Team:SDU-Denmark/Tour42

From 2014.igem.org

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We have made the pTet-OneProt construct in order for us to synthesize a nutritional protein with the  
We have made the pTet-OneProt construct in order for us to synthesize a nutritional protein with the  
correct ratio of essential amino acids and the correct ratio between essential and non-essential  
correct ratio of essential amino acids and the correct ratio between essential and non-essential  
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amino acids. The device is found as <a href="http://parts.igem.org/Part:BBa_K1475000"> Bba_K1475000.</a><br><br>
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amino acids. The device is found as <a href="http://parts.igem.org/Part:BBa_K1475000"> Bba_K1475000.</a> Please note that this part was characterized with an illegal XbaI site between the promoter and RBS, this has no influence on the function of the brick, the XbaI site was later removed and the brick has been submitted to the registry.<br><br>
<span class="intro">The protein is self-designed,</span> so we wanted to test if the protein were expressed in <i>E. coli</i> K12 MG1655, by
<span class="intro">The protein is self-designed,</span> so we wanted to test if the protein were expressed in <i>E. coli</i> K12 MG1655, by
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<div class="popupImg alignCenter" style="width:500px">
<div class="popupImg alignCenter" style="width:500px">
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   <img src="https://static.igem.org/mediawiki/2014/9/99/2014SDUoneprot5.png" style="width:500px" />
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   <img src="https://static.igem.org/mediawiki/2014/4/4e/2014SDUWestern_blot_with_GroL.jpg" style="width:500px" />
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Figure 1: Western blot showing <i>E. coli</i> wild-type and <i>E. coli</i> expressing OneProt at OD at 0.3, 0.8, 1.8 and an overnight  
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Figure 1: Western blot showing <i>E. coli</i> wild-type at OD600 at 0.3, 0.8 and 1.8 and <i>E. coli</i> expressing OneProt at OD600 at 0.3, 0.8, 1.8 and an overnight culture.  
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culture. The membrane has been exposed for 10 minutes.
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</div>
</div>
<p>
<p>
<br>
<br>
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<span class="intro">The protein has a</span> 3xFLAG tag and since bonds are showing, OneProt is expressed. However, from this
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<span class="intro">The protein has a 3xFLAG tag</span> and since bonds are showing, OneProt is expressed. However, from this
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western blot, we cannot see if the protein has been cut, just that it is expressed.
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western blot, we cannot see if the protein has been cut, just that it is expressed.<br><br>
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</p>
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<p>
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<a class="popupImg alignRight" style="width:250px" target="_blank" href="https://static.igem.org/mediawiki/2014/7/7d/2014SDUresults7.PNG" title="Figure 2: Coomassie staining of <i>E. coli</i> expressing OneProt at early exponential phase (OD600=0.3), late exponential phase (OD600=1.5), stationary phase (OD=2.5) and an overnight culture using an empty vector as control.">
<a class="popupImg alignRight" style="width:250px" target="_blank" href="https://static.igem.org/mediawiki/2014/7/7d/2014SDUresults7.PNG" title="Figure 2: Coomassie staining of <i>E. coli</i> expressing OneProt at early exponential phase (OD600=0.3), late exponential phase (OD600=1.5), stationary phase (OD=2.5) and an overnight culture using an empty vector as control.">
   <img src="https://static.igem.org/mediawiki/2014/7/7d/2014SDUresults7.PNG" style="width:250px" />
   <img src="https://static.igem.org/mediawiki/2014/7/7d/2014SDUresults7.PNG" style="width:250px" />
Figure 2: Coomassie staining of <i>E. coli</i> expressing OneProt at early exponential phase (OD600=0.3), late exponential phase (OD600=1.5), stationary phase (OD=2.5) and an overnight culture using an empty vector as control.
Figure 2: Coomassie staining of <i>E. coli</i> expressing OneProt at early exponential phase (OD600=0.3), late exponential phase (OD600=1.5), stationary phase (OD=2.5) and an overnight culture using an empty vector as control.
</a>
</a>
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<span class="intro">In order to check that</span> we had the protein expressed in its full length, we did a coomassie stain on a SDS-
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In order to check that we had the protein expressed in its full length, we did a coomassie stain on a SDS-
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page. Here we also wanted to receive information on the expression of the protein at different growth  
