Team:SDU-Denmark/Tour52

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<h2>Characterization</h2>
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<h2>Expression</h2>
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<h4></h4>
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<h4>Can we control it?</h4>
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<p>
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<span class="intro">Characterization of our biobricks</span> is a chance for us to prove that our design works as intended. We invite you along on a journey through our attempts to obtain proof of concept; to show that Bacteriorganic Rubber is a true possibility. This page will slowly guide you through our results, but keep in mind that not everything is presented below. For all the details, consult our protocol page where you will find a comprehensive picture of our project.
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<span class="intro">Characterization of our biobricks</span> is a chance for us to prove that our design works as intended. These next two pages will slowly guide you through our results, but keep in mind that not everything is presented below. For all the details, consult our protocol page where you will find a comprehensive picture of our project.
</p>
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<span class="intro">The question of design</span> and therefore of function is two-fold: How do we - and can we - control the expression of our system? And do the expressed proteins work as intended? These are the questions this page sets out to answer. Specifically, we have characterized our regulable biobricks (<span class="specialWord">LacI/Plac</span> and <span class="specialWord">AraC/Para</span>) and our central genes <span class="specialWord">(dxs(B. subtilis)</span> and <span class="specialWord">HRT2</span>)
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<span class="intro">The question of design</span> and therefore of function is two-fold: How do we - and can we - control the expression of our system? And do the expressed proteins work as intended? These are the questions this page sets out to answer. Specifically, we have characterized our regulable biobricks (<span class="specialWord">LacI/Plac</span> and <span class="specialWord">AraC/Para</span>)
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<br>
<p>
<p>
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<h4>Characterization of LacI/Plac</h4>
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<h2>Characterization of LacI/Plac</h2>
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<span class="intro">The <span class="specialWord">dxs</span> gene</span> is placed under the control of the lactose promoter (see <a class="dialogLink" href="https://2013.igem.org/Team:SDU-Denmark/Tour33">Design</a>). We assayed the inducible capabilities of our design and, as part of the experiment, we tested the ability to suppress expression prior to induction. The assay was carried out by measuring protein levels of Dxs fused to GFP using
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<span class="tooltipLink">FACS.</span> <span class="tooltip"><span class="tooltipHeader">FACS</span>Fluorescence Activated Cell Sorting</span>
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<span class="intro">The <span class="specialWord">dxs</span> gene</span> is placed under the control of the lactose promoter (see Design). We assayed the inducible capabilities of our design and, as part of the experiment, we tested the ability to suppress expression prior to induction. The assay was carried out by measuring protein levels of Dxs fused to GFP using Fluorescence Activated Cell Sorting (FACS).
 
