BBa_J23100">BBa_J23100</a></li>
+        <li><a href="http://parts.igem.org/Part:BBa_J23112">BBa_J23112</a></li>
+        <li><a href="http://parts.igem.org/Part:BBa_J23118">BBa_J23118</a></li>
+      </ul>
+      These parts are a family of constitutive promoters. They were present in plasmid
+      <a href="http://parts.igem.org/Part:BBa_J61002">BBa_J61002</a>). We analyzed the influence of the mentioned
+      promoters on the expression of RFP, both on protein level (fluorescence spectroscopy) and mRNA level (RT-qPCR).
+    </div>
+  </div>
+  <div class="columns">
+    <div class="column">
+      <h3 class="title is-3">Materials and Methods</h3>
+      <div class="notification">
+        <p>Forward and reverse primers were designed for RFP mRNA amplification:</p>
          <ul>
-           <li><a href="http://parts.igem.org/Part:BBa_J23100">BBa_J23100</a></li>
+           <li>forward primer: <code>atggcttcctccgaagacg</code></li>
-           <li><a href="http://parts.igem.org/Part:BBa_J23112">BBa_J23112</a></li>
+           <li>reverse primer: <code>actcaccgtcttgcaggg</code></li>
-          <li><a href="http://parts.igem.org/Part:BBa_J23118">BBa_J23118</a></li>
          </ul>
-         These parts are a family of constitutive promoters. They were present in plasmid
+         <p>The expected amplification product has a length of 352 bp.</p>
-        <a href="http://parts.igem.org/Part:BBa_J61002">BBa_J61002</a>). We analyzed the influence of the mentioned
-        promoters on the expression of RFP, both on protein level (fluorescence spectroscopy) and mRNA level (RT-qPCR).
        </div>
      </div>
+  </div>
-    <div class="columns">
+  <div class="columns">
-      <div class="column">
+    <div class="column">
-         <h3 class="title is-3">Materials and Methods</h3>
+      <p>
-         <div class="notification">
+         Lyophilized biobricks were resuspended in 10 &micro;L ddH<sub>2</sub>O according to the provided protocol. A
-          <p>Forward and reverse primers were designed for RFP mRNA amplification:</p>
+         vial of competent cells was retrieved from storage at -70 &deg;C and thawed on ice. Then, 1 &micro;L plasmid
-          <ul>
+        DNA was added to 80 &micro;L electrocompetent <em>E. coli </em>MG1655 and gently mixed.
-            <li>forward primer: <code>atggcttcctccgaagacg</code></li>
+        <br />
-            <li>reverse primer: <code>actcaccgtcttgcaggg</code></li>
+        The cell-DNA suspension was transferred to a chilled electroporation cuvette. Electroporation was performed at
-          </ul>
+V. Cells were immediately recovered in 1 mL pre-warmed 2YT medium and incubated at 37 &deg;C for 1 h.
-          <p>The expected amplification product has a length of 352 bp.</p>
+        Aliquots of the recovery medium were plated out on agar plates containing 100 &micro;g/mL ampicillin.
-         </div>
+        <br />The plates were incubated overnight at 37 &deg;C. Single colonies were picked from the plates and grown
-       </div>
+        overnight in 5 mL 2YT medium supplemented with 100 &micro;g/mL ampicillin in glass test tubes at 37 &deg;C and
+rpm.
+        <br />
+        The optical density (OD<sub>600</sub>) of the overnight cultures was determined and a 5 mL main culture in 2YT
+        medium supplemented with 100 &micro;g/mL ampicillin in glass test tubes was inoculated with a starting OD<sub
+      >600</sub
+      >
+        of 0.2. After 3 h of growth fluorescence of 50 &micro;L of the cell suspension was measured (excitation: 540
+        nm and emission: 590 nm).
+        <br />
+        Also, 500 &micro;L of culture was centrifuged and the pellet was used for RNA extraction. This was done using
+        the Zymo QuickRNA Fungal/Bacterial Mini Prep Kit following the user manual. In addition to that,
+        <em>E. coli </em>MG1655 was cultured and harvested for RNA isolation without the plasmid BBa_J61002 to ensure,
+        that our primers specifically bind to RFP mRNA.
