Team:SDU-Denmark/Tour53

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<h2>Submitted Parts</h2>
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<h2>Rubber results</h2>
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<h4>And so, the iGEM parts registry grows</h4>
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<h4>Did we indeed make rubber?</h4>
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<p>
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<span class="intro">Now that we control</span> the expression of the genes it is time to characterize our central genes <span class="specialWord">(dxs (B. subtilis)</span> and <span class="specialWord">HRT2)</span>. We invite you along on a journey through our attempts to obtain proof of concept; to show that Bacteriorganic Rubber is a true possibility.
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</p>
<p>
<p>
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<span class="intro">Browse through the tabs</span> below to get a complete picture of our submitted parts. Feel free to follow the links to parts registry, where you can find information on sequencing, characterization, etc.  
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<h2>Characterization of dxs <span class="specialWord">(B. subtilis)</span></h2>
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<span class="intro">Functionality assay</span><br>
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To optimize the flow through the MEP pathway, the dxs gene was overexpressed, the expectation being increased levels of IPP and DMAPP
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(see <a class="dialogLink" href="https://2013.igem.org/Team:SDU-Denmark/Tour31" title"">Specification</a>).
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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.  
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</p>
</p>
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<p>
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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
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<span class="sourceReference">established.</span>
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<span class="tooltip">
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  <span class="tooltipHeader">Source:</span>
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Fisher AJ, Rosenstiel TN, Shirk MC, Fall R. Nonradioactive assay for cellular dimethylallyl diphosphate. Anal Biochem. 2001 May 15;292(2):272-9.
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<a href="http://www.ncbi.nlm.nih.gov/pubmed/11355861" target="_blank">(Link)</a>
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</span>
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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) <b>(Fig. 1)</b>. 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.
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</p>
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<p>
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<a class="popupImg alignRight" style="width:300px" href="https://static.igem.org/mediawiki/2013/1/12/SDU2013_Characterization_Dxs_1.png" title="Figure 1 - Isoprene production after 2 min hydrolyzation of standard DMAPP solutions (Sigma-Aldrich). 4M H2SO4 was used to hydrolyze 1 mL solutions containing different DMAPP amounts. The produced isoprene was detected using headspace gas chromatography with flame ionization detector. Measurements are single values and shows linearity from 0-1000 pmol DMAPP. The isoprene detection in the sample containing 0 pmol DMAPP was subtracted from the peak heights.">
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  <img src="https://static.igem.org/mediawiki/2013/1/12/SDU2013_Characterization_Dxs_1.png" style="width:300px" />
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Figure 1.
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</a>
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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.
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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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</p><p>
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<span class="specialWord">"...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<span class="tooltipLink">...</span><span class="tooltip"><span class="tooltipHeader">Mail</span>Translated from danish</span>".</span>
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</p><p>
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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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</p>
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<p>
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<span class="intro">Growth Experiment</span><br>
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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.">
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  <img src="https://static.igem.org/mediawiki/2013/6/68/SDU2013_Characterization_Dxs_2.png" style="width:300px" />
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Figure 2.
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</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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</p>
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<p>
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Two triplicates of MG1655 carrying no vector, empty pSB1C3 vector,
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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-<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-<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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</p>
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<br>
<br>
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<p>
 
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<div class="accordion" style="width:650px;">
 
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  <div class="accordionTitel">Coding</div>
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<p>
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  <div class="pane" >
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<h2>Characterization of HRT2</h2>
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<span class="intro"><a class="dialogLink" href="http://parts.igem.org/Part:BBa_K1088000">BBa_K1088000 dxs (B. subtilis)</a></span><br>
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<span class="intro">Rubber purification</span><br>
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The BioBrick contains the coding region of the dxs gene derived from the Gram-positive bacteria Bacillus subtilis. Dxs is the first enzyme in the MEP pathway converting pyruvate and <span class="tooltipLink">GAP</span> <span class="tooltip"><span class="tooltipHeader">GAP</span>Glyceraldehyde-3-phosphate</span> into <span class="tooltipLink">DXP.</span> <span class="tooltip"><span class="tooltipHeader">DXP</span>1-Deoxy-D-xylulose 5-phosphate</span> Has been sequenced.
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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.