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A <a class="dialogLink" href="http://parts.igem.org/Part:BBa_C0012">lacI:LVA basic part</a> was available at parts registry, and we added a promotor and a terminator, producing a device. The GFP fusion devices with and without the  
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A <a class="dialogLink" href="http://parts.igem.org/Part:BBa_C0012">LacI:LVA basic part</a> was available at Parts Registry, and we added a promotor and a terminator, producing a device. The GFP fusion devices with and without the  
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<a class="dialogLink" href="http://parts.igem.org/Part:BBa_K1088020">lacI:LVA device</a> were assayed to check for expression control. One triplicate of MG1655 and two triplicates of each MG1655 strains carrying either  
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<a class="dialogLink" href="http://parts.igem.org/Part:BBa_K1088020">LacI:LVA device</a> were assayed to check for expression control. One triplicate of MG1655 and two triplicates of each MG1655 strains carrying either  
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<a class="dialogLink" href="http://parts.igem.org/Part:BBa_K1088008">pSB1C3-Plac-dxs <span class="specialWord">(B. subtilits)</span>-GFP</a> or  
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<a class="dialogLink" href="http://parts.igem.org/Part:BBa_K1088008">pSB1C3-Plac-dxs (B. subtilits)-GFP</a> or  
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<a class="dialogLink" href="http://parts.igem.org/Part:BBa_K1088009">pSB1C3-Pcon-lacI:LVA-term-Plac-dxs <span class="specialWord">(B. subtilits)</span>-GFP</a> were grown from OD<sub>600</sub> 0.005 to approximately 0.2. At this OD the MG1655 triplicate and one triplicate of each strain carrying constructs were induced with 1 mM IPTG at time 0 min. FACS measurements were done at times: -30, 0, 30, 60, 90, 120, and 150 min, thus showing the expression of GFP in each strain - both when induced and when not induced <b>(Fig. 1)</b>.  
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<a class="dialogLink" href="http://parts.igem.org/Part:BBa_K1088009">pSB1C3-Pcon-lacI:LVA-term-Plac-dxs (B. subtilits)-GFP</a> were grown from OD<sub>600</sub>=0.005 to approximately OD<sub>600</sub>=0.2. At this OD the MG1655 triplicate and one triplicate of each strain carrying constructs were induced with 1 mM IPTG at time 0 min. FACS measurements were done at times: -30, 0, 30, 60, 90, 120, and 150 min, thus showing the expression of GFP in each strain - both when induced and when not induced <b>(Fig. 1)</b>.  
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<span class="intro">The result</span> shows that strains lacking LacI:LVA do not repress expression from the lactose promoter. Even without induction, there is clear expression of GFP. Conversely, strains expressing LacI:LVA repress the promoter until induction. Approximately 90 min after induction of the strain expressing LacI:LVA, protein level is at its maximum. Still, maximum protein level is lower than that of the strain lacking LacI:LVA. Also, the fraction of flourescent cells is lower in the samples of pSB1C3-LacI:LVA-Plac-dxs <span class="specialWord">(B. subtilits)</span>-GFP compared to pSB1C3-Plac-dxs <span class="specialWord">(B. subtilits)</span>-GFP, indicating that expression of GFP is somewhat repressed, despite induction.
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<span class="intro">The results</span> show that strains lacking LacI:LVA do not repress expression from the lactose promoter. Even without induction, there is clear expression of GFP. Conversely, strains expressing LacI:LVA repress the promoter until induction. Approximately 90 min after induction of the strain expressing LacI:LVA, protein level is at its maximum. Still, maximum protein level is lower than that of the strain lacking LacI:LVA. Also, the fraction of flourescent cells is lower in the samples of pSB1C3-<span class="specialWord">lacI:LVA</span>-Plac-<span class="specialWord">dxs (B. subtilits)</span>-GFP compared to pSB1C3-Plac-<span class="specialWord">dxs (B. subtilits)</span>-GFP, indicating that expression of GFP is somewhat repressed, despite induction.
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<span class="intro">This experience</span> has been added to the experience of the part encoding the  
<span class="intro">This experience</span> has been added to the experience of the part encoding the  
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<a class="dialogLink" href="http://parts.igem.org/Part:BBa_C0012">lacI:LVA basic part</a> on parts registry
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<a class="dialogLink" href="http://parts.igem.org/Part:BBa_C0012">LacI:LVA basic part</a> on Parts Registry
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<br>
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<a class="popupImg alignCenter" style="width:800px" href="https://static.igem.org/mediawiki/2013/0/05/SDU2013_Characterization_LacIPlac_1.2.png" title="Figure 1 - FACS results and growth curves of +/-lacI:LVA carrying strains. One triplicate of MG1655 (WT) and two triplicates of MG1655 strains carrying either pSB1C3-Plac-dxs(B. subtilits)-GFP (BBa_K1088008) (-lacI:LVA) or pSB1C3-Pcon-lacI:LVA-term-Plac-dxs(B. subtilits)-GFP (BBa_K1088009) (+lacI:LVA) were grown from OD<sub>600</sub> 0.005 to approximately 0.2. At this OD the MG1655 triplicate and one triplicate of each strain carrying constructs were induced with 1 mM IPTG at time 0 min. FACS measurements were done at times: -30, 0, 30, 60, 90, 120, and 150 min.
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<a class="popupImg alignCenter" style="width:800px" href="https://static.igem.org/mediawiki/2013/0/05/SDU2013_Characterization_LacIPlac_1.2.png" title="Figure 1 - FACS results and growth curves of +/-<i>lacI:LVA</i> carrying strains. One triplicate of MG1655 (WT) and two triplicates of MG1655 strains carrying either pSB1C3-Plac-dxs(B. subtilits)-GFP (BBa_K1088008) (-<i>lacI:LVA</i>) or pSB1C3-Pcon-<i>lacI:LVA</i>-term-Plac-<i>dxs (B. subtilits)</i>-GFP (BBa_K1088009) (+<i>lacI:LVA</i>) were grown from OD<sub>600</sub> 0.005 to approximately 0.2. At this OD the MG1655 triplicate and one triplicate of each strain carrying constructs were induced with 1 mM IPTG at time 0 min. FACS measurements were done at times: -30, 0, 30, 60, 90, 120, and 150 min.
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A) Growth curve shows that WT grows slightly faster than strains bearing plasmids. B) Per cent of population above fluorescence threshold. None of the WT cells were fluorescent, almost all of the -lacI:LVA cells were constitutively fluorescent, and only cells overexpressing LacI:LVA weren’t fluorescent when not induced. Upon induction increasingly per cent of +lacI:LVA became fluorescent and reaches a maximum of 70-75 per cent after 90 min. C) Mean GFP fluorescence of entire population. The -lacI:LVA cells became increasingly more fluorescent over time, both with and without induction. Though, the induced cells were slightly more fluorescent, which is probably because of the relief of repression from LacI naturally present in the cells. For the +lacI:LVA cells the results from B is reflected.">
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<b>A)</b> Growth curve shows that WT grows slightly faster than strains bearing plasmids. <b>B)</b> Per cent of population above fluorescence threshold. None of the WT cells were fluorescent, almost all of the -<i>lacI:LVA</i> cells were constitutively fluorescent, and only cells overexpressing <i>lacI:LVA</i> weren’t fluorescent when not induced. Upon induction increasingly per cent of +<i>lacI:LVA</i> became fluorescent and reaches a maximum of 70-75 per cent after 90 min. <b>C)</b> Mean GFP fluorescence of entire population. The -<i>lacI:LVA</i> cells became increasingly more fluorescent over time, both with and without induction. Though, the induced cells were slightly more fluorescent, which is probably because of the relief of repression from LacI naturally present in the cells. For the +<i>lacI:LVA</i> cells the results from B is reflected.">
   <img src="https://static.igem.org/mediawiki/2013/0/05/SDU2013_Characterization_LacIPlac_1.2.png" style="width:800px" />
   <img src="https://static.igem.org/mediawiki/2013/0/05/SDU2013_Characterization_LacIPlac_1.2.png" style="width:800px" />
Figure 1.  
Figure 1.  
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<span class="intro">Although lacI:LVA was shown to be functional</span>, we found the prolonged response time and lowered maximum protein level after induction to be less than optimal. That is to say that the performance of LacI:LVA as a repressor did not live up to our expectations when comparing it to the constitutively active strains, nor when comparing it to the wildtype. Therefore, we built a device of similar design to the <a class="dialogLink" href="http://parts.igem.org/Part:BBa_K1088020">lacI:LVA device</a> with natural lacI:  
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<span class="intro">Although LacI:LVA was shown to be functional</span>, we found the prolonged and delayed response time together with lowered maximum protein level after induction to be less than optimal. In summary the performance of LacI:LVA as a repressor did not live up to our expectations when comparing it to the constitutively active strains, nor when comparing it to the wildtype. Therefore, we built a device of similar design to the <a class="dialogLink" href="http://parts.igem.org/Part:BBa_K1088020">LacI:LVA device</a> with natural LacI:  
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<a class="dialogLink" href="http://parts.igem.org/Part:BBa_K1088019">LacI(N).</a> To test its qualities compared to LacI:LVA, a FACS was analysed. The experimental setup was similar to the previous. Two triplicates of MG1655 strains carrying either
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<a class="dialogLink" href="http://parts.igem.org/Part:BBa_K1088019">lacI(N).</a> To test its qualities compared to LacI:LVA, a FACS was analysed. The experimental setup was similar to the previous. Two triplicates of MG1655 strains carrying either
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<a class="dialogLink" href="http://parts.igem.org/Part:BBa_K1088026">pSB1C3-Pcon-<span class="specialWord">lacI(N)</span>-term-Plac-<span class="specialWord">dxs (B. subtilits)</span>-GFP</span></a> or
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<a class="dialogLink" href="http://parts.igem.org/Part:BBa_K1088026">pSB1C3-Pcon-lacI(N)-term-Plac-dxs <span class="specialWord">(B. subtilits)</span>-GFP</span></a> or
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<a class="dialogLink" href="http://parts.igem.org/Part:BBa_K1088009">pSB1C3-Pcon-<span class="specialWord">lacI:LVA</span>-term-Plac-<span class="specialWord">dxs (B. subtilits)</span>-GFP</span></a>
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<a class="dialogLink" href="http://parts.igem.org/Part:BBa_K1088009">pSB1C3-Pcon-lacI:LVA-term-Plac-dxs <span class="specialWord">(B. subtilits)</span>-GFP</span></a>
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were used and an additional measurement at time 180 min was performed <b>(Fig. 2)</b>.
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were used and an additional measurement at time 180 min was taken.
 
