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− | /*The following code describes the | + | /*The following code describes the 2nd table*/ |
#table2 tbody tr:nth-child(odd) td:nth-child(1), | #table2 tbody tr:nth-child(odd) td:nth-child(1), | ||
#table2 tbody tr:nth-child(odd) td:nth-child(4) { | #table2 tbody tr:nth-child(odd) td:nth-child(4) { | ||
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background: none; | background: none; | ||
} | } | ||
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− | #table3 tbody tr:nth-child(1 | + | /*The following code describes the 3rd table*/ |
− | + | #table3 tbody tr:nth-child(4n+1) td:nth-child(4), | |
− | #table3 tbody tr:not(:nth-child(4n+1)) td:nth-child(3) | + | #table3 tbody tr:not(:nth-child(4n+1)) td:nth-child(3) { |
width: 210px; | width: 210px; | ||
white-space: normal; | white-space: normal; | ||
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+ | |||
+ | #table3 tbody tr:nth-child(1) td:nth-child(1), | ||
+ | #table3 tbody tr:nth-child(5) td:nth-child(1) { | ||
+ | background: #D1E0F0; | ||
+ | } | ||
+ | |||
+ | /*The following code describes the 4th table*/ | ||
+ | #table4 tbody tr:nth-child(odd) td:nth-child(1), | ||
+ | #table4 tbody tr:nth-child(odd) td:nth-child(4) { | ||
+ | background: #F0F5FA; | ||
+ | } | ||
+ | |||
+ | #table4 tbody tr:nth-child(even) td:nth-child(1), | ||
+ | #table4 tbody tr:nth-child(even) td:nth-child(4) { | ||
+ | background: #D1E0F0; | ||
+ | } | ||
+ | |||
+ | #table4 tbody tr:nth-child(odd):hover td:nth-child(1), | ||
+ | #table4 tbody tr:nth-child(odd):hover td:nth-child(4) { | ||
+ | background: none; | ||
+ | } | ||
+ | |||
+ | #table4 tbody tr:nth-child(even):hover td:nth-child(1), | ||
+ | #table4 tbody tr:nth-child(even):hover td:nth-child(4) { | ||
+ | background: none; | ||
+ | } | ||
+ | |||
+ | |||
/*The following content describes collapsible buttons*/ | /*The following content describes collapsible buttons*/ | ||
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<li><a class="aBG" href="#aAmylase">Alpha amylase production</a></li> | <li><a class="aBG" href="#aAmylase">Alpha amylase production</a></li> | ||
<li><a class="aBG" href="#improving">Improving alpha amylase production</a></li> | <li><a class="aBG" href="#improving">Improving alpha amylase production</a></li> | ||
+ | <li><a class="aBG" href="#discussion">Final discussion</a></li> | ||
+ | <li><a class="aBG" href="#ref">References</a></li> | ||
</ul> | </ul> | ||
</div> | </div> | ||
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<h2 class="headline">Introduction</h2> | <h2 class="headline">Introduction</h2> | ||
</div> | </div> | ||
− | <p> | + | <p>On this page the main findings and interpretations of the results of our project can be found. In our |
project we aimed to achieve four different goals. These four proof of concepts are summarized in | project we aimed to achieve four different goals. These four proof of concepts are summarized in | ||
<strong>Figure 1</strong>. | <strong>Figure 1</strong>. | ||
Line 93: | Line 123: | ||
</div> | </div> | ||
<p>As we plan on feeding our GMO with ammonia extracted from the MOF, an essential part of our project | <p>As we plan on feeding our GMO with ammonia extracted from the MOF, an essential part of our project | ||
− | is that our GMO should be able to grow in the presence of ammonia as its only nitrogen source. | + | is that our GMO should be able to grow in the presence of ammonia as its only nitrogen source. Therefore, |
− | + | we grew 6 different <em>Saccharomyces </em>strains under different ammonia concentrations to | |
see if the presence of ammonia would affect their growth. In the end we were able to identify the | see if the presence of ammonia would affect their growth. In the end we were able to identify the | ||
best performing strains. The protocol can be found in the <a | best performing strains. The protocol can be found in the <a | ||
href="https://2021.igem.org/Team:Groningen/Experiments">Experiments page</a>.</p> | href="https://2021.igem.org/Team:Groningen/Experiments">Experiments page</a>.</p> | ||
<figure> | <figure> | ||
− | <img src="https://static.igem.org/mediawiki/2021/ | + | <img src="https://static.igem.org/mediawiki/2021/8/86/T--Groningen--Results_Figure2_1.png"> |