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<br><br>
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The rubber was purified according to self-written
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<span class="intro"><a class="dialogLink" href="http://parts.igem.org/Part:BBa_K1088003">BBa_K1088003 HRT2</a></span><br>
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The BioBrick contains the coding region of the HRT2 gene derived from Hevea brasiliensis and codon-optimized for Escherichia coli. HRT2 is the prenyl transferase that polymerizes <span class="tooltipLink">IPP</span> <span class="tooltip"><span class="tooltipHeader">IPP</span>Isopentenyl pyrophosphate</span> and <span class="tooltipLink">DMAPP</span> <span class="tooltip"><span class="tooltipHeader">DMAPP</span>Dimethylallyl pyrophosphate</span> into rubber. Has been sequenced.
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<a class="pdfFile" href="https://static.igem.org/mediawiki/2013/4/48/IGEM2013_SOP0030_v01.pdf" target="_blank">SOP0030 - Rubber purification.</a><br>
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<br><br>
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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).
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<span class="intro"><a class="dialogLink" href="http://parts.igem.org/Part:BBa_K1088004">BBa_K1088004 ispG</a></span><br>
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</p>
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The BioBrick contains the coding region of the ispG gene derived from the Gram-negative bacteria Escherichia coli. ispG is the sixth enzyme in the MEP pathway converting <span class="tooltipLink">MEcPP</span> <span class="tooltip"><span class="tooltipHeader">MEcPP</span>2-C-methyl-D-erythritol 2,4-cyclopyrophosphate</span> into <span class="tooltipLink">HMB-PP.</span> <span class="tooltip"><span class="tooltipHeader">HMB-PP</span>(E)-4-Hydroxy-3-methyl-but-2-enyl pyrophosphate</span> Has been sequenced.
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<br><br>
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<span class="intro"><a class="dialogLink" href="http://parts.igem.org/Part:BBa_K1088005">BBa_K1088005 araC</a></span><br>
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<p>
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The BioBrick contains the coding region of the araC gene derived from the Gram-negative bacteria Escherichia coli. AraC is a DNA-binding protein that regulates the transcription of operons involved in arabinose metabolism. With glucose present AraC functions as a repressor, and without glucose and with arabinose present it functions as an activator.
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<br><br>
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<span class="intro"><a class="dialogLink" href="http://parts.igem.org/Part:BBa_K1088018">BBa_K1088018 lacI</a></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 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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The BioBrick contains the coding region of the lacI gene derived from the Gram-negative bacteria Escherichia coli. LacI is a DNA-binding protein that inhibits the transcription from the lac promoter when allolactose or <span class="tooltipLink">IPTG</span> <span class="tooltip"><span class="tooltipHeader">IPTG</span>Isopropyl β-D-1-thiogalactopyranoside</span> is absent. Has been sequenced.
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</p>
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<p>
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<h4>The H<sup>1</sup> NMR test</h4>
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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
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<span class="sourceReference">our strain!</span>
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<span class="tooltip">
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  <span class="tooltipHeader">Source:</span>
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Donald L. Pavia and Gary M. Lampman. Introduction to Spectroscopy, International Edition 4e (Book) ISBN-13: 9780538734189 / ISBN-10: 0538734183
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<a href="http://edu.cengage.co.uk/catalogue/product.aspx?isbn=0538734183" target="_blank">(Link)</a>
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</span>
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</p>
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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>
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<b>Figure 3</b> 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 <span class="tooltipLink">TMS</span><span class="tooltip"><span class="tooltipHeader">TMS</span>Tetramethylsilane</span> which is a calibrating standard.
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</p>
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<p>
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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>
<br>
<br>
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<p>
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  </div>
 
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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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  <div class="accordionTitel">Regulatory devices</div>
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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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  <div class="pane" >
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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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<span class="intro"><a class="dialogLink" href="http://parts.igem.org/Part:BBa_K1088017">BBa_K1088017 Pcon-araC-term</a></span><br>
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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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araC is being expressed from a constitutively active promoter. A terminator is put behind the coding region to prevent transcription of genes downstream of the activator. The device was used to check if we could enhance the control of the arabinose promoter. Has been sequenced.
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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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<br><br>
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<b> &nbsp; </b>
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<span class="intro"><a class="dialogLink" href="http://parts.igem.org/Part:BBa_K1088019">BBa_K1088019 Pcon-lacI(N)-term</a></span><br>
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</div>
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lacI is being expressed from a constitutively active promoter. A terminator is put behind the coding region to prevent transcription of genes downstream of the repressor. The device was used to enable us to control the lactose promoter. This device proved be most effective together for expression control.