</p><p>
</p><p>
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Both strains repress expression when uninduced. The strain expressing lacI:LVA reaches its maximum percentage of fluorescent cells approximately 150 min after induction, whereas the strain overexpressing natural lacI reaches its maximum after a mere 30 to 60 min. The maximum protein levels in the strain overexpressing natural lacI reaches a level approximately 3 times higher than the strain expressing lacI:LVA. Clearly, the function of the natural lacI is superior to that of lacI:LVA.
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Both strains repress expression when uninduced. The strain expressing LacI:LVA reaches its maximum percentage of fluorescent cells approximately 150 min after induction, whereas the strain overexpressing natural LacI reaches its maximum after a mere 30 to 60 min. The maximum protein levels in the strain overexpressing natural LacI reaches a level approximately 3 times higher than the strain expressing LacI:LVA. Clearly, the function of the natural LacI is superior to that of LacI:LVA.
</p>
</p>
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<br>
<p>
<p>
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<span class="intro">According to parts registry</span>, the LVA-tagged lacI part has been used 684 times in spite of its poor function. The natural lacI parts that have been submitted to the registry by previous teams are pending (and thus did not pass HQ quality control). We have therefore submitted the coding part of natural lacI to the registry for future teams to use, as an improvement upon lacI:LVA biobrick. Furthermore, our experience has been added to the experience of the part encoding the lacI:LVA basic part (BBa_C0012) on parts registry.
 
