Team:SDU-Denmark/Tour53

From 2013.igem.org

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<h4>Characterization of dxs <span class="specialWord">(B. subtilis)</span></h4>
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<h2>Characterization of dxs <span class="specialWord">(B. subtilis)</span></h2>
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<span class="intro">Optimization of the procedure is needed</span> before characterization of the dxs bricks can be completed using this approach. We suspect that the complexity of the bacterial samples is too high, and thus the reaction does not take place as fast as might be necessary for detection in our setup. Sonication of bacterial samples with and without addition of standard DMAPP, and subsequent measurements might shed some light on this hypothesis.  
<span class="intro">Optimization of the procedure is needed</span> before characterization of the dxs bricks can be completed using this approach. We suspect that the complexity of the bacterial samples is too high, and thus the reaction does not take place as fast as might be necessary for detection in our setup. Sonication of bacterial samples with and without addition of standard DMAPP, and subsequent measurements might shed some light on this hypothesis.  
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However, it should be noted that the GC wasn’t fully functional during the test period, consequently leading to broader peaks and thus lowered the sensitivity of the instrument. On the 3rd of October, we received a mail from Professor Lars Porskjær Christensen, Department of Chemistry-, Bio- and Environmental Technology, University of Southern Denmark:  
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However, it should be noted that the GC wasn’t fully functional during the test period, consequently leading to broader peaks and thus lowered the sensitivity of the instrument. On the 3<sup>rd</sup> of October, we received a mail from Professor Lars Porskjær Christensen, Department of Chemistry-, Bio- and Environmental Technology, University of Southern Denmark:  
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Unfortunately, with only 2 days left to wiki-freeze, there was no time for another round of testing.
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Unfortunately, with only 2 days left to wiki-freeze, there was no time for another round of testing. Since the European Jamboree in Lyon we have performed the experiment multiple times, but it still needs further optimization, as our results are inconclusive.  
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<a class="popupImg alignRight" style="width:300px" href="https://static.igem.org/mediawiki/2013/6/68/SDU2013_Characterization_Dxs_2.png" title="Figure 2 - Growth curve of dxs devices. Cells were started at OD<sub>600</sub>=0.005 and incubated at 37ºC and 180 rpm. 2 triplicates of MG1655 carrying no vector (WT), empty pSB1C3 vector (pSB1C3), pSB1C3-Plac-dxs(B.subtilis) (BBa_K1088011) (No LacI), pSB1C3-Pcon-lacI-Plac-dxs(B.subtilis) (BBa_K1088027) (LacI(N)), or pSB1C3-Pcon-lacI:LVA-Plac-dxs(B.subtilis) (BBa_K1088013) (LacI(LVA)). At time 2.5 hours one of each triplicate was induced with 1 mM IPTG. No growth change was observed.">
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<a class="popupImg alignRight" style="width:300px" href="https://static.igem.org/mediawiki/2013/6/68/SDU2013_Characterization_Dxs_2.png" title="Figure 2 - Growth curve of Dxs devices. Cells were started at OD<sub>600</sub>=0.005 and incubated at 37ºC and 180 rpm. 2 triplicates of MG1655 carrying no vector (WT), empty pSB1C3 vector (pSB1C3), pSB1C3-Plac-<i>dxs (B.subtilis)</i> (BBa_K1088011) (No LacI), pSB1C3-Pcon-<i>lacI</i>-Plac-<i>dxs (B.subtilis)</i> (BBa_K1088027) (LacI(N)), or pSB1C3-Pcon-<i>lacI:LVA</i>-Plac-<i>dxs (B.subtilis)</i> (BBa_K1088013) (LacI(LVA)). At time 2.5 hours one of each triplicate was induced with 1 mM IPTG. No growth change was observed.">
   <img src="https://static.igem.org/mediawiki/2013/6/68/SDU2013_Characterization_Dxs_2.png" style="width:300px" />
   <img src="https://static.igem.org/mediawiki/2013/6/68/SDU2013_Characterization_Dxs_2.png" style="width:300px" />
Figure 2.  
Figure 2.  
</a>
</a>
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Carrying the dxs devices and expressing the gene could impair the growth, and hence be important in production purpose. To test if the growth of MG1655 bacteria is impaired when carrying and expressing our dxs devices, we measured the growth rate with OD<sub>600</sub> measurements.  
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Carrying the Dxs devices and expressing the gene could impair the growth, and hence be important in production purpose. To test if the growth of MG1655 bacteria is impaired when carrying and expressing our Dxs devices, we measured the growth rate with OD<sub>600</sub> measurements.  
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<a class="dialogLink" href="http://parts.igem.org/Part:BBa_K1088011">pSB1C3-Plac-dxs <span class="specialWord">(B. subtilis)</span></a>,  
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<a class="dialogLink" href="http://parts.igem.org/Part:BBa_K1088011">pSB1C3-Plac-<span class="specialWord">dxs (B. subtilis)</span></a>,  
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<a class="dialogLink" href="http://parts.igem.org/Part:BBa_K1088027">pSB1C3-Pcon-lacI-Plac-dxs <span class="specialWord">(B. subtilis)</span></a>, or  
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<a class="dialogLink" href="http://parts.igem.org/Part:BBa_K1088027">pSB1C3-Pcon-<span class="specialWord">lacI</span>-Plac-<span class="specialWord">dxs (B. subtilis)</span></a>, or  
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<a class="dialogLink" href="http://parts.igem.org/Part:BBa_K1088013">pSB1C3-Pcon-lacI:LVA-Plac-dxs <span class="specialWord">(B. subtilis)</span></a> were started from ONC at time 0 hours, OD<sub>600</sub>=0.005, and grown at 37ºC and 180 rpm. OD<sub>600</sub> measurements were done every half an hour, and 1 of each triplicates was induced at time 2.5 hours. All strains grew at the same pace and induction didn’t impair growth rate <b>(Fig. 2)</b>.  
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<a class="dialogLink" href="http://parts.igem.org/Part:BBa_K1088013">pSB1C3-Pcon-<span class="specialWord">lacI:LVA</span>-Plac-<span class="specialWord">dxs (B. subtilis)</span></a> were started from ONC at time 0 hours, OD<sub>600</sub>=0.005, and grown at 37ºC and 180 rpm. OD<sub>600</sub> measurements were done every half an hour, and 1 of each triplicates was induced at time 2.5 hours. All strains grew at the same pace and induction didn’t impair growth rate <b>(Fig. 2)</b>.  
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<h4>Characterization of HRT2</h4>
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<h2>Characterization of HRT2</h2>
<span class="intro">Rubber purification</span><br>
<span class="intro">Rubber purification</span><br>
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<span class="intro">Two different methods</span> were tested to evaluate the most efficient extraction method. In the first method, we washed with acetone and extract with n-hexane in a soxhlet extractor. The second method excluded the time-consuming soxhlet steps and washed with acetone for a shorter duration of time (15 min shake at 37 deg). Rubber was extracted by adding hexane to the cell suspension, then centrifuging the sample. The supernatent was saved (hexane solution). We tested the methods of rubber extraction on a positive control made from MG1655, mixed with polyisoprene. Both the soxhlet and the non-soxhlet method were evaluated using H<sup>1</sup>-NMR. We deemed the results to be similar, and since the required time for non-soxhlet extraction was significantly less, we chose to use the non-soxleth method.
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<span class="intro">Two different methods</span> were tested to evaluate the most efficient extraction method. In the first method, we washed with acetone and extract with n-hexane in a soxhlet extractor. The second method excluded the time-consuming soxhlet steps and washed with acetone for a shorter duration of time (15 min shake at 37ºC). Rubber was extracted by adding hexane to the cell suspension, then centrifuging the sample. The supernatent was saved (hexane solution). We tested the methods of rubber extraction on a positive control made from MG1655, mixed with polyisoprene. Both the soxhlet and the non-soxhlet method were evaluated using H<sup>1</sup>-NMR. We deemed the results to be similar, and since the required time for non-soxhlet extraction was significantly less, we chose to use the non-soxleth method.
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<h4>The H<sup>1</sup> NMR test</h4>
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<div class="imageGallery galleryMedium alignCenter">
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<a class="galleryImg" target="_blank" href="https://static.igem.org/mediawiki/2013/1/1b/SDU2013_Characterization_NMR_1.png" title="Figure 3 - Spectrum illustrating the pure polyisoprene standard (Mw = 38 kDa) H NMR spectrum. The peaks at 5.12, 2.04 and 1.68 indicates the (A), (B) and (C) protons of the isoprene monomer respectively. This sample was not dried in vacuum oven, and therefore we have a large peak at aprox. 1.55 corresponding to water.">
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<img src="https://static.igem.org/mediawiki/2013/1/1b/SDU2013_Characterization_NMR_1.png">Figure 3.</a>
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<a class="galleryImg" target="_blank" href="https://static.igem.org/mediawiki/2013/f/f3/SDU2013_Characterization_NMR_2.png" title="Figure 4 - Spectrum illustrating the WT + polyisoprene standard, rubber purified, H NMR spectrum. The peaks at 5.12, 2.04 and 1.68 indicates the (A), (B) and (C) protons of the isoprene monomer respectively in this spectrum as well as the aforementioned pure polyisoprene H NMR spectrum. The different solvents of the rubber purification procedure is seen as well, however these are not interfering with out isoprene proton shifts.">