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<br><br>
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</p>
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<span class="intro"><a class="dialogLink" href="http://parts.igem.org/Part:BBa_K1088020">BBa_K1088020 Pcon-lacI:LVA-term</a></span><br>
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lacI:LVA (BBa_C0012) is being expressed from a constitutively active promoter. A terminator is put behind the coding region to prevent transcription of genes downstream of the repressor. The LVA-tag is a tag for degradation, and thus there is increased turnover of the protein. The device is meant to enable us to control the lactose promoter. However natural LacI proved to be more effective than the LVA-tagged. Has been sequenced.
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<br>
<br>
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<p>
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  </div>
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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.
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</p>
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  <div class="accordionTitel">Reporter fusions</div>
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<p>
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  <div class="pane">
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<span class="intro"><a class="dialogLink" href="http://parts.igem.org/Part:BBa_K1088006">BBa_K1088006 Pcon-dxs (B. subtilis)-amilCP</a></span><br>
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<div class="imageGallery gallerySmall alignRight">
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This device expresses the dxs gene derived from  B. subtilis linked to GFP, and is under the control of the lactose promoter. AmilCP proved to be a poor fusion protein for the Dxs protein. Has been sequenced.
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<br><br>
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<a class="galleryImg" target="_blank" href="https://static.igem.org/mediawiki/2013/0/06/SDU2013_Characterization_NMR_4.png" title="Figure 6 - Spectrum illustrating WT bacteria, which have undergone rubber purification. Notice that none of the peaks for the isoprene units are present in this spectrum. However it should be noticed that the vacuum oven was malfunctioning, and therefore this result might be due to insensitivity of the spectrometer, since the noise from the solvents are far greater than before.">
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<span class="intro"><a class="dialogLink" href="http://parts.igem.org/Part:BBa_K1088007">BBa_K1088007 Plac-dxs (E. coli)-GFP</a></span><br>
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<img src="https://static.igem.org/mediawiki/2013/0/06/SDU2013_Characterization_NMR_4.png">Fig. 6</a>
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This device expresses the dxs gene derived from  E. coli linked to GFP, and is under the control of the lactose promoter. The device was used to check the expression level of the E. coli dxs gene under various conditions. Has been sequenced.
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<br><br>
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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.">
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<span class="intro"><a class="dialogLink" href="http://parts.igem.org/Part:BBa_K1088008">BBa_K1088008 Plac-dxs (B. subtilis):GFP</a></span><br>
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<img src="https://static.igem.org/mediawiki/2013/9/9a/SDU2013_Characterization_NMR_5.png">Fig. 7</a>
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This device expresses the dxs gene derived from  B. subtilis linked to GFP, and is under the control of the lactose promoter. The device is was used to check the expression level of the B.subtilis dxs gene under various conditions. Has been sequenced.
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<br><br>
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</div>
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<span class="intro"><a class="dialogLink" href="http://parts.igem.org/Part:BBa_K1088009">BBa_K1088009 Pcon-lacI:LVA-Plac-dxs (B. subtilis)-GFP</a></span><br>
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This device expresses the dxs gene derived from  B. subtilis linked to GFP, and is under the control of the lactose promoter. The device to check the expression level of the B.subtilis dxs gene under various conditions. Our LacI:LVA (BBa_K1088020) device (with a constitutive promoter was added to optimize the expression control through the lactose promoter. Natural LacI proved to be more efficient, though. Has been sequenced.
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<br><br>
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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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<span class="intro"><a class="dialogLink" href="http://parts.igem.org/Part:BBa_K1088010">BBa_K1088010 Pcon-lacI:LVA-term-Plac-dxs (E. coli)-GFP</a></span><br>
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</p>
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This device expresses the dxs gene derived from  E. coli linked to GFP, and is under the control of the lactose promoter. The device is meant for us to check the expression level of the dxs gene under various conditions. Our LacI:LVA (BBa_K1088020) device (with a constitutive promoter was added to optimize the expression control through the lactose promoter.
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<br><br>
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<p>
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<span class="intro"><a class="dialogLink" href="http://parts.igem.org/Part:BBa_K1088026">BBa_K1088026 Pcon-lacI(N)-Plac-dxs (B. subtilis)-GFP</a></span><br>
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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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This device expresses the dxs gene derived from  B. subtilis linked to GFP, and is under the control of the lactose promoter. The device is meant for us to check the expression level of the dxs gene under various conditions. Furthermore the lacI gene with a constitutive promoter has been added to optimize the expression control through the lactose promoter. This device proved to have the most efficient expression control (see results for more detail). Has been sequenced. 
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</p>
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<br>
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  </div>
 
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<p>
 +
<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.