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</p>
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<a class="popupImg alignCenter" style="width:800px"  href="https://static.igem.org/mediawiki/2013/c/c6/SDU2013_Characterization_LacIPlac_2.png"
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title="Figure 2 - FACS results and growth curves of <i>lacI(N)</i> and <i>lacI:LVA</i> carrying strains. Two triplicates of MG1655 strains carrying either pSB1C3-Pcon-<i>lacI(N)</i>-term-Plac-<i>dxs (B. subtilits)</i>-GFP (BBa_K1088026) (<i>lacI(N)</i>) or pSB1C3-Pcon-<i>lacI:LVA</i>-term-Plac-<i>dxs (B. subtilits)</i>-GFP (BBa_K1088009) (lacI:LVA) were grown from OD<sub>600</sub> 0.005 to approximately 0.2. At this OD the one triplicate of each strain were induced with 1 mM IPTG at time 0 min. FACS measurements were done at times: -30, 0, 30, 60, 90, 120, 150 and 180 min.
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<b>A)</b> <i>lacI(lva)</i> and <i>lacI(N)</i> strains grew at same pace both with and without induction. <b>B)</b> Per cent of population above fluorescence threshold. Both <i>lacI(lva)</i> and <i>lacI(N)</i> represses the expression, when not induced with IPTG. <i>lacI(lva)</i> reaches a maximum of merely 70-75 % after 150 min, which is both a poor response time and maximum cell per cent fluorescent compared to <i>lacI(N)</i>. <i>lacI(N)</i> reaches a maximum just below 100 % at a time between 30 and 60 min. <b>C)</b> Mean GFP fluorescence of entire population. These results reflect what is seen in B, and clearly indicates that overexpression of natural LacI instead of LacI:LVA is the better, when a quick response and high level of expression is wanted.">
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<p>
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<a class="popupImg alignCenter" style="width:800px"
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href="https://static.igem.org/mediawiki/2013/c/c6/SDU2013_Characterization_LacIPlac_2.png"
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title="Figure 2 - FACS results and growth curves of lacI(N) and lacI:LVA carrying strains. Two triplicates of MG1655 strains carrying either pSB1C3-Pcon-lacI(N)-term-Plac-dxs(B. subtilits)-GFP (BBa_K1088026) (lacI(N)) or pSB1C3-Pcon-lacI:LVA-term-Plac-dxs(B. subtilits)-GFP (BBa_K1088009) (lacI:LVA) were grown from OD<sub>600</sub> 0.005 to approximately 0.2. At this OD the one triplicate of each strain were induced with 1 mM IPTG at time 0 min. FACS measurements were done at times: -30, 0, 30, 60, 90, 120, 150 and 180 min.
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A)  lacI(lva) and lacI(N) strains grew at same pace both with and without induction. B) Per cent of population above fluorescence threshold. Both lacI(lva) and lacI(N) represses the expression, when not induced with IPTG. lacI(lva) reaches a maximum of merely 70-75 % after 150 min, which is both a poor response time and maximum cell per cent fluorescent compared to lacI(N). lacI(N) reaches a maximum just below 100 % at a time between 30 and 60 min. C) Mean GFP fluorescence of entire population. These results reflect what is seen in B, and clearly indicates that overexpression of natural LacI instead of LacI:LVA is the better, when a quick response and high level of expression is wanted.">
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   <img src="https://static.igem.org/mediawiki/2013/c/c6/SDU2013_Characterization_LacIPlac_2.png"  
   <img src="https://static.igem.org/mediawiki/2013/c/c6/SDU2013_Characterization_LacIPlac_2.png"  
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</p>
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<span class="intro">To test its qualities compared</span> to LacI:LVA further, another FACS was performed. The experimental setup was similar to the previous. Dublicates of MG1655 strains carrying either pSB1C3-Pcon-<span class="specialWord">lacI(N)</span><span class="specialWord">-term-Plac-dxs (B. subtilis)</span>-GFP or pSB1C3-Pcon-<span class="specialWord">lacI:LVA</span>-term-Plac-<span class="specialWord">dxs (B. subtilis)</span>-GFP were induced with IPTG concentrations rangingfrom 0.05-0.5mM <b>(Fig. 3)</b>. After 180 min upon induction, the strains expressing LacI:LVA with the IPTG concentrations ranging from 0.05-0.5mM reached approximately 25-30 % cells above the fluorescent threshold. Comparing this observation to the strains overexpressing the natural LacI, even the least induced (0.05mM) reached a higher percentage of cells above the fluorescent threshold after 180 min. All of the cells in the culture overexpressing natural LacI induced with 0.125-0.5mM IPTG are above the fluorescent threshold within 180 min. In general, the response time is faster for higher concentrations of IPTG.
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<h4>Characterization of AraC/Para</h4>
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</p>
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<br>
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<p>
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<a class="popupImg alignRight" style="width:200px" href="https://static.igem.org/mediawiki/2013/8/88/SDU2013_Characterization_AraCPara_1.png" title="
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<a class="popupImg alignCenter" style="width:800px" href="https://static.igem.org/mediawiki/2013/7/73/SDU2013_Expression_FACS3.png" title="Figure 3 - FACS results and growth curves of <i>lacI(N)</i> and <i>lacI:LVA</i> carrying strains. Dublicates of MG1655 carrying either pSB1C3-Pcon-<i>lacI(N)</i>-term-Plac-<i>dxs (B. subtilis)</i>-GFP (BBa_K1088026) (<i>lacI(N)</i>) or pSB1C3-Pcon-<i>lacI:LVA</i>-term-Plac-<i>dxs (B. subtilis)</i>-GFP (BBa_K1088009) (<i>lacI(lva)</i>) were grown from OD<sub>600</sub>=0.005 to approximately OD<sub>600</sub>=0.2. At this OD, the one triplicate of each strain was induced with 0.05, 0.125, 0.25, or 0.5 mM IPTG at time 0 min. FACS measurements were done at times: -30, 0, 30, 60, 90, 120, 150 and 180 min. <b> A)</b> <i>lacI(lva)</i> and <i>lacI(N)</i> strains grew at the same pace <b> B)</b> Percentage of population above fluorescenct threshold. Upon induction of lacI(lva) with different concentrations of IPTG, the percentage of bacteria becoming fluorescent slowly increases. After approximately 180 min, 25-30 % of <i>lacI(lva)</i> induced bacteria in the range from 0.05-0.5 mM IPTG are above the fluorescenct threshold. After 180 min, there is a slightly higher percentage of bacteria above the fluorescenct threshold than for the lacI(lva) strain induced with 10-fold higher IPTG concentration. All <i>lacI(N)</i> induced with IPTG concentrations from 0.125-0.5mM become fluorescent within 180 min, and cultures with higher IPTG concentrations reach this point sooner. These results support what was seen in figure 2">
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  <img src="https://static.igem.org/mediawiki/2013/7/73/SDU2013_Expression_FACS3.png"  
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Figure 3 - A) Normalized intensity of HRT2 mRNA using intensity of 5S rRNA as reference. Duplicates of MG1655 strains carrying either pSB1C3-Para-HRT2 (BBa_K1088024) (-araC) or pSB1C3-Pcon-araC-term-Para-HRT2 (BBa_K1088016) (+araC) were induced with 0.2 % arabinose at time t=0 min (OD<sub>600</sub>=0.8), and samples were taken at times: -2 min, 15 min, and 30 min. The normalized intensity of sample -araC -2min were set to 1 and the other samples are relative to that. Within 15 min of induction the expression levels are at its maximum in both strains, and overexpression of AraC does not seem to be necessary for expression control of the arabinose promoter.
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style="width:800px">
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B) Northern blot result reflecting diagram.
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">
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  <img src="https://static.igem.org/mediawiki/2013/8/88/SDU2013_Characterization_AraCPara_1.png" style="width:200px" />
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Figure 3.  
Figure 3.  
</a>
</a>
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<span class="intro">The HRT2 gene is under the control</span> of the arabinose promoter (see Design). We assayed the inducible capabilities of our design and, as part of the experiment, we tested the ability to suppress expression prior to induction. The assay was carried out by measuring the mRNA levels of HRT2 using the Northern blotting technique.
 