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<img src="https://static.igem.org/mediawiki/2013/f/f3/SDU2013_Characterization_NMR_2.png">Figure 4.</a>
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<a class="galleryImg" target="_blank" href="https://static.igem.org/mediawiki/2013/0/04/SDU2013_Characterization_NMR_3.png" title="Figure 5 - Spectrum illustrating our PT+DXS part. In this spectrum it is important to notice the same peaks as before, 5.12, 2.04, and 1.68 which reveals the presence of our polyisoprene. The same solvents as the previous spectrum is present, but the focus should be put towards the isoprene presence, proving the effect of HRT2/PT.">
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<img src="https://static.igem.org/mediawiki/2013/0/04/SDU2013_Characterization_NMR_3.png">Figure 5.</a>
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<b> &nbsp; </b>
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</div>
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<span class="intro">The H<sup>1</sup> NMR test</span><br>
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We now seemed able to detect rubber that was extracted from a mixture of bacterial debris and polyisoprene, and felt ready for a test of our production system. H<sup>1</sup> NMR allowed us a certain specificity and although a significant amount of polyisoprene was needed to enable detection, it was preferred over C<sup>13</sup>NMR. All samples were run an a bruker 400 mHz NMR spectrometer. We also knew that we could control the expression of the genes in both of our plasmids. We were ready to put our abilities to extract rubber through the non-soxhlet method and detect it using H<sup>1</sup> NMR to the test. In other words, we were ready to find the rubber present in  
We now seemed able to detect rubber that was extracted from a mixture of bacterial debris and polyisoprene, and felt ready for a test of our production system. H<sup>1</sup> NMR allowed us a certain specificity and although a significant amount of polyisoprene was needed to enable detection, it was preferred over C<sup>13</sup>NMR. All samples were run an a bruker 400 mHz NMR spectrometer. We also knew that we could control the expression of the genes in both of our plasmids. We were ready to put our abilities to extract rubber through the non-soxhlet method and detect it using H<sup>1</sup> NMR to the test. In other words, we were ready to find the rubber present in  
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<span class="intro">During the first test</span> of our CPS bacteria (MG1655 transformed with both plasmids - see <a class="dialogLink" href="https://2013.igem.org/Team:SDU-Denmark/Tour33" target="_blank">Design</a>), bacteria were grown to late exponential phase at 37 deg, then induced with 1mM IPTG, 0.2% Ara, and 1mM MgCl2. It was then moved to room temperature for a further 4 hours of growth. Non-soxhlet extraction was used to purify any rubber present in the sample, which was then compared to a positive control (WT mixed with polyisopren - also undergone non-soxhlet extraction) and a standard isoprene solution.
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<span class="intro">During the first test</span> of our CPS bacteria (MG1655 transformed with both plasmids - see <a class="dialogLink" href="https://2013.igem.org/Team:SDU-Denmark/Tour33" target="_blank">Design</a>), bacteria were grown to late exponential phase at 37ºC, then induced with 1mM IPTG, 0.2% Ara, and 1mM MgCl2. It was then transfered to room temperature for another 4 hours of growth. Non-soxhlet extraction was used to purify any rubber present in the sample, which was then compared to a positive control (WT mixed with polyisopren - also undergone non-soxhlet extraction) and a standard isoprene solution.
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<p>
Our rubber extraction of WT + polyisoprene <b>(Fig. 4)</b> gives the same peak placement as the pure polyisoprene ((A), (B) and (C)). However, the integration of the 3 peaks shows a relationship of approximately 1:5:4. The discrepancy can be explained by the impurities in the area between 0-2.5 ppm. Such impurities might add additional integration value to the peaks assigned to the polyisoprene peaks (B) and (C) - hereby causing a disruption of the true relationship. Some peaks from the solvents used to purify the rubber (Acetone, Ethanol, and Hexane), as well as a small amount of water (1.56 ppm) are seen, too.
Our rubber extraction of WT + polyisoprene <b>(Fig. 4)</b> gives the same peak placement as the pure polyisoprene ((A), (B) and (C)). However, the integration of the 3 peaks shows a relationship of approximately 1:5:4. The discrepancy can be explained by the impurities in the area between 0-2.5 ppm. Such impurities might add additional integration value to the peaks assigned to the polyisoprene peaks (B) and (C) - hereby causing a disruption of the true relationship. Some peaks from the solvents used to purify the rubber (Acetone, Ethanol, and Hexane), as well as a small amount of water (1.56 ppm) are seen, too.
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</p>
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<div class="imageGallery galleryMedium alignCenter">
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<a class="galleryImg" target="_blank" href="https://static.igem.org/mediawiki/2013/1/1b/SDU2013_Characterization_NMR_1.png" title="Figure 3 - Spectrum illustrating the pure polyisoprene standard (Mw = 38 kDa) H NMR spectrum. The peaks at 5.12, 2.04 and 1.68 indicates the (A), (B) and (C) protons of the isoprene monomer respectively. This sample was not dried in vacuum oven, and therefore we have a large peak at aprox. 1.55 corresponding to water.">
 +
<img src="https://static.igem.org/mediawiki/2013/1/1b/SDU2013_Characterization_NMR_1.png">Figure 3.</a>
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<a class="galleryImg" target="_blank" href="https://static.igem.org/mediawiki/2013/f/f3/SDU2013_Characterization_NMR_2.png" title="Figure 4 - Spectrum illustrating the WT + polyisoprene standard, rubber purified, H NMR spectrum. The peaks at 5.12, 2.04 and 1.68 indicates the (A), (B) and (C) protons of the isoprene monomer respectively in this spectrum as well as the aforementioned pure polyisoprene H NMR spectrum. The different solvents of the rubber purification procedure is seen as well, however these are not interfering with out isoprene proton shifts.">
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<img src="https://static.igem.org/mediawiki/2013/f/f3/SDU2013_Characterization_NMR_2.png">Figure 4.</a>
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<a class="galleryImg" target="_blank" href="https://static.igem.org/mediawiki/2013/0/04/SDU2013_Characterization_NMR_3.png" title="Figure 5 - Spectrum illustrating our HRT2+DXS part. In this spectrum it is important to notice the same peaks as before, 5.12, 2.04, and 1.68 which reveals the presence of our polyisoprene. The same solvents as the previous spectrum is present, but the focus should be put towards the isoprene presence, proving the effect of HRT2.">
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<img src="https://static.igem.org/mediawiki/2013/0/04/SDU2013_Characterization_NMR_3.png">Figure 5.</a>
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<b> &nbsp; </b>
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<span class="intro">The conclusion arrives in figure 5</span>, named DXS+PT, which is our double plasmid CPS bacteria, and displays the same peaks (A), (B) and (C) as both the WT + polyisoprene and pure polyisoprene. This is a strong indication of the presence of rubber - specifically, this is an indication that our CPS bacteria produces rubber: Bacteriorganic Rubber. Peak distortion of the spectrum due to solvents are the same as seen in the rubber purification from WT + polyisoprene.
<span class="intro">The conclusion arrives in figure 5</span>, named DXS+PT, which is our double plasmid CPS bacteria, and displays the same peaks (A), (B) and (C) as both the WT + polyisoprene and pure polyisoprene. This is a strong indication of the presence of rubber - specifically, this is an indication that our CPS bacteria produces rubber: Bacteriorganic Rubber. Peak distortion of the spectrum due to solvents are the same as seen in the rubber purification from WT + polyisoprene.
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  <img src="https://static.igem.org/mediawiki/2013/0/06/SDU2013_Characterization_NMR_4.png">Fig. 6</a>
  <img src="https://static.igem.org/mediawiki/2013/0/06/SDU2013_Characterization_NMR_4.png">Fig. 6</a>
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<a class="galleryImg" target="_blank" href="https://static.igem.org/mediawiki/2013/9/9a/SDU2013_Characterization_NMR_5.png" title="Figure 7 - Spectrum illustrating PT+DXS, which have undergone rubber purification. The peak at 5.12 is vaguely present, and the rest of the peaks assigned to isoprene are hidden in the noise from the solvent and impurities. The peak at 5.12 is a triplet that corresponds to the previously observed shape of the 5.12 peak, and we expect that it is a indicator of the presence of polyisoprene production.">
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<a class="galleryImg" target="_blank" href="https://static.igem.org/mediawiki/2013/9/9a/SDU2013_Characterization_NMR_5.png" title="Figure 7 - Spectrum illustrating HRT2+DXS, which have undergone rubber purification. The peak at 5.12 is vaguely present, and the rest of the peaks assigned to isoprene are hidden in the noise from the solvent and impurities. The peak at 5.12 is a triplet that corresponds to the previously observed shape of the 5.12 peak, and we expect that it is a indicator of the presence of polyisoprene production.">
  <img src="https://static.igem.org/mediawiki/2013/9/9a/SDU2013_Characterization_NMR_5.png">Fig. 7</a>