 +
</p><p>
 +
<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.
 +
</p>
 +
<br>
 +
<p>
 +
<div class="imageGallery galleryMedium alignCenter">
 +
<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.">
 +
<img src="https://static.igem.org/mediawiki/2013/7/77/SDU2013_Rubber_WT2.png">
 +
Figure 8.
 +
</a>
 +
<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.">
 +
<img src="https://static.igem.org/mediawiki/2013/3/38/SDU2013_Rubber_CPS2.png">
 +
Figure 9.
 +
</a>
-
  <div class="accordionTitel">Constitutively active production devices</div>
+
<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.">
-
  <div class="pane">
+
<img src="https://static.igem.org/mediawiki/2013/b/b4/SDU2013_Rubber_HRT2.png">
 +
Figure 10.
 +
</a>
-
<span class="intro"><a class="dialogLink" href="http://parts.igem.org/Part:BBa_K1088011">BBa_K1088011 Plac-dxs(B. subtilis)</a></span><br>
+
<b> &nbsp; </b>
-
This device expresses the dxs gene derived from  B. subtilis, and is under the control of the lactose promoter. The device is meant for us to increase the amount of IPP and DMAPP in the cell.
+
 
-
<br><br>
+
</div>
-
<span class="intro"><a class="dialogLink" href="http://parts.igem.org/Part:BBa_K1088012">BBa_K1088012 Plac-dxs(E. coli)</a></span><br>
+
 
-
This device expresses the dxs gene (BBa_K118000) derived from  E. coli, and is under the control of the lactose promoter. The device is meant for us to increase the amount of IPP and DMAPP in the cell. Has been sequenced.
+
</p>
<br>
<br>
 +
<p>
-
  </div>
+
<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.
 +
</p><p>
 +
<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>
-
  <div class="accordionTitel">Regulable production devices</div>
+
<div class="imageGallery galleryMedium alignCenter">
-
  <div class="pane current">
+
 +
<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>
-
<span class="intro"><a class="dialogLink" href="http://parts.igem.org/Part:BBa_K1088013">BBa_K1088013 Pcon-lacI:LVA-term-Plac-dxs(B. subtilis)</a></span><br>
+
<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.">
-
This device expresses the dxs gene derived from  B. subtilis, and is under the control of the lactose promoter. The device is meant for us to increase the amount of <span class="tooltipLink">IPP</span> <span class="tooltip"><span class="tooltipHeader">IPP</span>Isopentenyl pyrophosphate</span> and <span class="tooltipLink">DMAPP</span> <span class="tooltip"><span class="tooltipHeader">DMAPP</span>Dimethylallyl pyrophosphate</span> in the cell. Furthermore the lacI:LVA gene with a constitutive promoter was added to optimize the expression control through the lactose promoter. Natural LacI proved to be more efficient, though. Has been sequenced.
+
  <img src="https://static.igem.org/mediawiki/2013/f/ff/SDU2013_Rubber_Int32.png">
-
<br><br>
+
Figure 12.
-
<span class="intro"><a class="dialogLink" href="http://parts.igem.org/Part:BBa_K1088014">BBa_K1088014 Pcon-lacI:LVA-term-Plac-dxs(E. coli)</a></span><br>
+
</a>
-
This device expresses the dxs gene derived from  E. coli, and is under the control of the lactose promoter. The device is meant for us to increase the amount of <span class="tooltipLink">IPP</span> <span class="tooltip"><span class="tooltipHeader">IPP</span>Isopentenyl pyrophosphate</span> and <span class="tooltipLink">DMAPP</span> <span class="tooltip"><span class="tooltipHeader">DMAPP</span>Dimethylallyl pyrophosphate</span> in the cell. Furthermore the lacI-LVA gene with a constitutive promoter has been added to optimize the expression control through the lactose promoter.
+
-
<br><br>
+
-
<span class="intro"><a class="dialogLink" href="http://parts.igem.org/Part:BBa_K1088015">BBa_K1088015 Pcon-lacI:LVA-term-Plac-dxs(B. subtilis)-ispG</a></span><br>
+
-
This device expresses the dxs gene derived from B. subtilis, and is under the control of the lactose promoter.The device was build to increase the amount of <span class="tooltipLink">IPP</span> <span class="tooltip"><span class="tooltipHeader">IPP</span>Isopentenyl pyrophosphate</span> and <span class="tooltipLink">DMAPP</span> <span class="tooltip"><span class="tooltipHeader">DMAPP</span>Dimethylallyl pyrophosphate</span> in the cell if the first rate limiting step was overcome.  Furthermore the lacI:LVA gene with a constitutive promoter has been added to optimize the expression control through the lactose promoter (see results for description LacI-LVA efficiency).