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<p>
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<span class="intro">According to Parts Registry</span>, the LVA-tagged LacI part has been used <span class="intro">684 times</span> despite of its poor function. The natural LacI parts that have been submitted to the Registry by previous teams are pending (and thus did not pass HQ quality control). We have therefore submitted the coding sequence of natural LacI to the Registry for future teams to use as an improvement to the LacI:LVA biobrick. Furthermore, our experience has been added to the experience of the part encoding the LacI:LVA basic part (<a class="dialogLink" href="http://parts.igem.org/Part:BBa_C0012">BBa_C0012</a>) on Parts Registry.
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<span class="intro">To test whether overexpression of AraC improved expression control</span>, devices with and without the  
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<a class="dialogLink" href="http://parts.igem.org/Part:BBa_K1088017">araC device</a> were assayed. Duplicates of MG1655 strains carrying either
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<a class="dialogLink" href="http://parts.igem.org/Part:BBa_K1088024">pSB1C3-Para-HRT2</a> or
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<a class="dialogLink" href="http://parts.igem.org/Part:BBa_K1088016">pSB1C3-Pcon-araC-term-Para-HRT2</a> were grown to late-exponential phase: OD<sub>600</sub>=0.8. At this OD, the strains were induced with 0.2 % arabinose at time t=0 min, and samples were taken at times: -2 min, 15 min, and 30 min. Total RNA purified from the samples were run on a gel, blotted onto a membrane, and hybridized with probes specific for HRT2 mRNA and 5S rRNA (loading control), respectively.  
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</p>
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<span class="intro">The results prove that we are capable</span> of inducing our HRT2 devices with arabinose. There is only little expression before induction and within the first 15 min, expression is at its maximum. Overexpression of AraC does not seem to have an effect on the expression levels after 15 min compared to natural levels of AraC. However, it is inconclusive whether AraC might contribute to an effect at times less than 15 min after induction. <b>(Fig. 3)</b>.
 
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<span class="intro">This experience</span> has been added to the experience of the part encoding the <a class="dialogLink" href="http://parts.igem.org/Part:BBa_I13453">arabinose promoter</a> on parts registry.
 
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<h4>Characterization of dxs <span class="specialWord">(B. subtilis)</span></h4>
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<h2>Characterization of AraC/Para</h2>
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<span class="intro">Functionality assay</span><br>
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<a class="popupImg alignRight" style="width:200px" href="https://static.igem.org/mediawiki/2013/8/88/SDU2013_Characterization_AraCPara_1.png" title="
-
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.  
+
-
</p>
+
Figure 4 - <b>A)</b> Normalized intensity of <i>HRT2</i> mRNA using intensity of 5S rRNA as reference. Duplicates of MG1655 strains carrying either pSB1C3-Para-<i>HRT2</i> (BBa_K1088024) (-araC) or pSB1C3-Pcon-<i>araC</i>-term-Para-<i>HRT2</i> (BBa_K1088016) (+<i>araC</i>) were induced with 0.2 % arabinose at time t=0 min (OD<sub>600</sub>=0.8), and samples were taken at times: -2 min, 15 min, and 30 min. The normalized intensity of sample -<i>araC</i> -2min were set to 1 and the other samples are relative to that. Within 15 min of induction the expression levels are at its maximum in both strains, and overexpression of AraC does not seem to be necessary for expression control of the arabinose promoter.
 +
<b>B)</b> Northern blot result reflecting diagram.
-
 