  <img src="https://static.igem.org/mediawiki/2013/9/9a/SDU2013_Characterization_NMR_5.png">Fig. 7</a>
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<span class="intro">A second round of testing</span> was done to validate the first experiment. We wanted to include a negative test (WT), in order to exclude the possibility of a naturally occurring polyisoprenoid compounds in E. coli. We performed rubber purification on WT, our CPS bacteria as well as a strain containing only the pSB1K3-AraC-Para-PT device. Bacteria were grown and rubber extracted as described above. The three samples where unfortunately not dried properly in the vacuum oven due to apparatus malfunction.
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<span class="intro">A second round of testing</span> was done to validate the first experiment. We wanted to include a negative test (WT), in order to exclude the possibility of a naturally occurring polyisoprenoid compounds in <span class="specialWord">E. coli</span>. We performed rubber purification on WT, our CPS bacteria as well as a strain containing only the pSB1K3-<span class="specialWord">araC</span>-Para-<span class="specialWord">HRT2</span> device. Bacteria were grown and rubber extracted as described above. The three samples where unfortunately not dried properly in the vacuum oven due to apparatus malfunction.
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The test provided the following results: <b>Fig 6</b> lacks the characteristic peaks of polyisoprene at 5.12, 2.04 and 1.68. It can be concluded from their absence that there is not rubber (nor any other compound that might provide a similar chemical shift values) in the wildtype. In the CPS <b>(Fig. 7)</b>, we observe very a slight peak at 5.12, indicating the presence of (A) hydrogen. This peak has the same splitting pattern as the first round of H<sup>1</sup>-NMR, but it has a very low intensity. The (B) and (C) peaks are hidden in the background noise, which is most likely due to cell debris and solvents, which did not evaporate appropriately. We suspect that the machinery has a decreased sensitivity towards the isoprene peaks due to the high amount of solvent seen from the assigned peaks, but the peak at 5.12 (with the recognizable splitting pattern) is a strong indication that rubber is once again present in our CPS bacteria (containing both PT and dxs plasmids).
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The test provided the following results: <b>Fig 6</b> lacks the characteristic peaks of polyisoprene at 5.12, 2.04 and 1.68. It can be concluded from their absence that there is not rubber (nor any other compound that might provide a similar chemical shift values) in the wildtype. In the CPS <b>(Fig. 7)</b>, we observe very a slight peak at 5.12, indicating the presence of (A) hydrogen. This peak has the same splitting pattern as the first round of H<sup>1</sup>-NMR, but it has a very low intensity. The (B) and (C) peaks are hidden in the background noise, which is most likely due to cell debris and solvents, which did not evaporate appropriately. We suspect that the machinery has a decreased sensitivity towards the isoprene peaks due to the high amount of solvent seen from the assigned peaks, but the peak at 5.12 (with the recognizable splitting pattern) is a strong indication that rubber is once again present in our CPS bacteria (containing both HRT2 and Dxs plasmids).
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<span class="intro">The future of our H<sup>1</sup> NMR test rubber characterization experiments</span> will see us replicate our results. Specifically, we must redo the second rubber extraction and rerun the H<sup>1</sup> NMR to verify the rubber presence in our CPS bacteria. Also, this particular rerun allows us to check that the WT is indeed lacking any sign of a peak at 5.12.
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<span class="intro">When we advanced to the finals in Boston</span>, we wanted to verify that our previous experiments where replicable as well as investigate the quantity and yield of rubber. To assess this we did a third round of H<sup>1</sup>-NMR on MG1655 (WT), and MG1655 carrying pSB1C3-Pcon-<span class="specialWord">araC</span>-term-Para-<span class="specialWord">HRT2</span> (HRT2), or pSB1C3-Pcon-<span class="specialWord">lacI(N)</span>-term-<span class="specialWord">dxs (B. subtilis)</span> and pSB1K3-Pcon-<span class="specialWord">araC</span>-term-Para-<span class="specialWord">HRT2</span> (Dxs + HRT2). 1 L LB media was inoculated with the strains, starting from OD<sub>600</sub>=0.05. The cultures were grown at 37ºC with shaking for 5 hours; the temperature was then lowered to 20ºC and cultures were induced with 1mM IPTG, 0,2% arabinose, and 1mM MgCl<sub>2</sub> ON. Rubber purification was commenced the following day, and the final yield was solubilized in 2 mL d-chloroform and analyzed by H<sup>1</sup>-NMR.  
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In addition, we must investigate the rubber yield in terms of weight to further evaluate the two purification methods mentioned above.
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<span class="intro">From the spectrum reflecting</span> the WT strain, none of the peaks originating from polyisoprene were observed <b>(fig. 8)</b>. From the spectrum reflecting the Dxs + HRT2 strain, all three peaks originating from polyisoprene were observed <b>(fig. 9)</b>. Lastly, from the spectrum reflecting the HRT2 strain <b>(fig. 10)</b>, the peaks potentially originating from polyisoprene could not be conclusively identified indicating the decreased rubber production performance of this strain compared to the Dxs + HRT2 strain. This emphasizes the importance of excess IPP and DMAPP (rubber precursors) available as a consequence of Dxs overexpression, and serves as an indirect proof of the effect of MEP pathway optimization.  
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<div class="imageGallery galleryMedium alignCenter">
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<a class="galleryImg" target="_blank" href="https://static.igem.org/mediawiki/2013/7/77/SDU2013_Rubber_WT2.png" title="Figure 8 - Rubber purified MG1655 (WT) H<sup>1</sup>-NMR spectrum. The three characteristic polyisoprene peaks (5.12, 2.04, and 1.68 ppm) are all absent.">
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<img src="https://static.igem.org/mediawiki/2013/7/77/SDU2013_Rubber_WT2.png">
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Figure 8.
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</a>
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<a class="galleryImg" target="_blank" href="https://static.igem.org/mediawiki/2013/3/38/SDU2013_Rubber_CPS2.png" title="Figure 9 - Rubber purified MG1655 carrying pSB1C3-Pcon-lacI(N)-term-dxs and pSB1K3-Pcon-araC-term-Para-HRT2 (Dxs + HRT2) H<sup>1</sup>-NMR spectrum. The three characteristic polyisoprene peaks (5.12, 2.04, and 1.68 ppm) are all present.">
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<img src="https://static.igem.org/mediawiki/2013/3/38/SDU2013_Rubber_CPS2.png">
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Figure 9.
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</a>
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<a class="galleryImg" target="_blank" href="https://static.igem.org/mediawiki/2013/b/b4/SDU2013_Rubber_HRT2.png" title="Figure 10 - Rubber purified MG1655 carrying pSB1C3-Pcon-lacI(N)-term-dxs (HRT2) H<sup>1</sup>-NMR spectrum. The three characteristic polyisoprene peaks (5.12, 2.04, and 1.68 ppm) cannot be concluded to be present.">
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<img src="https://static.igem.org/mediawiki/2013/b/b4/SDU2013_Rubber_HRT2.png">
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Figure 10.
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</a>
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<b> &nbsp; </b>
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<span class="intro">In order to determine rubber yield</span>, we compared the yield from 1L of our Dxs + HRT2 strain with 5 rubber purifications on WT + a selected amount of standard polyisoprene. Yields from all 5 purifications were solubilized in equal volumes of d-chloroform (2 mL), and the resulting TMS peaks could consequently be used for normalization of the polyisoprene peaks at 5.12 ppm. This peak was selected for quantification due to its localization in a region without background noise. From these 5 purifications, we calculated a standard curve to which we compared the integral value of the 5.12 ppm peak originating from our Dxs + HRT2 strain.
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 +
 +
<span class="intro">From the sample with the lowest</span> amount of polyisoprene added (0.2 mg), no peaks could be identified, indicating the detection limit of the H<sup>1</sup>-NMR setup. Together with normalized peak integrals from the other 4 samples, we calculated the standard curve depicted in <b>fig. 13</b>. Based on the standard curve and the integral originating from the sample containing 3.2 mg polyisoprene <b>(Fig. 13, 12)</b>, we roughly estimate our polyisoprene yields to be in the range of 0.5-5 mg polyisoprene/L <b>(fig. 11)</b>. We realize that this yield estimation is far from optimal, however, only the sensitivity of H<sup>1</sup>-NMR allowed us to give a rough estimate of our yield.
 +
 +
</p>
 +
<br>
<p>
<p>
 +
<div class="imageGallery galleryMedium alignCenter">
-
<span class="intro">MALDI-ToF</span><br>
+
<a class="galleryImg" target="_blank" href="https://static.igem.org/mediawiki/2013/6/68/SDU2013_Rubber_IntCPS2.png" title="Figure 11 - Integral of the 5.12 ppm peak originating from the rubber purified MG1655 carrying pSB1C3-Pcon-lacI(N)-term-dxs and pSB1K3-Pcon-araC-term-Para-HRT2 (Dxs + HRT2) relative to the TMS peak. The relative integral value equals 0.16.">
 +
<img src="https://static.igem.org/mediawiki/2013/6/68/SDU2013_Rubber_IntCPS2.png">
 +
Figure 11.
 +
</a>
 +
 