+
-
<br><br>
+
-
<span class="intro"><a class="dialogLink" href="http://parts.igem.org/Part:BBa_K1088016">BBa_K1088016 Pcon-araC-term-Para-HRT2-(3xFLAG)</a></span><br>
+
-
This device expresses the HRT2 gene derived from Hevea brasiliensis, and is under the control of the arabinose promoter. The device was made to enable the bacteria to polymerize <span class="tooltipLink">IPP</span> <span class="tooltip"><span class="tooltipHeader">IPP</span>Isopentenyl pyrophosphate</span> and <span class="tooltipLink">DMAPP</span> <span class="tooltip"><span class="tooltipHeader">DMAPP</span>Dimethylallyl pyrophosphate</span> into rubber. Furthermore the arabinose promoter regulator AraC has been added to check if it would enhance the expression control of arabinose promoter . It did not seem to improve expression control. Has been sequenced.
+
-
<br><br>
+
-
<span class="intro"><a class="dialogLink" href="http://parts.igem.org/Part:BBa_K1088024">BBa_K1088024 Para-HRT2-(3xFLAG)</a></span><br>
+
-
This device expresses the HRT2 gene derived from Hevea brasiliensis, and is under the control of the arabinose promoter. The device is meant to enable the bacteria to polymerize <span class="tooltipLink">IPP</span> <span class="tooltip"><span class="tooltipHeader">IPP</span>Isopentenyl pyrophosphate</span> and <span class="tooltipLink">DMAPP</span> <span class="tooltip"><span class="tooltipHeader">DMAPP</span>Dimethylallyl pyrophosphate</span> into rubber. Has been sequenced.
+
-
<br><br>
+
-
<span class="intro"><a class="dialogLink" href="http://parts.igem.org/Part:BBa_K1088027">BBa_K1088027 Pcon-lacI(N)-Plac-dxs (B. subtilis)</a></span><br>
+
-
This device expresses the dxs gene derived from  B. subtilis, and is under the control of the lactose promoter. The device is meant for us to increase the amount of <span class="tooltipLink">IPP</span> <span class="tooltip"><span class="tooltipHeader">IPP</span>Isopentenyl pyrophosphate</span> and <span class="tooltipLink">DMAPP</span> <span class="tooltip"><span class="tooltipHeader">DMAPP</span>Dimethylallyl pyrophosphate</span> in the cell. Furthermore the lacI gene with a constitutive promoter was added to optimize the expression control through the lactose promoter. This addition proved to have the most efficient expression control.
+
 +
<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>
<br>
 +
<p>
-
  </div>
+
<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>
-
</div>
+
<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.
 +
 
 +
<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>
 +
 +
<p>
 +
 +
<span class="intro">We tried several matrixes</span> including <span class="tooltipLink">MBT</span><span class="tooltip"><span class="tooltipHeader">MBT</span>2-mercaptobenzothiazole</span>, <span class="tooltipLink">DHB</span><span class="tooltip"><span class="tooltipHeader">DHB</span>2,5-dihydroxybenzoic acid</span>, <span class="tooltipLink">CHCA</span><span class="tooltip"><span class="tooltipHeader">CHCA</span>alpha-cyano-4-hydroxycinnamic acid</span>, <span class="tooltipLink">SA</span><span class="tooltip"><span class="tooltipHeader">SA</span>sinapinic acid</span> and <span class="tooltipLink">DT</span><span class="tooltip"><span class="tooltipHeader">DT</span>dithranol</span>, 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.
 +
<p>
 +
 +
<br>
 +
 +
<h2>Composite production system</h2>
 +
 +
<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>
 +
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>.
 +
 +
</p>
 +
 +
<p>
 +
<br>
 +
 +
<a class="popupImg alignCenter" style="width:800px" href="https://static.igem.org/mediawiki/2013/6/64/SDU2013_Characterization_CPS_1.png"
 +
 +
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"
 +
 +
style="width:800px">
 +
Figure 14.
 +
</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.
 +
 +
<br>
 +
 +
</p><p>
 +
 +
<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" />
 +
Figure 17.
 +
</a>
 +
 +
 +
 +
</p>
 +
 +
<br>
 +
 +
<p>
 +
 +
<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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</p>
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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.