+      </p>
+      <p>
+        Subsequently RT-qPCR was performed. For the reverse transcription 0.5 &micro;L random hexamers (stock = 50
+        &micro;M), 0.5 &micro;L dNTPs (stock = 10 mM each), 0.5 &micro;L RNA template and 5.5 &micro;L RNAse-free
+        water where mixed and incubated at 65 &deg;C for 5 min, followed by 5 min on ice.
+        <br />
+         The reverse transcription mix was prepared and added to each reaction. It composed of 2 &micro;L 5x SSIV
+        buffer, 0.5 &micro;L DTT and 0.5 &micro;L SSIV per reaction. The mixture was incubated at RT for 10 min,
+        followed by incubation at 50 &deg;C for another 10 min. The reaction was inactivated by incubation at 80
+        &deg;C for 10 min. QPCR was performed in triplicates using KAPA SYBR FAST qPCR Mix (2x). 10 &micro;L 2x KAPA
+        Mix, 0.4 &micro;L each forward and reverse primer (stock = 10 pmol/&micro;L), 1 &micro;L cDNA and 8.2 &micro;L
+        water were mixed. Initial denaturation was performed at 95 &deg;C for 3 min, followed by 44 cycles of
+        denaturation (95 &deg;C, 15 sec), annealing (58&nbsp;&deg;C, 30 sec) and elongation (68 &deg;C, 45 sec).
+       </p>
      </div>
+     <div class="column">
-     <div class="columns">
+       <p>
-       <div class="column">
+         SYBR, included in the KAPA mix, binds to double stranded DNA absorbing blue light and emitting green light.
-        <p>
+        Therefore, the fluorescence signal increases with the increasing amount of double stranded DNA. The higher the
-          Lyophilized biobricks were resuspended in 10 &micro;L ddH<sub>2</sub>O according to the provided protocol. A
+        concentration of the template in the sample, the faster the fluorescence exceeds the threshold value. The
-          vial of competent cells was retrieved from storage at -70 &deg;C and thawed on ice. Then, 1 &micro;L plasmid
+        number of cycles at which this happens is called threshold cycle (C<sub>t</sub>). The amount of RNA present in
-          DNA was added to 80 &micro;L electrocompetent <em>E. coli </em>MG1655 and gently mixed.
+        the sample can be calculated as follows: if sample A showed a C<sub>t</sub> of 8 and sample B showed a C<sub
-          <br />
+      >t</sub
-          The cell-DNA suspension was transferred to a chilled electroporation cuvette. Electroporation was performed at
+      >
-V. Cells were immediately recovered in 1 mL pre-warmed 2YT medium and incubated at 37 &deg;C for 1 h.
+        of 11, sample A contained 2<sup>3</sup>&nbsp;= 8 times more template.
-          Aliquots of the recovery medium were plated out on agar plates containing 100 &micro;g/mL ampicillin.
+      </p>
-          <br />The plates were incubated overnight at 37 &deg;C. Single colonies were picked from the plates and grown
+      <p>
-          overnight in 5 mL 2YT medium supplemented with 100 &micro;g/mL ampicillin in glass test tubes at 37 &deg;C and
+        A melting curve was run afterwards in a temperature range between 60 &deg;C and 93.9 &deg;C (in 1 &deg;C
-rpm.
+        steps), to verify, that one specific amplification product was generated.
-          <br />
+      </p>
-          The optical density (OD<sub>600</sub>) of the overnight cultures was determined and a 5 mL main culture in 2YT
+      <p>
-          medium supplemented with 100 &micro;g/mL ampicillin in glass test tubes was inoculated with a starting OD<sub
+        To validate, that the expected amplification product was generated, 5 &micro;L of the amplification mix was
-            >600</sub
+        added to 1 &micro;L of 6 x loading dye and loaded onto a 2 % agarose gel. A marker was also loaded
-          >
+        (ThermoFisher 1 kb Plus DNA ladder). In addition to that, the gel contained the nucleic acid stain GelRed to
-          of 0.2. After 3 h of growth fluorescence of 50 &micro;L of the cell suspension was measured (excitation: 540
+        visualize the nucleic acid when exposed under UV light. The gel was run at 80 V (1V/cm) for 1 h.
-          nm and emission: 590 nm).