 +
<a class="galleryImg" target="_blank" href="https://static.igem.org/mediawiki/2013/f/ff/SDU2013_Rubber_Int32.png" title="Figure 12 - Integral of the 5.12 ppm peak originating from the rubber purified MG1655 (WT) with addition of 3.2 mg polyisoprene standard relative to the TMS peak. The relative integral value equals 0.09.">
 +
<img src="https://static.igem.org/mediawiki/2013/f/ff/SDU2013_Rubber_Int32.png">
 +
Figure 12.
 +
</a>
 +
 
 +
<a class="galleryImg" target="_blank" href="https://static.igem.org/mediawiki/2013/2/20/SDU2013_Rubber_Standardcurve.png" title="Figure 13 - Standard curve of normalized integrals for known amounts of polyisoprene purified from MG1655 (WT) ONC. The standard curve displays a linear relationship between the normalized 5.12 ppm peak integrals (relative to the TMS peak) and amount of polyisoprene.">
 +
<img src="https://static.igem.org/mediawiki/2013/2/20/SDU2013_Rubber_Standardcurve.png">
 +
Figure 13.
 +
</a>
 +
<b> &nbsp; </b>
 +
</div>
 +
 
 +
 
 +
</p>
 +
<br>
 +
<p>
 +
 
 +
<span class="intro">When we tried to weigh our yield</span>, we observed a many-fold increased yield compared to our input amount, and we assume that this conflicting reading is due to the presence of cell debris and other impurities adding to the output reading. This situation forced us to seek other yield calculation strategies, such as H<sup>1</sup>-NMR.
 +
 
 +
</p>
 +
 
 +
 
 +
<br>
 +
 
 +
<p>
 +
 
 +
 
 +
<h4>MALDI-ToF</h4>
We thoroughly studied the literature concerning polyisoprene on MALDI-ToF and found that the formation of adducts by adding AgNO<sub>3</sub><sup>-</sup> would make it possible to ionize the long alkene chain, despite its lack of functional groups that can be ionized.
We thoroughly studied the literature concerning polyisoprene on MALDI-ToF and found that the formation of adducts by adding AgNO<sub>3</sub><sup>-</sup> would make it possible to ionize the long alkene chain, despite its lack of functional groups that can be ionized.
 +
<span class="sourceReference">be ionized.</span>
 +
<span class="tooltip">
 +
  <span class="tooltipHeader">Source:</span>
 +
 