page. Here we also wanted to receive information on the expression of the protein at different growth  
stages of <i>E. coli</i>. We analyzed samples from early exponential phase (OD600=0.3), late exponential phase  
stages of <i>E. coli</i>. We analyzed samples from early exponential phase (OD600=0.3), late exponential phase  
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(PSC1C3) was used.<br><br>
(PSC1C3) was used.<br><br>
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OneProt has a molecular weight of approximately 53.7 kDa. Unfortunately, there is no clear bond at this
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<span class="intro">OneProt has a molecular weight</span> of approximately 53.7 kDa. Unfortunately, there is no clear bond at this length. However, there is a bond at approximately 25 kDa, which is not detected in the control. We cannot  
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length. However, there is a bond at approximately 25 kDa, which is not detected in the control. We cannot  
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conclude what gives rise to the band, but it might be a cellular response to an unfolded protein.<br><br>
conclude what gives rise to the band, but it might be a cellular response to an unfolded protein.<br><br>
-
To test what effect the expression of OneProt have on <i>E. coli</i> we set up a growth experiment
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<span class="intro">To test what effect</span> the expression of OneProt have on <i>E. coli</i> we set up a growth experiment
measuring OD over time on the growth of <i>E. coli</i> K12 MG1655 WT, odor-free <i>E. coli</i> YYC912, <i>E. coli</i> K12  
measuring OD over time on the growth of <i>E. coli</i> K12 MG1655 WT, odor-free <i>E. coli</i> YYC912, <i>E. coli</i> K12  
expressing OneProt and with an empty vector.<br><br>
expressing OneProt and with an empty vector.<br><br>
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</p>
</p>
<p>
<p>
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<a class="popupImg alignLeft" style="width:250px" target="_blank" href="https://static.igem.org/mediawiki/2014/e/e5/2014SDUresults8.png" title="Figure 3: Growth curve illustrating the growth of <i>E. coli</i> K12 MG1655 WT, odor-free <i>E. coli</i> YYC912, <i>E. coli</i> K12 expressing OneProt and with an empty vector.">
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<a class="popupImg alignLeft" style="width:250px" target="_blank" href="https://static.igem.org/mediawiki/2014/b/bf/2014SDUGrowth_-_WT%2C_YYC912%2C_Oneprot%2C_Empty_vector.png" title="Figure 3: Growth curve illustrating the growth of <i>E. coli</i> K12 MG1655 WT, odor-free <i>E. coli</i> YYC912, <i>E. coli</i> K12 expressing OneProt and with an empty vector.">
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   <img src="https://static.igem.org/mediawiki/2014/e/e5/2014SDUresults8.png" style="width:250px" />
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   <img src="https://static.igem.org/mediawiki/2014/b/bf/2014SDUGrowth_-_WT%2C_YYC912%2C_Oneprot%2C_Empty_vector.png" style="width:250px" />
Figure 3: Growth curve illustrating the growth of <i>E. coli</i> K12 MG1655 WT, odor-free <i>E. coli</i> YYC912, <i>E. coli</i> K12 expressing OneProt and with an empty vector.
Figure 3: Growth curve illustrating the growth of <i>E. coli</i> K12 MG1655 WT, odor-free <i>E. coli</i> YYC912, <i>E. coli</i> K12 expressing OneProt and with an empty vector.
</a>
</a>
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From the growth curve, it is shown that the expression of OneProt stresses the metabolism a lot compared
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<span class="intro">From the growth curve, it is shown</span> that the growth-rate of neither the strain expressing the OneProt protein nor the YYC912 strain is affected a lot compared to the <i>E. coli</i> K12 wild-type.<br><br>
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to the <i>E. coli</i> K12 wild-type. In addition to this, the metabolism of YYC912 is also quite stressed compared to
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the K12 wild-type. Despite the stressed metabolism of the two strains, the expression of OneProt increases
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over time as does the growth of YYC912.<br><br>
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By comparing the growth curve of <i>E. coli</i> K12 expressing OneProt, TetR(+LVA), TetR(no LVA) and odor-free <i>E. coli</i> YYC912 it is seen that the metabolism of <i>E. coli</i> expressing OneProt and TetR is stressed compared to  
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<span class="intro">By comparing the growth curve</span> of <i>E. coli</i> K12 expressing OneProt, TetR(+LVA), TetR(no LVA) and odor-free <i>E. coli</i> YYC912 it is seen that the growth-rate of <i>E. coli</i> expressing TetR is lowered compared to  