+      </p>
-          <br />
-          Also, 500 &micro;L of culture was centrifuged and the pellet was used for RNA extraction. This was done using
-          the Zymo QuickRNA Fungal/Bacterial Mini Prep Kit following the user manual. In addition to that,
-          <em>E. coli </em>MG1655 was cultured and harvested for RNA isolation without the plasmid BBa_J61002 to ensure,
-          that our primers specifically bind to RFP mRNA.
-         </p>
-        <p>
-          Subsequently RT-qPCR was performed. For the reverse transcription 0.5 &micro;L random hexamers (stock = 50
-          &micro;M), 0.5 &micro;L dNTPs (stock = 10 mM each), 0.5 &micro;L RNA template and 5.5 &micro;L RNAse-free
-          water where mixed and incubated at 65 &deg;C for 5 min, followed by 5 min on ice.
-          <br />
-          The reverse transcription mix was prepared and added to each reaction. It composed of 2 &micro;L 5x SSIV
-          buffer, 0.5 &micro;L DTT and 0.5 &micro;L SSIV per reaction. The mixture was incubated at RT for 10 min,
-          followed by incubation at 50 &deg;C for another 10 min. The reaction was inactivated by incubation at 80
-          &deg;C for 10 min. QPCR was performed in triplicates using KAPA SYBR FAST qPCR Mix (2x). 10 &micro;L 2x KAPA
-          Mix, 0.4 &micro;L each forward and reverse primer (stock = 10 pmol/&micro;L), 1 &micro;L cDNA and 8.2 &micro;L
-          water were mixed. Initial denaturation was performed at 95 &deg;C for 3 min, followed by 44 cycles of
-          denaturation (95 &deg;C, 15 sec), annealing (58&nbsp;&deg;C, 30 sec) and elongation (68 &deg;C, 45 sec).
-        </p>
-      </div>
-      <div class="column">
-        <p>
-          SYBR, included in the KAPA mix, binds to double stranded DNA absorbing blue light and emitting green light.
-          Therefore, the fluorescence signal increases with the increasing amount of double stranded DNA. The higher the
-          concentration of the template in the sample, the faster the fluorescence exceeds the threshold value. The
-          number of cycles at which this happens is called threshold cycle (C<sub>t</sub>). The amount of RNA present in
-          the sample can be calculated as follows: if sample A showed a C<sub>t</sub> of 8 and sample B showed a C<sub
-            >t</sub
-          >
-          of 11, sample A contained 2<sup>3</sup>&nbsp;= 8 times more template.
-        </p>
-        <p>
-          A melting curve was run afterwards in a temperature range between 60 &deg;C and 93.9 &deg;C (in 1 &deg;C
-          steps), to verify, that one specific amplification product was generated.
-        </p>
-        <p>
-          To validate, that the expected amplification product was generated, 5 &micro;L of the amplification mix was
-          added to 1 &micro;L of 6 x loading dye and loaded onto a 2 % agarose gel. A marker was also loaded
-          (ThermoFisher 1 kb Plus DNA ladder). In addition to that, the gel contained the nucleic acid stain GelRed to
-          visualize the nucleic acid when exposed under UV light. The gel was run at 80 V (1V/cm) for 1 h.
-        </p>
-      </div>
      </div>
+  </div>
-    <div class="columns is-centered">
+  <div class="columns is-centered">
-      <div class="column is-three-quarters">
+    <div class="column is-three-quarters">
-        <h3 class="title is-3">
+      <h3 class="title is-3">
-          Results
+        Results
-        </h3>
+      </h3>
-        <h3 class="title is-4">
+      <h3 class="title is-4">
-          Results of fluorescence spectroscopy
+        Results of fluorescence spectroscopy
-        </h3>
+      </h3>
-        Investigating the grown cell cultures by fluorescence spectroscopy clearly showed emission at 590nm (excitation
+      Investigating the grown cell cultures by fluorescence spectroscopy clearly showed emission at 590nm (excitation
-        at 540 nm) indicating a successful expression of RFP.
+      at 540 nm) indicating a successful expression of RFP.