 +
Hyuneui L, Yeonhee L, Seunghee H, Youngsook Y, and Kang-Jin K. Investigation of Polystyrene, Polyisoprene, and Poly(2-vinylpyridine) using Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry. Bull. Korean Chem. Soc. 1999, Vol. 20, No. 7 p. 853-856. <a href="http://pdf.lookchem.com/pdf/32/19391c15-f5be-4e7d-8b1d-7becdc30c921.pdf" target="_blank">(Link)</a>
 +
 +
</span>
</p>
</p>
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<p>
<p>
 +
<br>
-
<h4>Composite production system</h4>
+
<h2>Composite production system</h2>
-
<span class="intro">Growth experiments</span><br>
+
 
-
Carrying the HRT2, <span class="specialWord">dxs</span> and both (CPS) devices and expressing the genes could impair the growth, and hence be important in production purpose. To test if the growth of MG1655 bacteria is impaired when carrying and expressing our devices, we measured the growth rate with OD<sub>600</sub> measurements.  
+
<h4>Growth experiments</h4>
 +
Carrying the <span class="specialWord">HRT2</span>, <span class="specialWord">dxs</span> and both (CPS) devices and expressing the genes could impair the growth, and hence be important in production purpose. To test if the growth of MG1655 bacteria is impaired when carrying and expressing our devices, we measured the growth rate with OD<sub>600</sub> measurements.  
</p><p>
</p><p>
-
Four different MG1655 strains carrying either no plasmid (WT), <a class="dialogLink" href="http://parts.igem.org/Part:BBa_K1088016" title="">pSB1C3-Pcon-araC-term-Para-HRT2</a> (HRT2), <a class="dialogLink" href="http://parts.igem.org/Part:BBa_K1088024" title="">pSB1C3-Pcon-lacI(N)-term-Plac-dxs(B.subtilis)</a> (Dxs), or pSB1K3-Pcon-araC-term-Para-HRT2 and pSB1C3-Pcon-lacI(N)-term-Plac-dxs(B.subtilis) (CPS), were grown from OD<sub>600</sub>=0.05 and induced with 1 mM IPTG and/or 0.2% arabinose  during growth. The first experiment showed impaired growth of the bacteria only carrying construct expressing HRT2. We thought that the reason why the strains expressing HRT2 as well as Dxs didn’t show impaired growth rate could be due to a phenomenon called inclusion bodies. When overexpressing too many proteins, there is a risk of misfolding and hence loss of function. A way to lower this risk is to lower the temperature. Therefore we set up a growth experiment where the temperature was lowered from 37 to 20ºC at time of induction. This did not prove to make a difference, and CPS strains with and without IPTG and/or arabinose induction grew at same pace as WT <b>(fig 8)</b>
+
Four different MG1655 strains carrying either no plasmid (WT), <a class="dialogLink" href="http://parts.igem.org/Part:BBa_K1088016" title="">pSB1C3-Pcon-<span class="specialWord">araC</span>-term-Para-<span class="specialWord">HRT2</span></a> (HRT2), <a class="dialogLink" href="http://parts.igem.org/Part:BBa_K1088024" title="">pSB1C3-Pcon-<span class="specialWord">lacI(N)</span>-term-Plac-<span class="specialWord">dxs (B.subtilis)</span></a> (Dxs), or pSB1K3-Pcon-<span class="specialWord">araC</span>-term-Para-<span class="specialWord">HRT2</span> and pSB1C3-Pcon-<span class="specialWord">lacI(N)</span>-term-Plac-<span class="specialWord">dxs (B.subtilis)</span> (CPS), were grown from OD<sub>600</sub>=0.05 and induced with 1 mM IPTG and/or 0.2% arabinose  during growth. The first experiment showed impaired growth of the bacteria only carrying construct expressing HRT2. We thought that the reason why the strains expressing HRT2 as well as Dxs didn’t show impaired growth rate could be due to a phenomenon called inclusion bodies. When overexpressing too many proteins, there is a risk of misfolding and hence loss of function. A way to lower this risk is to lower the temperature. Therefore we set up a growth experiment where the temperature was lowered from 37 to 20ºC at time of induction. This did not prove to make a difference, and CPS strains with and without IPTG and/or arabinose induction grew at same pace as WT. However, we want to verify these results by a more thorough investigation and consequently setup a new growth experiment including OD<sub>600</sub> measurements, counts of colony forming units (CFU) and finally scanning electron microscopy <b>(fig. 14)</b>.
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+
</p>
</p>
<p>
<p>
 +
<br>
<a class="popupImg alignCenter" style="width:800px" href="https://static.igem.org/mediawiki/2013/6/64/SDU2013_Characterization_CPS_1.png"  
<a class="popupImg alignCenter" style="width:800px" href="https://static.igem.org/mediawiki/2013/6/64/SDU2013_Characterization_CPS_1.png"  
-
title="Figure 8 - Growth experiments of MG1655 strains carrying either no plasmid (WT), pSB1C3-Pcon-araC-term-Para-HRT2 (BBa_1088016; HRT2), pSB1C3-Pcon-lacI(N)-term-Plac-dxs(B.subtilis) (BBa_1088024; Dxs), or pSB1K3-Pcon-araC-term-Para-HRT2 and pSB1C3-Pcon-lacI(N)-term-Plac-dxs (B. subtilis) (CPS). - and + indicates absence and presence, respectively of 1 mM IPTG and 0.2 % arabinose, respectively as well. The dashed line indicates time of induction with IPTG and/or arabinose. All strains were started at OD600=0.05 from an overnight culture, and were shaked with 180 rpm. A) The HRT2 strain grew slightly slower than the other strains at 37ºC, and upon induction some of the culture even seemed to die, though it catched up with the rest in the stationary phase. B) The CPS strain didn’t reflect what was seen in A when only the HRT2 expression was induced with arabinose. In this experiment WT grew slightly faster than CPS. C) WT and CPS was grown for 3 hours at 37ºC, and along with induction the temperature was lowered to 20ºC. This was done due to a hypothesis about a phenomenon called protein clouding (see main text for details). All strains grew at the same pace, in this experiment.">
+
title="Figure 14 - Growth experiments of MG1655 strains carrying either no plasmid (WT), pSB1C3-Pcon-araC-term-Para-HRT2 (BBa_1088016; HRT2), pSB1C3-Pcon-lacI(N)-term-Plac-dxs (B.subtilis) (BBa_1088024; Dxs), or pSB1K3-Pcon-araC-term-Para-HRT2 and pSB1C3-Pcon-lacI(N)-term-Plac-dxs (B. subtilis) (CPS). - and + indicates absence and presence, respectively of 1 mM IPTG and 0.2 % arabinose, respectively. The dashed line indicates time of induction with IPTG and/or arabinose. All strains were initiated at OD<sub>600</sub>=0.05 from an overnight culture, and were shaked with 180 rpm. <b>A)</b> The HRT2 strain grew slightly slower than the other strains at 37ºC, and upon induction we observed an initial decrease in OD<sub>600</sub> readings, even though it catched up with the other strains in the stationary phase. <b>B)</b> The CPS strain didn’t reflect what was seen in A when only the HRT2 expression was induced with arabinose. In this experiment the WT strain grew slightly faster than the CPS strain. <b>C)</b> WT and CPS strains were grown for 3 hours at 37ºC, and upon induction the temperature was lowered to 20ºC. This was done due to a hypothesis concerning a phenomenon called protein clouding (see main text for details). All strains grew at the same pace in this experiment.">
   <img src="https://static.igem.org/mediawiki/2013/6/64/SDU2013_Characterization_CPS_1.png"  
   <img src="https://static.igem.org/mediawiki/2013/6/64/SDU2013_Characterization_CPS_1.png"  
style="width:800px">
style="width:800px">
-
Figure
+
Figure 14.
</a>
</a>
 +
</p>
 +
<br>
 +
<p>
 +
<h4>Cell viability</h4>
 +
<span class="intro">Three different MG1655 strains</span>; WT, HRT2 and CPS (similar to above), were grown under equal conditions as when cultures were grown for rubber purification. 1 L of bacterial cultures were initiated at OD<sub>600</sub>=0.05, from overnight cultures and grown at 37°C and shaking at 180 rpm. After 5 hours of growth, cultures were induced with 1 mM IPTG, 0.2% arabinose and 1 mM MgCl2 and the temperature was lowered to 20°C to prevent the formation of inclusion bodies. The OD<sub>600</sub> was measured each hour throughout the experiment and sequential dilution series were plated out at time 4, 5, 6, 7 and 8 hours after initiation. Furthermore, samples for electron microscopy were taken from the induced ON cultures.
 +
 +
</p><p>
 +
 +
<span class="intro">OD<sub>600</sub> measurements illustrate</span> a slight impairment of growth for the HRT2 strain compared with the WT strain, whereas the CPS strain shows heavily decreased Growth <b> (Fig. 15A) </b>. CFU counts reflect the number of viable cells in a culture. We observed a steady amount of CFU counts for the WT and HRT2 strains throughout the experiment similar to the CPS strain, although this strain presents with a lower amount of CFU counts <b>(Fig. 15B)</b>.
 +
 +
</p><p>
 +
 +
<span class="intro">We notice that</span> the growth rate of the CPS strains is controversial to the growth experiment presented earlier, which we cannot directly explain. Importantly, the viability of the CPS strain does not decrease upon induction in both growth experiments, which indicates that the amount of produced rubber is not toxic to the bacteria. This might be due to the low amounts of rubber produced in the cells as validated by H<sup>1</sup>-NMR.
 +
 +
</p><p>
 +
<br>
 +
<a class="popupImg alignCenter" style="width:600px" href="https://static.igem.org/mediawiki/2013/e/ed/SDU2013_Rubber_9.png" title="Figure 15 - Cell viability experiment of MG1655 strains carrying either no plasmid (WT), pSB1K3-Pcon-araC-term-Para-HRT2 (HRT2) or pSB1K3-Pcon-araC-term-Para-HRT2 and pSB1C3-Pcon-lacI(N)-term-Plac-dxs(B.subtilis) (CPS). The three strains were grown under equal conditions as when cultures were grown for rubber purification. 1 L of bacterial cultures were initiated at OD<sub>600</sub>=0.05, from ON cultures and grown at 37°C and shaking at 180 rpm. After 5 hours of growth, cultures were induced with 1 mM IPTG, 0.2% arabinose and 1 mM MgCl<sub>2</sub>, indicated by the dashed line, and the temperature was lowered to 20°C to prevent the formation of inclusion bodies. The OD<sub>600</sub> was measured each hour throughout the experiment and sequential dilution series were plated out at time 4, 5, 6, 7 and 8 hours after initiation. <b>A)</b> OD<sub>600</sub> measurements illustrate a slight impairment of growth for the HRT2 strain compared with the WT strain, whereas the CPS strain shows heavily decreased growth. <b>B)</b> A steady amount of CFU counts for the WT and HRT2 strains throughout the experiment similar to the CPS strain was observed, although the CPS strain presents with a lower amount of CFU counts.">
 +
  <img src="https://static.igem.org/mediawiki/2013/e/ed/SDU2013_Rubber_9.png" style="width:600px" />
 +
Figure 15.
 +
</a>
 +
<br>
 +
</p><p>
 +
 +
<span class="intro">Additionally, images from</span> an equal dilution and time point in the CFU assay are displayed below <b>(Fig. 16)</b>. The difference in size between the colonies from the three different strains confirm the decreased growth rate of the CPS strain compared to the WT and HRT2 strains.
 +
 +
</p><p>
 +
<br>
 +
<a class="popupImg alignCenter" style="width:800px" href="https://static.igem.org/mediawiki/2013/f/ff/SDU2013_Rubber_10.png" title="Figure 16 - Images from an equal dilution (10<sup>6</sup>) and time point (6 hours) in the CFU assay. The difference in size between the colonies from <b>A)</b> WT, <b>B)</b> HRT2 and <b>C)</b> CPS strains confirm the decreased growth rate of the CPS strain compared to the WT and HRT2 strains. ">
 +
  <img src="https://static.igem.org/mediawiki/2013/f/ff/SDU2013_Rubber_10.png" style="width:800px" />
 +
Figure 16.
 +
</a>
 +
<br>
 +
</p><p>
 +
<span class="intro">Lastly, scanning electron microcopy</span> indicate no noticeable difference in cell morphology between the three strains upon induction <b>(Fig. 17)</b>. This emphasizes our prior conclusion that the bacteria tolerate the amount of rubber produced in our molecular biology production system.
 +
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<br>
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</p><p>
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<a class="popupImg alignCenter" style="width:500px" href="https://static.igem.org/mediawiki/2013/2/29/SDU2013_Rubber_11.jpg" title="Figure 17 - Scanning electron microcopy indicate no noticeable difference in cell morphology between the WT, HRT2 and CPS MG1655 strains upon induction. This emphasizes our prior conclusion that the bacteria tolerate the amount of rubber produced in our molecular biology production system.">
 +
  <img src="https://static.igem.org/mediawiki/2013/2/29/SDU2013_Rubber_11.jpg" style="width:500px" />
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Figure 17.
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<span class="intro">Now that you have viewed</span> our most valuable results describing our constructs ability to produce rubber, we invite you to dig further on, to see a comprehensive list of the parts we have submitted to Parts Registry. 
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Latest revision as of 02:44, 29 October 2013