<figcaption>Growth under different ammonia concentrations of different wild type yeast | <figcaption>Growth under different ammonia concentrations of different wild type yeast | ||
(Saccharomyces cerevisiae and Saccharomyces paradoxus) strains. The strains tested are a. sYB76 | (Saccharomyces cerevisiae and Saccharomyces paradoxus) strains. The strains tested are a. sYB76 | ||
(NCYC3597, S. cerevisiae) b. sYB77 (NCYC3566, S. cerevisiae) c. sYB78 (NCYC3598, S. cerevisiae) | (NCYC3597, S. cerevisiae) b. sYB77 (NCYC3566, S. cerevisiae) c. sYB78 (NCYC3598, S. cerevisiae) | ||
d. sYB79 (NCYC3635, S. paradoxus) e. sYB83 (NCYC3647, S. paradoxus) f. sYB85 (NCYC3687, S. | d. sYB79 (NCYC3635, S. paradoxus) e. sYB83 (NCYC3647, S. paradoxus) f. sYB85 (NCYC3687, S. | ||
− | paradoxus)</figcaption> | + | paradoxus) g. Control (no cells)</figcaption> |
</figure> | </figure> | ||
− | <p>As can be seen from <strong>Figure 2</strong> there are 4 strains that outperformed in the | + | <p>As can be seen from <strong>Figure 2</strong>, there are 4 strains that outperformed in the |
− | experiment | + | experiment. These strains were chosen for further experimentation. The doubling time of the chosen |
strains can be seen in<strong> Table 1</strong>.</p> | strains can be seen in<strong> Table 1</strong>.</p> | ||
<p class="caption">Doubling time (T<sub>D</sub>) for each strain in different ammonia concentrations in | <p class="caption">Doubling time (T<sub>D</sub>) for each strain in different ammonia concentrations in | ||
the exponential phase.</p> | the exponential phase.</p> | ||
− | <div class="table_div" id="table1"> | + | <div class="table_div" style="overflow-x: auto;" id="table1"> |
<table class="table_content"> | <table class="table_content"> | ||
<thead> | <thead> | ||
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<p>For our second proof of concept, we wanted to demonstrate that it is possible to use Golden Gate | <p>For our second proof of concept, we wanted to demonstrate that it is possible to use Golden Gate | ||
Assembly to clone heterologous genes for alpha-amylase in <em>Saccharomyces spp</em>. Going from the | Assembly to clone heterologous genes for alpha-amylase in <em>Saccharomyces spp</em>. Going from the | ||
− | gene of interest to the cassette plasmid expressed in <em>Saccharomyces spp.</em> is a long process | + | gene of interest to the cassette plasmid expressed in <em>Saccharomyces spp.</em> is a long process. |
− | + | Throughout it there were some checkpoints that we used to confirm the success of the cloning: GFP | |
screening; DNA concentration measurements with a Nanodrop; basic parts sequencing; and digestion of | screening; DNA concentration measurements with a Nanodrop; basic parts sequencing; and digestion of | ||
the final cassette plasmids.</p> | the final cassette plasmids.</p> | ||
<h3>GFP screening</h3> | <h3>GFP screening</h3> | ||
<p>The empty plasmid (pRS426__ConLS'-GFPdropout-ConRE'-URA3-2micron-Kan) used in the Golden Gate | <p>The empty plasmid (pRS426__ConLS'-GFPdropout-ConRE'-URA3-2micron-Kan) used in the Golden Gate | ||
− | cassette plasmid assemblies contains a <strong>GFP | + | cassette plasmid assemblies contains a <strong>GFP dropout.</strong> This way, when the assembled |
− | plasmid is inserted correctly, the GFP insert will | + | plasmid is inserted correctly, the GFP insert will dropout and white colonies will be formed. |
− | Whenever the plasmid was not inserted correctly the GFP | + | Whenever the plasmid was not inserted correctly, the GFP dropout will still be present and colonies |
− | will appear green when examined | + | will appear green when examined under UV-light (<strong>Figure 3</strong>). With this simple technique |
we make sure that the chosen colonies have uptaken the assembled plasmid without the need of | we make sure that the chosen colonies have uptaken the assembled plasmid without the need of | ||
performing sequencing or digestion experiments prior to plasmid isolation. This GFP screening was | performing sequencing or digestion experiments prior to plasmid isolation. This GFP screening was | ||
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<figure> | <figure> | ||