-        <table class="table">
+      <table class="table">
-          <thead>
+        <thead>
-            <tr>
+        <tr>
-              <th>
+          <th>
-                Promoter
+            Promoter
-              </th>
+          </th>
-              <th>
+          <th>
-                Emission at 590 nm
+            Emission at 590 nm
-              </th>
+          </th>
-            </tr>
+        </tr>
-          </thead>
+        </thead>
-          <tbody>
+        <tbody>
-            <tr>
+        <tr>
-              <td>
+          <td>
-                <p>BBa_J23100</p>
+            <p>BBa_J23100</p>
-              </td>
+          </td>
-              <td>
+          <td>
-                <p>192 +- 13</p>
+            <p>192 +- 13</p>
-              </td>
+          </td>
-            </tr>
+        </tr>
-            <tr>
+        <tr>
-              <td>
+          <td>
-                <p>BBa_J23112</p>
+            <p>BBa_J23112</p>
-              </td>
+          </td>
-              <td>
+          <td>
-                <p>30 +- 1</p>
+            <p>30 +- 1</p>
-              </td>
+          </td>
-            </tr>
+        </tr>
-            <tr>
+        <tr>
-              <td>
+          <td>
-                <p>BBa_J23118</p>
+            <p>BBa_J23118</p>
-              </td>
+          </td>
-              <td>
+          <td>
-                <p>345 +- 4</p>
+            <p>345 +- 4</p>
-              </td>
+          </td>
-            </tr>
+        </tr>
-          </tbody>
+        </tbody>
-        </table>
+      </table>
-        <h3 class="title is-4">
+      <h3 class="title is-4">
-          Results of fluorescence spectroscopy
+        Results of RT-qPCR
-        </h3>
+      </h3>
-        <div class="has-text-centered" style="width: 75%; margin:0 auto;">
+      <div class="has-text-centered" style="width: 75%; margin:0 auto;">
-          <canvas id="mRFP"></canvas>
+        <canvas id="mRFP"></canvas>
-        </div>
+      </div>
-        <small>
+      <small>
-          Figure1 - Fluorescence signal over cycle number for amplification of the target cDNA of
+        Figure1 - Fluorescence signal over cycle number for amplification of the target cDNA of
-          <em>E. coli</em> cells containing the plasmid BBa_J61002 with different promoters (100, 112, 118). Means and
+        <em>E. coli</em> cells containing the plasmid BBa_J61002 with different promoters (100, 112, 118). Means and
-          standard deviation of technical triplicates are shown.
+        standard deviation of technical triplicates are shown.
-        </small>
+      </small>
-        <script>
+      <script>
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-        <small>
+      <small>
-          Figure2 - Fluorescence signal over cycle number for amplification of the target cDNA of <em>E. coli</em> cells
+        Figure2 - Fluorescence signal over cycle number for amplification of the target cDNA of <em>E. coli</em> cells
-          without the plasmid BBa_J61002. Means and standard deviation of technical triplicates are shown.
+        without the plasmid BBa_J61002. Means and standard deviation of technical triplicates are shown.
-        </small>
+      </small>
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+      <script>
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-        <br />
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-        <br />
+      <br />
-        <p>
+      <br />
-          Figure 1 shows the fluorescence signal over cycle number for amplification of the target cDNA of
+      <p>
-          <em>E. coli</em> cells containing the plasmid BBa_J61002 with different promoters (100, 112, 118). As
+        Figure 1 shows the fluorescence signal over cycle number for amplification of the target cDNA of
-          expected, the <em>E. coli </em>without the plasmid (-) showed amplification of unspecific targets (figure 2),
+        <em>E. coli</em> cells containing the plasmid BBa_J61002 with different promoters (100, 112, 118). As
-          which can be derived from high C<sub>t</sub> values. This indicates, that the primers we designed bind to RFP
+        expected, the <em>E. coli </em>without the plasmid (-) showed amplification of unspecific targets (figure 2),
-          cDNA specifically.
+        which can be derived from high C<sub>t</sub> values. This indicates, that the primers we designed bind to RFP
-        </p>
+        cDNA specifically.