Rubber results

Did we indeed make rubber?

Now that we control the expression of the genes it is time to characterize our central genes (dxs (B. subtilis) and HRT2). We invite you along on a journey through our attempts to obtain proof of concept; to show that Bacteriorganic Rubber is a true possibility.

Characterization of dxs (B. subtilis)

Functionality assay
To optimize the flow through the MEP pathway, the dxs gene was overexpressed, the expectation being increased levels of IPP and DMAPP (see Specification). To examine if overexpression indeed resultats in an increase in substrate, we attempted to assay the levels of DMAPP using a headspace gas chromatography (GC)-technique.

DMAPP was hydrolyzed in acid to the volatile hydrocarbon gas isoprene. The gas was subsequently analyzed with headspace GC. A linear relationship between amount of detected isoprene and DMAPP concentration has previously been established. Source: Fisher AJ, Rosenstiel TN, Shirk MC, Fall R. Nonradioactive assay for cellular dimethylallyl diphosphate. Anal Biochem. 2001 May 15;292(2):272-9. (Link) We were capable of producing a standard curve by reacting DMAPP with acid for 2 min (instead of the 60 min specified in the previous study) (Fig. 1). At this time, we obtained optimal peak detection for standard solutions. We were, however, incapable of detecting isoprene, even in high concentrations of bacterial samples treated with acid. The test was expanded to include acid hydrolyzation for 2, 30, 60 or 90 min, yet we could not detect isoprene, and therefore not detect DMAPP.