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the wild-type, which means that it might be difficult to have OneProt expressed in high amounts controlled  
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the other strains, which means that it might be difficult to have OneProt expressed in high amounts controlled  
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by pTet (+/-LVA). It is, however, shown that the cells are growing despite of their stressed metabolism
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by pTet (+/-LVA), with the TetR repressors overexpressed at present levels. However, as argued on the previous page, the levels of TetR would have to be lowered, which would probably alleviate the attenuated growth-rate. <br><br>
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and it is possible that the expression of OneProt can be controlled by pTet, which in turn is controlled by TetR, although
+
-
the TetR(+LVA) seems more favorable. It can also be seen that the growth of <i>E. coli</i> YYC912 is comprised
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compared to the K12 wild-type which also contributes to possible difficulties in expressing OneProt in the
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-
odor-free YYC912 strain. However, the possibility still exists.<br><br>
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</p>
</p>
<p>
<p>
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<a class="popupImg alignRight" style="width:400px" target="_blank" href="https://static.igem.org/mediawiki/2014/4/44/2014SDUresults9.png" title="Figure 4: Growth curves showing <i>E. coli</i> K12 MG 1655 expressing OneProt, TetR(+LVA), TetR(no LVA), wild-type and odor-free <i>E. coli</i> YYC912.">
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<a class="popupImg alignRight" style="width:400px" target="_blank" href="https://static.igem.org/mediawiki/2014/c/cb/2014SDUDouble_growth_curve_-_revideret_daniel-01-01.PNG" title="Figure 4: Growth curves showing <i>E. coli</i> K12 MG 1655 expressing OneProt, TetR(+LVA), TetR(no LVA), wild-type and odor-free <i>E. coli</i> YYC912.">
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   <img src="https://static.igem.org/mediawiki/2014/4/44/2014SDUresults9.png" style="width:400px" />
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   <img src="https://static.igem.org/mediawiki/2014/c/cb/2014SDUDouble_growth_curve_-_revideret_daniel-01-01.PNG" style="width:400px" />
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Figure 4: Growth curves showing <i>E. coli</i> K12 expressing OneProt, TetR(+LVA), TetR(no LVA), wild-type and odor-free <i>E. coli</i> YYC912.
+
Figure 4: Growth curves showing <i>E. coli</i> K12 MG1655 expressing OneProt, TetR(+LVA), TetR(no LVA), wild-type and odor-free <i>E. coli</i> YYC912.
</a>
</a>
-
Because OneProt is self-designed, we wanted to test if the protein has any toxicity. To do so, we fed  
+
<span class="intro">Because OneProt is self-designed,</span> we wanted to test if the protein has any toxicity. To do so, we fed  
<i>Caenorhabditis elegans</i> (<i>C. elegans</i>) with <i>E. coli</i> K12 MG1655 containing an empty vector and a vector  
<i>Caenorhabditis elegans</i> (<i>C. elegans</i>) with <i>E. coli</i> K12 MG1655 containing an empty vector and a vector  
expressing OneProt on separate plates. On both plates, 20 <i>C. elegans</i> were tested. Articles recommend  
expressing OneProt on separate plates. On both plates, 20 <i>C. elegans</i> were tested. Articles recommend  
-
using heat chock assay for 7 hours: 1 hour at 35° C followed by 1 hour at 22°C,   
+
using heat shock assay for 7 hours: 1 hour at 35° C followed by 1 hour at 22°C,   
<span class="sourceReference">repeated.</span>
<span class="sourceReference">repeated.</span>
<span class="tooltip">
<span class="tooltip">
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<a href="http://www.sciencedirect.com/science/article/pii/S016895251300022X" target="_blank">(Link)</a></span><br><br>
<a href="http://www.sciencedirect.com/science/article/pii/S016895251300022X" target="_blank">(Link)</a></span><br><br>
-
After approximately 5 hours no effects on <i>C. elegans</i> was detectable. Therefore we decided to stress  
+
<span class="intro">After approximately 5 hours</span> no effects on <i>C. elegans</i> was detectable. Therefore we decided to stress  
<i>C. elegans</i> a little more, incubating them in 2 hours at 35°C followed by 1 hour at 22°C, repeated. After 7  
<i>C. elegans</i> a little more, incubating them in 2 hours at 35°C followed by 1 hour at 22°C, repeated. After 7  
-
hours, every <i>C. elegans</i> on both plates were alive. Thus we conclude that the protein has no toxic effect.<br><br>
+
hours, every <i>C. elegans</i> on both plates were alive. Thus we conclude that the strain expressing OneProt at presents levels doesn't display toxic effects on <i>C. elegans</i>. This suggest that there are no obvious toxic effects towards eukaryotic cells such as human cells. However, the resulst are far from conclusive, and extensive testing is necessarry to confirm this suggestion.<br><br>
</p>
</p>