-        <br />
+      </p>
-        <p>
+      <br />
-          To quantify the expression levels of RFP under different promoters, the cDNA of a house-keeping gene was
+      <p>
-          amplified as well. After we spoke to Dr. Trachtmann, who is an expert in performing and evaluating RT-qPCR
+        To quantify the expression levels of RFP under different promoters, the cDNA of a house-keeping gene was
-          experiments, we chose a GTPase-activation protein (GAP) as a housekeeping gene. The specific primers to
+        amplified as well. After we spoke to Dr. Trachtmann, who is an expert in performing and evaluating RT-qPCR
-          amplify the desired house-keeping gene were kindly provided by Dr. Trachtmann.
+        experiments, we chose a GTPase-activation protein (GAP) as a housekeeping gene. The specific primers to
-        </p>
+        amplify the desired house-keeping gene were kindly provided by Dr. Trachtmann.
+      </p>
-        <div class="has-text-centered" style="width: 75%; margin:0 auto;">
+      <div class="has-text-centered" style="width: 75%; margin:0 auto;">
-          <canvas id="gap"></canvas>
+        <canvas id="gap"></canvas>
-        </div>
+      </div>
-        <small>
+      <small>
-          Figure 3 - Fluorescence signal over cycle number for amplification of the house keeping gene GAP cDNA of
+        Figure 3 - Fluorescence signal over cycle number for amplification of the house keeping gene GAP cDNA of
-          <em>E. coli</em> cells containing the plasmid BBa_J61002 with different promoters (100, 112, 118). Means and
+        <em>E. coli</em> cells containing the plasmid BBa_J61002 with different promoters (100, 112, 118). Means and
-          standard deviation of technical triplicates are shown.
+        standard deviation of technical triplicates are shown.
-        </small>
+      </small>
-        <script>
+      <script>
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-        <small>
+      <small>
-          Figure 4 - Fluorescence signal over cycle number for amplification of the house keeping gene GAP cDNA of
+        Figure 4 - Fluorescence signal over cycle number for amplification of the house keeping gene GAP cDNA of
-          <em>E. coli</em> cells without the plasmid BBa_J61002. Means and standard deviation of technical triplicates
+        <em>E. coli</em> cells without the plasmid BBa_J61002. Means and standard deviation of technical triplicates
-          are shown
+        are shown
-        </small>
+      </small>
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-                   * whether to interpet the plus/minus values, relative to the value itself (default) or absolute
+                 * whether to interpet the plus/minus values, relative to the value itself (default) or absolute
-                   * @default false
+                 * @default false
-                   */
+                 */
-                  absoluteValues: false
+                absoluteValues: false
-                }
                }
              }
-           };
+           }
-         </script>
+         };
-        <br />
+      </script>
-        <br />
+      <br />
-        <p>
+      <br />
-          Figure 3 and 4 showed that GAP is present in all samples. Under the assumption, that GAP is expressed under
+      <p>
-          constant rates, one is able to quantify the expression of the target gene relatively to the expression of the
+        Figure 3 and 4 showed that GAP is present in all samples. Under the assumption, that GAP is expressed under
-          house-keeping gene. Analytik Jena provided a qPCR device, which came with a software to interpret our results
+        constant rates, one is able to quantify the expression of the target gene relatively to the expression of the
-          using the ddCt-method. Using this method, revealed the results shown in figure 5.
+        house-keeping gene. Analytik Jena provided a qPCR device, which came with a software to interpret our results
-        </p>
+        using the ddCt-method. Using this method, revealed the results shown in figure 5.
+      </p>
-        <br />
+      <br />
-        <img
+      <img
-          style="margin: 0 auto; display: block; max-width: 700px"
+              style="margin: 0 auto; display: block; max-width: 700px"
-          src="https://2019.igem.org/wiki/images/thumb/5/50/T--Stuttgart--balkendiagramm_charaktereisierung.png/800px-T--Stuttgart--balkendiagramm_charaktereisierung.png"
+              src="https://2019.igem.org/wiki/images/thumb/5/50/T--Stuttgart--balkendiagramm_charaktereisierung.png/800px-T--Stuttgart--balkendiagramm_charaktereisierung.png"
-        />
+      />
-        <small>
+      <small>
-          Figure 5 - Relative quantification of RFP expression compared to the expression of the house-keeping gene GAP
+        Figure 5 - Relative quantification of RFP expression compared to the expression of the house-keeping gene GAP
-          in <em>E. coli </em>MG1655. Cells contained the plasmid BBa_J61002 with different promoters: BBa_J23100,
+        in <em>E. coli </em>MG1655. Cells contained the plasmid BBa_J61002 with different promoters: BBa_J23100,
-          BBa_J23112 and BBa_J23118. As a control <em>E. coli </em>MG1655 were cultivated without the plasmid.