Figure 1. Optimization of the procedure is needed before characterization of the dxs bricks can be completed using this approach. We suspect that the complexity of the bacterial samples is too high, and thus the reaction does not take place as fast as might be necessary for detection in our setup. Sonication of bacterial samples with and without addition of standard DMAPP, and subsequent measurements might shed some light on this hypothesis. However, it should be noted that the GC wasn’t fully functional during the test period, consequently leading to broader peaks and thus lowered the sensitivity of the instrument. On the 3rd of October, we received a mail from Professor Lars Porskjær Christensen, Department of Chemistry-, Bio- and Environmental Technology, University of Southern Denmark:

"...The GC has now been repaired and the sensitivity has been improved considerably. The GC-peaks should be very sharp now. This may be the reason that you have not observed any release of isoprene from your samples...MailTranslated from danish".

Unfortunately, with only 2 days left to wiki-freeze, there was no time for another round of testing. Since the European Jamboree in Lyon we have performed the experiment multiple times, but it still needs further optimization, as our results are inconclusive.

Growth Experiment
Figure 2. Carrying the Dxs devices and expressing the gene could impair the growth, and hence be important in production purpose. To test if the growth of MG1655 bacteria is impaired when carrying and expressing our Dxs devices, we measured the growth rate with OD600 measurements.

Two triplicates of MG1655 carrying no vector, empty pSB1C3 vector, pSB1C3-Plac-dxs (B. subtilis), pSB1C3-Pcon-lacI-Plac-dxs (B. subtilis), or pSB1C3-Pcon-lacI:LVA-Plac-dxs (B. subtilis) were started from ONC at time 0 hours, OD600=0.005, and grown at 37ºC and 180 rpm. OD600 measurements were done every half an hour, and 1 of each triplicates was induced at time 2.5 hours. All strains grew at the same pace and induction didn’t impair growth rate (Fig. 2).


Characterization of HRT2

Rubber purification
To discover rubber, it is useful to isolate the rubber from the rest of the bacterial cells. This will allow us to remove as many variables in the detection assays as possible. The rubber was purified according to self-written SOP0030 - Rubber purification.
The method of purification was based on a literature search and chemical evaluation of solubility of the polyisoprene. Cells were sonicated in ethanol suspension, washed in acetone, and extracted in n-hexane (both steps were overnight).

Two different methods were tested to evaluate the most efficient extraction method. In the first method, we washed with acetone and extract with n-hexane in a soxhlet extractor. The second method excluded the time-consuming soxhlet steps and washed with acetone for a shorter duration of time (15 min shake at 37ºC). Rubber was extracted by adding hexane to the cell suspension, then centrifuging the sample. The supernatent was saved (hexane solution). We tested the methods of rubber extraction on a positive control made from MG1655, mixed with polyisoprene. Both the soxhlet and the non-soxhlet method were evaluated using H1-NMR. We deemed the results to be similar, and since the required time for non-soxhlet extraction was significantly less, we chose to use the non-soxleth method.

The H1 NMR test

We now seemed able to detect rubber that was extracted from a mixture of bacterial debris and polyisoprene, and felt ready for a test of our production system. H1 NMR allowed us a certain specificity and although a significant amount of polyisoprene was needed to enable detection, it was preferred over C13NMR. All samples were run an a bruker 400 mHz NMR spectrometer. We also knew that we could control the expression of the genes in both of our plasmids. We were ready to put our abilities to extract rubber through the non-soxhlet method and detect it using H1 NMR to the test. In other words, we were ready to find the rubber present in our strain! Source: Donald L. Pavia and Gary M. Lampman. Introduction to Spectroscopy, International Edition 4e (Book) ISBN-13: 9780538734189 / ISBN-10: 0538734183 (Link)

During the first test of our CPS bacteria (MG1655 transformed with both plasmids - see Design), bacteria were grown to late exponential phase at 37ºC, then induced with 1mM IPTG, 0.2% Ara, and 1mM MgCl2. It was then transfered to room temperature for another 4 hours of growth. Non-soxhlet extraction was used to purify any rubber present in the sample, which was then compared to a positive control (WT mixed with polyisopren - also undergone non-soxhlet extraction) and a standard isoprene solution.

Figure 3 illustrates the peaks given by pure polyisoprene are found at 5.12 (A), 2.04(B) and 1.68(C)in the ratio 1(A):4(B):3(C). Additionally, a peak is visible at 1.56 indicating water (the standard was not dried in a vacuum oven ON like the rubber extraction was). The peak at 0.00 ppm is the defining peak of the ppm axis and represents TMSTMSTetramethylsilane which is a calibrating standard.

Our rubber extraction of WT + polyisoprene (Fig. 4) gives the same peak placement as the pure polyisoprene ((A), (B) and (C)). However, the integration of the 3 peaks shows a relationship of approximately 1:5:4. The discrepancy can be explained by the impurities in the area between 0-2.5 ppm. Such impurities might add additional integration value to the peaks assigned to the polyisoprene peaks (B) and (C) - hereby causing a disruption of the true relationship. Some peaks from the solvents used to purify the rubber (Acetone, Ethanol, and Hexane), as well as a small amount of water (1.56 ppm) are seen, too.



The conclusion arrives in figure 5, named DXS+PT, which is our double plasmid CPS bacteria, and displays the same peaks (A), (B) and (C) as both the WT + polyisoprene and pure polyisoprene. This is a strong indication of the presence of rubber - specifically, this is an indication that our CPS bacteria produces rubber: Bacteriorganic Rubber. Peak distortion of the spectrum due to solvents are the same as seen in the rubber purification from WT + polyisoprene.

A second round of testing was done to validate the first experiment. We wanted to include a negative test (WT), in order to exclude the possibility of a naturally occurring polyisoprenoid compounds in E. coli. We performed rubber purification on WT, our CPS bacteria as well as a strain containing only the pSB1K3-araC-Para-HRT2 device. Bacteria were grown and rubber extracted as described above. The three samples where unfortunately not dried properly in the vacuum oven due to apparatus malfunction.

The test provided the following results: Fig 6 lacks the characteristic peaks of polyisoprene at 5.12, 2.04 and 1.68. It can be concluded from their absence that there is not rubber (nor any other compound that might provide a similar chemical shift values) in the wildtype. In the CPS (Fig. 7), we observe very a slight peak at 5.12, indicating the presence of (A) hydrogen. This peak has the same splitting pattern as the first round of H1-NMR, but it has a very low intensity. The (B) and (C) peaks are hidden in the background noise, which is most likely due to cell debris and solvents, which did not evaporate appropriately. We suspect that the machinery has a decreased sensitivity towards the isoprene peaks due to the high amount of solvent seen from the assigned peaks, but the peak at 5.12 (with the recognizable splitting pattern) is a strong indication that rubber is once again present in our CPS bacteria (containing both HRT2 and Dxs plasmids).