+
">
-
<p>
+
   <img src="https://static.igem.org/mediawiki/2013/8/88/SDU2013_Characterization_AraCPara_1.png" style="width:200px" />
-
 
+
-
 
+
-
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
+
-
<span class="sourceReference">established.</span>
+
-
<span class="tooltip">
+
-
  <span class="tooltipHeader">Source:</span>
+
-
  Alison J. Fisher et. al; Nonradioactive Assay for Cellular Dimethyllyl Diphosphate
+
-
</span>
+
-
 
+
-
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. 4)</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.
+
-
 
+
-
</p>
+
-
 
+
-
 
+
-
<p>
+
-
 
+
-
 
+
-
<a class="popupImg alignRight" style="width:300px" href="https://static.igem.org/mediawiki/2013/1/12/SDU2013_Characterization_Dxs_1.png" title="Figure 4 - 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.">
+
-
   <img src="https://static.igem.org/mediawiki/2013/1/12/SDU2013_Characterization_Dxs_1.png" style="width:300px" />
+
Figure 4.  
Figure 4.  
</a>
</a>
-
<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.
 
-
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:
 
-
 
-
</p><p>
 
-
 
-
<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>
 
-
 
-
</p><p>
 
-
 
-
Unfortunately, with only 2 days left to wiki-freeze, there was no time for another round of testing.
 
 +
<span class="intro">The HRT2 gene is under the control</span> of the arabinose promoter (see <a class="dialogLink" href="https://2013.igem.org/Team:SDU-Denmark/Tour33">Design</a>). We assayed the inducible capabilities of our design and, as part of the experiment, we tested the ability to suppress expression prior to induction. The assay was carried out by measuring the mRNA levels of HRT2 using the Northern blotting technique.
</p>
</p>
Line 226: Line 162:
<p>
<p>
-
<span class="intro">Growth Experiment</span><br>
 
-
<a class="popupImg alignRight" style="width:300px" href="https://static.igem.org/mediawiki/2013/6/68/SDU2013_Characterization_Dxs_2.png" title="Figure 5 - 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.">
+
<span class="intro">To test whether overexpression of AraC improved expression control</span>, devices with and without the
-
  <img src="https://static.igem.org/mediawiki/2013/6/68/SDU2013_Characterization_Dxs_2.png" style="width:300px" />
+
-
Figure 5.
+
-
</a>
+
-
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.  
+
<a class="dialogLink" href="http://parts.igem.org/Part:BBa_K1088017">araC device</a> were assayed. Duplicates of MG1655 strains carrying either
 +
<a class="dialogLink" href="http://parts.igem.org/Part:BBa_K1088024">pSB1C3-Para-<span class="specialWord">HRT2</span></a> or
 +
<a class="dialogLink" href="http://parts.igem.org/Part:BBa_K1088016">pSB1C3-Pcon-<span class="specialWord">araC</span>-term-Para-<span class="specialWord">HRT2</span></a> were grown to late-exponential phase: OD<sub>600</sub>=0.8. At this OD, the strains were induced with 0.2 % arabinose at time t=0 min, and samples were taken at times: -2 min, 15 min, and 30 min. Total RNA purified from the samples were run on a gel, blotted onto a membrane, and hybridized with probes specific for HRT2 mRNA and 5S rRNA (loading control), respectively.  
-
</p>
 
-
 
-
 
-
<p>
 
-
 
-
 
-
Two triplicates of MG1655 carrying no vector, empty pSB1C3 vector,
 
-
 
-
 
-
<a class="dialogLink" href="http://parts.igem.org/Part:BBa_K1088011">pSB1C3-Plac-dxs <span class="specialWord">(B. subtilis)</span></a>,
 
-
 
-
<a class="dialogLink" href="http://parts.igem.org/Part:BBa_K1088027">pSB1C3-Pcon-lacI-Plac-dxs <span class="specialWord">(B. subtilis)</span></a>, or
 
-
 
-
<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. 5)</b>.
 
</p>
</p>
-
 
-
 
-
 
-
 
-
 
-
<br>
 
-
 
Line 264: Line 177:
<p>
<p>
-
<h4>Characterization of HRT2</h4>
+
<span class="intro">The results prove that we are capable</span> of inducing our HRT2 devices with arabinose. There is only little expression before induction and within the first 15 min, expression is at its maximum. Overexpression of AraC does not seem to have an effect on the expression levels after 15 min compared to natural levels of AraC. However, it is inconclusive whether AraC might contribute to an effect at times less than 15 min after induction. <b>(Fig. 4)</b>.
-
<span class="intro">Rubber purification</span><br>
 
-
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 SOP0031 - 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).
 