+        BBa_J23112 and BBa_J23118. As a control <em>E. coli </em>MG1655 were cultivated without the plasmid.
-        </small>
+      </small>
-        <br />
+      <br />
-        <br />
+      <br />
-        <p>
+      <p>
-          RFP expression was about 7-fold higher than the expression of GAP in cells containing promoter BBa_J23100.
+        RFP expression was about 7-fold higher than the expression of GAP in cells containing promoter BBa_J23100.
-          Compared to that, the expression of RFP in cells containing promoter BBa_J23112 was about 55-fold higher than
+        Compared to that, the expression of RFP in cells containing promoter BBa_J23112 was about 55-fold higher than
-          expression of the house-keeping gene. The highest expression of RFP compared to GAP was observed in cells
+        expression of the house-keeping gene. The highest expression of RFP compared to GAP was observed in cells
-          containing promoter BBa_J23118. Here, RFP expression was 74-fold compared to GAP expression.
+        containing promoter BBa_J23118. Here, RFP expression was 74-fold compared to GAP expression.
-        </p>
+      </p>
-        <br />
+      <br />
-        <br />
+      <br />
-        <div class="has-text-centered" style="width: 75%; margin:0 auto;">
+      <div class="has-text-centered" style="width: 75%; margin:0 auto;">
-          <canvas id="mrfpschmelz"></canvas>
+        <canvas id="mrfpschmelz"></canvas>
-        </div>
+      </div>
-        <small>
+      <small>
-          Figure 6: Melting curve of the amplification mix of the target cDNA (RFP) of <em>E. coli</em> cells containing
+        Figure 6: Melting curve of the amplification mix of the target cDNA (RFP) of <em>E. coli</em> cells containing
-          the plasmid BBa_J61002 with different promoters (100, 112, 118). Means and standard deviation of technical
+        the plasmid BBa_J61002 with different promoters (100, 112, 118). Means and standard deviation of technical
-          triplicates are shown.
+        triplicates are shown.
-        </small>
+      </small>
-        <script>
+      <script>
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+      </script>
-        <br />
+      <br />
-        <div class="has-text-centered" style="width: 75%; margin:0 auto;">
+      <div class="has-text-centered" style="width: 75%; margin:0 auto;">
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+        <canvas id="gapschmelz"></canvas>
-        </div>
+      </div>
-        <small>
+      <small>
-          Figure 7 - Melting curve of the amplification mix of the house keeping gene GAP cDNA of <em>E. coli</em> cells
+        Figure 7 - Melting curve of the amplification mix of the house keeping gene GAP cDNA of <em>E. coli</em> cells
-          containing the plasmid BBa_J61002 with different promoters (100, 112, 118). Means and standard deviation of
+        containing the plasmid BBa_J61002 with different promoters (100, 112, 118). Means and standard deviation of
-          technical triplicates are shown.
+        technical triplicates are shown.
-        </small>
+      </small>
-        <script>
+      <script>
-          var gapschmelzconfig = {
+        var gapschmelzconfig = {
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+          type: 'line',
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-        <small>
+      <small>
-          Figure 8 - Melting curve of the amplification mix of the house keeping gene GAP cDNA of <em>E. coli</em> cells
+        Figure 8 - Melting curve of the amplification mix of the house keeping gene GAP cDNA of <em>E. coli</em> cells
-          without the plasmid BBa_J61002. Means and standard deviation of technical triplicates are shown.
+        without the plasmid BBa_J61002. Means and standard deviation of technical triplicates are shown.
-        </small>
+      </small>
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+      <script>
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-        <small>
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-          Figure 9: Melting curve of the amplification mix of the target cDNA (RFP) of <em>E. coli</em> cells without
+        Figure 9: Melting curve of the amplification mix of the target cDNA (RFP) of <em>E. coli</em> cells without
-          the plasmid BBa_J61002. Means and standard deviation of technical triplicates are shown.
+        the plasmid BBa_J61002. Means and standard deviation of technical triplicates are shown.