Latest revision as of 03:53, 18 October 2014

OneProt

The pTet expression system and limonene synthase construct is evolved around one thing: the OneProt. We have made the pTet-OneProt construct in order for us to synthesize a nutritional protein with the correct ratio of essential amino acids and the correct ratio between essential and non-essential amino acids. The device is found as Bba_K1475000. Please note that this part was characterized with an illegal XbaI site between the promoter and RBS, this has no influence on the function of the brick, the XbaI site was later removed and the brick has been submitted to the registry.

The protein is self-designed, so we wanted to test if the protein were expressed in E. coli K12 MG1655, by the use of Western blotting. The western blot was blottet with E. coli K12 MG1655 wild-type and E. coli expressing OneProt at different OD measures.

Figure 1: Western blot showing E. coli wild-type at OD600 at 0.3, 0.8 and 1.8 and E. coli expressing OneProt at OD600 at 0.3, 0.8, 1.8 and an overnight culture.


The protein has a 3xFLAG tag and since bonds are showing, OneProt is expressed. However, from this western blot, we cannot see if the protein has been cut, just that it is expressed.

Figure 2: Coomassie staining of E. coli expressing OneProt at early exponential phase (OD600=0.3), late exponential phase (OD600=1.5), stationary phase (OD=2.5) and an overnight culture using an empty vector as control. In order to check that we had the protein expressed in its full length, we did a coomassie stain on a SDS- page. Here we also wanted to receive information on the expression of the protein at different growth stages of E. coli. We analyzed samples from early exponential phase (OD600=0.3), late exponential phase (OD600=1.5), stationary phase (OD=2.5) and an overnight culture. As a control, E. coli with an empty vector (PSC1C3) was used.

OneProt has a molecular weight of approximately 53.7 kDa. Unfortunately, there is no clear bond at this length. However, there is a bond at approximately 25 kDa, which is not detected in the control. We cannot conclude what gives rise to the band, but it might be a cellular response to an unfolded protein.

To test what effect the expression of OneProt have on E. coli we set up a growth experiment measuring OD over time on the growth of E. coli K12 MG1655 WT, odor-free E. coli YYC912, E. coli K12 expressing OneProt and with an empty vector.

Figure 3: Growth curve illustrating the growth of E. coli K12 MG1655 WT, odor-free E. coli YYC912, E. coli K12 expressing OneProt and with an empty vector. From the growth curve, it is shown that the growth-rate of neither the strain expressing the OneProt protein nor the YYC912 strain is affected a lot compared to the E. coli K12 wild-type.

By comparing the growth curve of E. coli K12 expressing OneProt, TetR(+LVA), TetR(no LVA) and odor-free E. coli YYC912 it is seen that the growth-rate of E. coli expressing TetR is lowered compared to the other strains, which means that it might be difficult to have OneProt expressed in high amounts controlled by pTet (+/-LVA), with the TetR repressors overexpressed at present levels. However, as argued on the previous page, the levels of TetR would have to be lowered, which would probably alleviate the attenuated growth-rate.

Figure 4: Growth curves showing E. coli K12 MG1655 expressing OneProt, TetR(+LVA), TetR(no LVA), wild-type and odor-free E. coli YYC912. Because OneProt is self-designed, we wanted to test if the protein has any toxicity. To do so, we fed Caenorhabditis elegans (C. elegans) with E. coli K12 MG1655 containing an empty vector and a vector expressing OneProt on separate plates. On both plates, 20 C. elegans were tested. Articles recommend using heat shock assay for 7 hours: 1 hour at 35° C followed by 1 hour at 22°C, repeated. Source: Mosbech, M., et al.: Functional Loss of Two Ceramide Synthases Elicits Autophagy-Dependent Lifespan Extension in C. elegans.: PLoS ONE, 2013. 8 vol:7. (Link)   Source: Rodriguez, M., et al.:Worms under stress: C. elegans stress response and its relevance to complex human disease and aging. Trends in Genetics, 2013. Vol: 29, 6, p. 367-374. (Link)

After approximately 5 hours no effects on C. elegans was detectable. Therefore we decided to stress C. elegans a little more, incubating them in 2 hours at 35°C followed by 1 hour at 22°C, repeated. After 7 hours, every C. elegans on both plates were alive. Thus we conclude that the strain expressing OneProt at presents levels doesn't display toxic effects on C. elegans. This suggest that there are no obvious toxic effects towards eukaryotic cells such as human cells. However, the resulst are far from conclusive, and extensive testing is necessarry to confirm this suggestion.

Figure 5: Picture of C. elegans fed with E. coli K12 MG1655 expressing OneProt.