</p>
</p>
Line 274: Line 184:
<p>
<p>
-
 
+
<span class="intro">This experience</span> has been added to the experience of the part encoding the <a class="dialogLink" href="http://parts.igem.org/Part:BBa_I13453">arabinose promoter</a> on Parts Registry.  
-
<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.
+
-
 
+
</p>
</p>
-
<p>
 
-
 
-
 
-
<div class="imageGallery galleryMedium alignCenter">
 
-
 
-
<a class="galleryImg" target="_blank" href="https://static.igem.org/mediawiki/2013/1/1b/SDU2013_Characterization_NMR_1.png" title="Figure 6 - 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">Fig. 6</a>
 
-
 
-
<a class="galleryImg" target="_blank" href="https://static.igem.org/mediawiki/2013/f/f3/SDU2013_Characterization_NMR_2.png" title="Figure 7 - 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.">
 
-
<img src="https://static.igem.org/mediawiki/2013/f/f3/SDU2013_Characterization_NMR_2.png">Fig. 7</a>
 
-
 
-
<a class="galleryImg" target="_blank" href="https://static.igem.org/mediawiki/2013/0/04/SDU2013_Characterization_NMR_3.png" title="Figure 8 - 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.">
 
-
<img src="https://static.igem.org/mediawiki/2013/0/04/SDU2013_Characterization_NMR_3.png">Fig. 8</a>
 
-
 
-
.
 
-
</div>
 
-
 
-
<span class="intro">The H<sup>1</sup> NMR test</span><br>
 
-
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 our strain!
 
-
 
-
</p>
 
-
<span class="intro">During the first test</span> of our CPS bacteria (MG1655 transformed with both plasmids - see Design), 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.
 
-
 
-
<p>
 
-
Figure 6 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.
 
-
</p>
 
-
 
-
<p>
 
-
Our rubber extraction of WT + polyisoprene 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.
 
-
</p>
 
-
 
-
<p>
 
-
<span class="intro">The conclusion arrives in figure 8</span>, named DXS+PT, which is our double plasmid CPS bacteria, 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.
 
-
</p>
 
-
 
-
 
-
 
-
 
-
<p>
 
-
 
-
<div class="imageGallery gallerySmall alignRight">
 
-
 
-
<a class="galleryImg" target="_blank" href="https://static.igem.org/mediawiki/2013/0/06/SDU2013_Characterization_NMR_4.png" title="Figure 9 - 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.">
 
-
<img src="https://static.igem.org/mediawiki/2013/0/06/SDU2013_Characterization_NMR_4.png">Fig. 9</a>
 
-
 
-
<a class="galleryImg" target="_blank" href="https://static.igem.org/mediawiki/2013/9/9a/SDU2013_Characterization_NMR_5.png" title="Figure 10 - 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.">
 
-
<img src="https://static.igem.org/mediawiki/2013/9/9a/SDU2013_Characterization_NMR_5.png">Fig. 10</a>
 
-
 
-
</div>
 
<br>
<br>
-
<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.
 
-
</p>
 
-
 
-
<p>
 
-
The test provided the following results: Fig 9 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. 10), 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).
 
-
 
-
</p>
 
-
<br>
 
-
 
-
 
-
<p>
 
-
<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.
 
-
In addition, we must investigate the rubber yield in terms of weight to further evaluate the two purification methods mentioned above.
 
-
 
-
</p>
 
-
 
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<br>
 
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<p>
 
-
 
-
 
-
<span class="intro">MALDI-ToF</span><br>
 
-
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.
 
-
 
-
 
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</p>
 
-
 
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<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>
 
-
 
-
 
-
<h4>Composite production system</h4>
 
-
<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.
 
-
</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 11)</b>
 
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</p>
 
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<p>
 
-
 
-
<a class="popupImg alignCenter" style="width:800px" href="https://static.igem.org/mediawiki/2013/6/64/SDU2013_Characterization_CPS_1.png"
 
-
 
-
title="Figure 11 - 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.">
 
-
  <img src="https://static.igem.org/mediawiki/2013/6/64/SDU2013_Characterization_CPS_1.png"
 
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style="width:800px">
 
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Figure 11 
 
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</a>
 
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</p>
 

Latest revision as of 01:31, 29 October 2013

Expression

Can we control it?

“And by what, O Socrates, is the soul nourished?” - Hippocrates
“By knowledge, of course, I said.” - Socrates

Characterization of our biobricks is a chance for us to prove that our design works as intended. These next two pages will slowly guide you through our results, but keep in mind that not everything is presented below. For all the details, consult our protocol page where you will find a comprehensive picture of our project.

The question of design and therefore of function is two-fold: How do we - and can we - control the expression of our system? And do the expressed proteins work as intended? These are the questions this page sets out to answer. Specifically, we have characterized our regulable biobricks (LacI/Plac and AraC/Para)


Characterization of LacI/Plac

The dxs gene is placed under the control of the lactose promoter (see Design). We assayed the inducible capabilities of our design and, as part of the experiment, we tested the ability to suppress expression prior to induction. The assay was carried out by measuring protein levels of Dxs fused to GFP using FACS. FACSFluorescence Activated Cell Sorting

A LacI:LVA basic part was available at Parts Registry, and we added a promotor and a terminator, producing a device. The GFP fusion devices with and without the LacI:LVA device were assayed to check for expression control. One triplicate of MG1655 and two triplicates of each MG1655 strains carrying either pSB1C3-Plac-dxs (B. subtilits)-GFP or pSB1C3-Pcon-lacI:LVA-term-Plac-dxs (B. subtilits)-GFP were grown from OD600=0.005 to approximately OD600=0.2. At this OD the MG1655 triplicate and one triplicate of each strain carrying constructs were induced with 1 mM IPTG at time 0 min. FACS measurements were done at times: -30, 0, 30, 60, 90, 120, and 150 min, thus showing the expression of GFP in each strain - both when induced and when not induced (Fig. 1).