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+                 /**
-                 xAxes: [
+                 * stroke color, or array of colors
-                  {
+                 * @default: derived from borderColor
-                    scaleLabel: {
+                 */
-                      display: true,
-                      labelString: 'temperature [°C]'
-                    },
-                    ticks: {
-                      maxTicksLimit: 10,
-                      precision: 0
-                    }
-                  }
-                ],
-                yAxes: [
-                  {
-                    scaleLabel: {
-                      display: true,
-                      labelString: 'ddRn/dT'
-                    },
-                    ticks: {
-                      min: -2,
-                      max: 20
-                    }
-                  }
-                ]
-              },
-              plugins: {
-                chartJsPluginErrorBars: {
-                  /**
-                   * stroke color, or array of colors
-                   * @default: derived from borderColor
-                   */
-                  /**
+                /**
-                   * bar width in pixel as number or string or bar width in percent based on the barchart bars width (max 100%), or array of such definition
+                 * bar width in pixel as number or string or bar width in percent based on the barchart bars width (max 100%), or array of such definition
-                   * @default 10
+                 * @default 10
-                   */
+                 */
-                  width: 10 | '10px' | '60%',
+                width: 10 | '10px' | '60%',
-                  /**
+                /**
-                   * lineWidth as number, or as string with pixel (px) ending
+                 * lineWidth as number, or as string with pixel (px) ending
-                   */
+                 */
-                  lineWidth: 2 | '2px',
+                lineWidth: 2 | '2px',
-                  /**
+                /**
-                   * lineWidth as number, or as string with pixel (px) ending, or array of such definition
+                 * lineWidth as number, or as string with pixel (px) ending, or array of such definition
-                   * @default 2
+                 * @default 2
-                   */
+                 */
-                  lineWidth: 2 | '2px',
+                lineWidth: 2 | '2px',
-                  /**
+                /**
-                   * whether to interpet the plus/minus values, relative to the value itself (default) or absolute
+                 * whether to interpet the plus/minus values, relative to the value itself (default) or absolute
-                   * @default false
+                 * @default false
-                   */
+                 */
-                  absoluteValues: false
+                absoluteValues: false
-                }
                }
              }
-           };
+           }
-         </script>
+         };
+      </script>
+      <br />
+      <br />
+      <p>
+        The melting curves showed that specific amplification products were generated (figure 6, figure 7, figure 8).
+        As E. Coli MG1655 does not contain any target sequences for RFP primers when no plasmid is present, the
+        melting curves show unspecific product generation (figure 9).
+      </p>
+      <br />
+      <br />
+      <h3 class="title is-4">
+        Results of agarose gel electrophoresis
+      </h3>
+      <p>
+        Exposing the gel under UV light, reveals that the expected amplification product was generated (about 350 bp).
+      </p>
+      <img
+              style="margin: 0 auto; display: block; max-width: 700px"
+              src="https://2019.igem.org/wiki/images/5/54/T--Stuttgart--gelbild_charakterisierung.png"
+      />
+      <small>
+        Figure 10: Amplification mix loaded onto a 2 % agarose gel reveals production of one amplification product
+        for cells containing the plasmid with different promoters.
+      </small>
+      <p>
          <br />
          <br />
-        <p>
+      <strong>Conclusion</strong> <br />
-          The melting curves showed that specific amplification products were generated (figure 6, figure 7, figure 8).
-          As E. Coli MG1655 does not contain any target sequences for RFP primers when no plasmid is present, the
-          melting curves show unspecific product generation (figure 9).