When we advanced to the finals in Boston, we wanted to verify that our previous experiments where replicable as well as investigate the quantity and yield of rubber. To assess this we did a third round of H1-NMR on MG1655 (WT), and MG1655 carrying pSB1C3-Pcon-araC-term-Para-HRT2 (HRT2), or pSB1C3-Pcon-lacI(N)-term-dxs (B. subtilis) and pSB1K3-Pcon-araC-term-Para-HRT2 (Dxs + HRT2). 1 L LB media was inoculated with the strains, starting from OD600=0.05. The cultures were grown at 37ºC with shaking for 5 hours; the temperature was then lowered to 20ºC and cultures were induced with 1mM IPTG, 0,2% arabinose, and 1mM MgCl2 ON. Rubber purification was commenced the following day, and the final yield was solubilized in 2 mL d-chloroform and analyzed by H1-NMR.

From the spectrum reflecting the WT strain, none of the peaks originating from polyisoprene were observed (fig. 8). From the spectrum reflecting the Dxs + HRT2 strain, all three peaks originating from polyisoprene were observed (fig. 9). Lastly, from the spectrum reflecting the HRT2 strain (fig. 10), the peaks potentially originating from polyisoprene could not be conclusively identified indicating the decreased rubber production performance of this strain compared to the Dxs + HRT2 strain. This emphasizes the importance of excess IPP and DMAPP (rubber precursors) available as a consequence of Dxs overexpression, and serves as an indirect proof of the effect of MEP pathway optimization.



In order to determine rubber yield, we compared the yield from 1L of our Dxs + HRT2 strain with 5 rubber purifications on WT + a selected amount of standard polyisoprene. Yields from all 5 purifications were solubilized in equal volumes of d-chloroform (2 mL), and the resulting TMS peaks could consequently be used for normalization of the polyisoprene peaks at 5.12 ppm. This peak was selected for quantification due to its localization in a region without background noise. From these 5 purifications, we calculated a standard curve to which we compared the integral value of the 5.12 ppm peak originating from our Dxs + HRT2 strain.

From the sample with the lowest amount of polyisoprene added (0.2 mg), no peaks could be identified, indicating the detection limit of the H1-NMR setup. Together with normalized peak integrals from the other 4 samples, we calculated the standard curve depicted in fig. 13. Based on the standard curve and the integral originating from the sample containing 3.2 mg polyisoprene (Fig. 13, 12), we roughly estimate our polyisoprene yields to be in the range of 0.5-5 mg polyisoprene/L (fig. 11). We realize that this yield estimation is far from optimal, however, only the sensitivity of H1-NMR allowed us to give a rough estimate of our yield.



When we tried to weigh our yield, we observed a many-fold increased yield compared to our input amount, and we assume that this conflicting reading is due to the presence of cell debris and other impurities adding to the output reading. This situation forced us to seek other yield calculation strategies, such as H1-NMR.


MALDI-ToF

We thoroughly studied the literature concerning polyisoprene on MALDI-ToF and found that the formation of adducts by adding AgNO3- would make it possible to ionize the long alkene chain, despite its lack of functional groups that can be ionized. be ionized. Source: Hyuneui L, Yeonhee L, Seunghee H, Youngsook Y, and Kang-Jin K. Investigation of Polystyrene, Polyisoprene, and Poly(2-vinylpyridine) using Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry. Bull. Korean Chem. Soc. 1999, Vol. 20, No. 7 p. 853-856. (Link)

We tried several matrixes including MBTMBT2-mercaptobenzothiazole, DHBDHB2,5-dihydroxybenzoic acid, CHCACHCAalpha-cyano-4-hydroxycinnamic acid, SASAsinapinic acid and DTDTdithranol, but didn't have the time to perform the tests on anything but our standard polyisoprene (Mw 38 kDa). Unfortunately, it appeared to be too large a molecule to be detected by the bruker MALDI-ToF machine. It was concluded that the machine's hardware settings could not match the requirements for such large molecules. However, the expected length that we expect HRT2 produces is in the 2-10 kDa range. This is within the limits of the machine, though we have yet to test this hypothesis as the machine is frequently occupied by other research groups.


Composite production system

Growth experiments

Carrying the HRT2, dxs and both (CPS) devices and expressing the genes could impair the growth, and hence be important in production purpose. To test if the growth of MG1655 bacteria is impaired when carrying and expressing our devices, we measured the growth rate with OD600 measurements.

Four different MG1655 strains carrying either no plasmid (WT), pSB1C3-Pcon-araC-term-Para-HRT2 (HRT2), pSB1C3-Pcon-lacI(N)-term-Plac-dxs (B.subtilis) (Dxs), or pSB1K3-Pcon-araC-term-Para-HRT2 and pSB1C3-Pcon-lacI(N)-term-Plac-dxs (B.subtilis) (CPS), were grown from OD600=0.05 and induced with 1 mM IPTG and/or 0.2% arabinose during growth. The first experiment showed impaired growth of the bacteria only carrying construct expressing HRT2. We thought that the reason why the strains expressing HRT2 as well as Dxs didn’t show impaired growth rate could be due to a phenomenon called inclusion bodies. When overexpressing too many proteins, there is a risk of misfolding and hence loss of function. A way to lower this risk is to lower the temperature. Therefore we set up a growth experiment where the temperature was lowered from 37 to 20ºC at time of induction. This did not prove to make a difference, and CPS strains with and without IPTG and/or arabinose induction grew at same pace as WT. However, we want to verify these results by a more thorough investigation and consequently setup a new growth experiment including OD600 measurements, counts of colony forming units (CFU) and finally scanning electron microscopy (fig. 14).


Figure 14.


Cell viability

Three different MG1655 strains; WT, HRT2 and CPS (similar to above), were grown under equal conditions as when cultures were grown for rubber purification. 1 L of bacterial cultures were initiated at OD600=0.05, from overnight cultures and grown at 37°C and shaking at 180 rpm. After 5 hours of growth, cultures were induced with 1 mM IPTG, 0.2% arabinose and 1 mM MgCl2 and the temperature was lowered to 20°C to prevent the formation of inclusion bodies. The OD600 was measured each hour throughout the experiment and sequential dilution series were plated out at time 4, 5, 6, 7 and 8 hours after initiation. Furthermore, samples for electron microscopy were taken from the induced ON cultures.

OD600 measurements illustrate a slight impairment of growth for the HRT2 strain compared with the WT strain, whereas the CPS strain shows heavily decreased Growth (Fig. 15A) . CFU counts reflect the number of viable cells in a culture. We observed a steady amount of CFU counts for the WT and HRT2 strains throughout the experiment similar to the CPS strain, although this strain presents with a lower amount of CFU counts (Fig. 15B).

We notice that the growth rate of the CPS strains is controversial to the growth experiment presented earlier, which we cannot directly explain. Importantly, the viability of the CPS strain does not decrease upon induction in both growth experiments, which indicates that the amount of produced rubber is not toxic to the bacteria. This might be due to the low amounts of rubber produced in the cells as validated by H1-NMR.


Figure 15.

Additionally, images from an equal dilution and time point in the CFU assay are displayed below (Fig. 16). The difference in size between the colonies from the three different strains confirm the decreased growth rate of the CPS strain compared to the WT and HRT2 strains.


Figure 16.

Lastly, scanning electron microcopy indicate no noticeable difference in cell morphology between the three strains upon induction (Fig. 17). This emphasizes our prior conclusion that the bacteria tolerate the amount of rubber produced in our molecular biology production system.

Figure 17.


Now that you have viewed our most valuable results describing our constructs ability to produce rubber, we invite you to dig further on, to see a comprehensive list of the parts we have submitted to Parts Registry.