The results show that strains lacking LacI:LVA do not repress expression from the lactose promoter. Even without induction, there is clear expression of GFP. Conversely, strains expressing LacI:LVA repress the promoter until induction. Approximately 90 min after induction of the strain expressing LacI:LVA, protein level is at its maximum. Still, maximum protein level is lower than that of the strain lacking LacI:LVA. Also, the fraction of flourescent cells is lower in the samples of pSB1C3-lacI:LVA-Plac-dxs (B. subtilits)-GFP compared to pSB1C3-Plac-dxs (B. subtilits)-GFP, indicating that expression of GFP is somewhat repressed, despite induction.

This experience has been added to the experience of the part encoding the LacI:LVA basic part on Parts Registry


Figure 1.


Although LacI:LVA was shown to be functional, we found the prolonged and delayed response time together with lowered maximum protein level after induction to be less than optimal. In summary the performance of LacI:LVA as a repressor did not live up to our expectations when comparing it to the constitutively active strains, nor when comparing it to the wildtype. Therefore, we built a device of similar design to the LacI:LVA device with natural LacI: LacI(N). To test its qualities compared to LacI:LVA, a FACS was analysed. The experimental setup was similar to the previous. Two triplicates of MG1655 strains carrying either pSB1C3-Pcon-lacI(N)-term-Plac-dxs (B. subtilits)-GFP or pSB1C3-Pcon-lacI:LVA-term-Plac-dxs (B. subtilits)-GFP were used and an additional measurement at time 180 min was performed (Fig. 2).

Both strains repress expression when uninduced. The strain expressing LacI:LVA reaches its maximum percentage of fluorescent cells approximately 150 min after induction, whereas the strain overexpressing natural LacI reaches its maximum after a mere 30 to 60 min. The maximum protein levels in the strain overexpressing natural LacI reaches a level approximately 3 times higher than the strain expressing LacI:LVA. Clearly, the function of the natural LacI is superior to that of LacI:LVA.


Figure 2.


To test its qualities compared to LacI:LVA further, another FACS was performed. The experimental setup was similar to the previous. Dublicates of MG1655 strains carrying either pSB1C3-Pcon-lacI(N)-term-Plac-dxs (B. subtilis)-GFP or pSB1C3-Pcon-lacI:LVA-term-Plac-dxs (B. subtilis)-GFP were induced with IPTG concentrations rangingfrom 0.05-0.5mM (Fig. 3). After 180 min upon induction, the strains expressing LacI:LVA with the IPTG concentrations ranging from 0.05-0.5mM reached approximately 25-30 % cells above the fluorescent threshold. Comparing this observation to the strains overexpressing the natural LacI, even the least induced (0.05mM) reached a higher percentage of cells above the fluorescent threshold after 180 min. All of the cells in the culture overexpressing natural LacI induced with 0.125-0.5mM IPTG are above the fluorescent threshold within 180 min. In general, the response time is faster for higher concentrations of IPTG.


Figure 3.


According to Parts Registry, the LVA-tagged LacI part has been used 684 times despite of its poor function. The natural LacI parts that have been submitted to the Registry by previous teams are pending (and thus did not pass HQ quality control). We have therefore submitted the coding sequence of natural LacI to the Registry for future teams to use as an improvement to the LacI:LVA biobrick. Furthermore, our experience has been added to the experience of the part encoding the LacI:LVA basic part (BBa_C0012) on Parts Registry.


Characterization of AraC/Para

Figure 4. The HRT2 gene is under the control of the arabinose promoter (see Design). We assayed the inducible capabilities of our design and, as part of the experiment, we tested the ability to suppress expression prior to induction. The assay was carried out by measuring the mRNA levels of HRT2 using the Northern blotting technique.

To test whether overexpression of AraC improved expression control, devices with and without the araC device were assayed. Duplicates of MG1655 strains carrying either pSB1C3-Para-HRT2 or pSB1C3-Pcon-araC-term-Para-HRT2 were grown to late-exponential phase: OD600=0.8. At this OD, the strains were induced with 0.2 % arabinose at time t=0 min, and samples were taken at times: -2 min, 15 min, and 30 min. Total RNA purified from the samples were run on a gel, blotted onto a membrane, and hybridized with probes specific for HRT2 mRNA and 5S rRNA (loading control), respectively.

The results prove that we are capable of inducing our HRT2 devices with arabinose. There is only little expression before induction and within the first 15 min, expression is at its maximum. Overexpression of AraC does not seem to have an effect on the expression levels after 15 min compared to natural levels of AraC. However, it is inconclusive whether AraC might contribute to an effect at times less than 15 min after induction. (Fig. 4).

This experience has been added to the experience of the part encoding the arabinose promoter on Parts Registry.