-        </p>
          <br />
-        <br />
+      We were surprised by the results of the characterization of the Anderson promoters as they do not meet our expectations. Actually, we expected that the use of different promoter strengths would result in different levels of mRNA in a way that weak promoters would result in weak fluorescence and low corresponding mRNA levels and strong promoters would lead to high corresponding mRNA levels. More precisely, we expected cells with the promoter BBa_J23100 to exhibit the highest mRNA level, as it was supposed to be the strongest promoter of the ones we characterized.<br>
-        <h3 class="title is-4">
+      When measuring fluorescence, we obtained the expected results: it was revealed, that cells with the promoter BBa_J23100 (strongest promoter) produced more RFP than cells with the promoter BBa_J23118. Cells with the weakest promoter (BBa_J23112) produced fewest amounts of RFP.<br>
-          Results of agarose gel electrophoresis
+      Possible reasons for the discrepancy between promoter strength, RFP amount and mRNA concentration could lie in the promoter library itself: Changes in the promoter sequence to generate the library could lead to impaired secondary structure of the mRNA. This in turn may influence the binding of ribosomes and the translation initiation efficiency significantly. <br>
-        </h3>
+      It may be possible, that the differences in fluorescence are mainly caused by changes on the translational level and not on transcriptional level, as to date promotor strength for these biobricks was only determined by fluorescence measurements.<br>
-        <p>
+      <br/>
-          Exposing the gel under UV light, reveals that the expected amplification product was generated (about 350 bp).
+      <br/>
-        </p>
+      </p>
-        <img
+      <script>
-          style="margin: 0 auto; display: block; max-width: 700px"
+        window.onload = function() {
-          src="https://2019.igem.org/wiki/images/5/54/T--Stuttgart--gelbild_charakterisierung.png"
+           var mRFPctx = document.getElementById('mRFP').getContext('2d');
-        />
+          window.myLine = new Chart(mRFPctx, mRFPconfig);
-        <small>
-          Figure 10 - Amplification mix loaded onto a 2 % agarose gel reveals production of one amplification product
-           for cells containing the plasmid with different promoters.
-        </small>
-        <script>
+           var wtmrfpctx = document.getElementById('wtmrfp').getContext('2d');
-           window.onload = function() {
+          window.myLine = new Chart(wtmrfpctx, wtmrfpconfig);
-            var mRFPctx = document.getElementById('mRFP').getContext('2d');
-            window.myLine = new Chart(mRFPctx, mRFPconfig);
-            var wtmrfpctx = document.getElementById('wtmrfp').getContext('2d');
+          var gapctx = document.getElementById('gap').getContext('2d');
-            window.myLine = new Chart(wtmrfpctx, wtmrfpconfig);
+          window.myLine = new Chart(gapctx, gapconfig);
-            var gapctx = document.getElementById('gap').getContext('2d');
+          var wtgapctx = document.getElementById('wtgap').getContext('2d');
-            window.myLine = new Chart(gapctx, gapconfig);
+          window.myLine = new Chart(wtgapctx, wtgapconfig);
-            var wtgapctx = document.getElementById('wtgap').getContext('2d');
+          var mrfpschmelzctx = document.getElementById('mrfpschmelz').getContext('2d');
-            window.myLine = new Chart(wtgapctx, wtgapconfig);
+          window.myLine = new Chart(mrfpschmelzctx, mrfpschmelzconfig);
-            var mrfpschmelzctx = document.getElementById('mrfpschmelz').getContext('2d');
+          var gapschmelzctx = document.getElementById('gapschmelz').getContext('2d');
-            window.myLine = new Chart(mrfpschmelzctx, mrfpschmelzconfig);
+          window.myLine = new Chart(gapschmelzctx, gapschmelzconfig);
-            var gapschmelzctx = document.getElementById('gapschmelz').getContext('2d');
+          var wtgapschmelzctx = document.getElementById('wtgapschmelz').getContext('2d');
-            window.myLine = new Chart(gapschmelzctx, gapschmelzconfig);
+          window.myLine = new Chart(wtgapschmelzctx, wtgapschmelzconfig);
-            var wtgapschmelzctx = document.getElementById('wtgapschmelz').getContext('2d');
+          var wtTmRFPSchmelzctx = document.getElementById('wtmRFPSchmelz').getContext('2d');
-            window.myLine = new Chart(wtgapschmelzctx, wtgapschmelzconfig);
+          window.myLine = new Chart(wtTmRFPSchmelzctx, wtmRFPSchmelzconfig);
+        };
-            var wtTmRFPSchmelzctx = document.getElementById('wtmRFPSchmelz').getContext('2d');
+      </script>
-            window.myLine = new Chart(wtTmRFPSchmelzctx, wtmRFPSchmelzconfig);
-          };
-        </script>
-      </div>
      </div>
    </div>
+</div>
 </html>

Difference between revisions of "Team:Stuttgart/Contribution"