<img src="https://static.igem.org/mediawiki/2021/2/26/T--Groningen--Results_Figure3.png"> | <img src="https://static.igem.org/mediawiki/2021/2/26/T--Groningen--Results_Figure3.png"> | ||
− | <figcaption>Screening in E. coli colonies with the cassette plasmid SP034. The green fluorescent | + | <figcaption>Screening in <i>E. coli</i> colonies with the cassette plasmid SP034. The green fluorescent |
colony can be found underneath the cross, the rest appears white coloured. Only white colonies | colony can be found underneath the cross, the rest appears white coloured. Only white colonies | ||
were selected for plasmid isolation.</figcaption> | were selected for plasmid isolation.</figcaption> | ||
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<div class="collapsible_content"> | <div class="collapsible_content"> | ||
<p>GFP screening smoothened the process, considering the amount of samples that we needed to | <p>GFP screening smoothened the process, considering the amount of samples that we needed to | ||
− | assemble. Generally, all the transformed E.coli with cassette plasmids (64 plates) showed a high | + | assemble. Generally, all the transformed <i>E.coli</i> with cassette plasmids (64 plates) showed a high |
efficiency of the Golden Gate assembly; a few countable green colonies were observed in contrast | efficiency of the Golden Gate assembly; a few countable green colonies were observed in contrast | ||
to hundreds of white colonies. One sample (SP007) didn’t show any white colonies, the | to hundreds of white colonies. One sample (SP007) didn’t show any white colonies, the | ||
Line 216: | Line 246: | ||
using a nanodrop spectrophotometer, measuring the DNA concentration at a spectrum of wavelengths. | using a nanodrop spectrophotometer, measuring the DNA concentration at a spectrum of wavelengths. | ||
</p> | </p> | ||
− | <p class="caption">DNA concentration and purity after E. coli transformation of cassette plasmid and | + | <p class="caption">DNA concentration and purity after <i>E. coli</i> transformation of cassette plasmid and |
plasmid isolation.</p> | plasmid isolation.</p> | ||
− | <div class="table_div" id="table2"> | + | <div class="table_div" style="overflow-x: auto;" id="table2"> |
<table class="table_content"> | <table class="table_content"> | ||
<thead> | <thead> | ||
Line 235: | Line 265: | ||
<td>22,6</td> | <td>22,6</td> | ||
<td>1,823</td> | <td>1,823</td> | ||
− | <td> SP034</td> | + | <td>SP034</td> |
<td>80</td> | <td>80</td> | ||
<td>0,001</td> | <td>0,001</td> | ||
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</button> | </button> | ||
<div class="collapsible_content"> | <div class="collapsible_content"> | ||
− | <p>The A260/280 ratio | + | <p>The A260/280 ratio represents the purity of the DNA, an ideal sample would have a value of |
− | approximately 1.8. Due to large variation in DNA concentration and A260/280 ratio, | + | approximately 1.8. Due to large variation in DNA concentration and A260/280 ratio, 20 μl of each |
− | cassette plasmid was used in the <em>Saccharomyces spp.</em> transformation instead of | + | cassette plasmid was used in the <em>Saccharomyces spp.</em> transformation instead of 5 μl. |
− | Overall the concentration of DNA in the samples after transformation and plasmid isolation were | + | Overall, the concentration of DNA in the samples after transformation and plasmid isolation were |
good and ensured the continuation with<em> Saccharomyces spp</em>. transformations.</p> | good and ensured the continuation with<em> Saccharomyces spp</em>. transformations.</p> | ||
</div> | </div> | ||
Line 508: | Line 538: | ||
href="https://2021.igem.org/Team:Groningen/Engineering">Engineering page</a>). pYTK001 (entry | href="https://2021.igem.org/Team:Groningen/Engineering">Engineering page</a>). pYTK001 (entry | ||
vector) with Part 3 and 3b was sequenced using two primers flanking the gene, the vector is used to | vector) with Part 3 and 3b was sequenced using two primers flanking the gene, the vector is used to | ||
− | compose the cassettes plasmids in further experiments. | + | compose the cassettes plasmids in further experiments. All sequencing results are summarized in |
− | + | <strong>Table 3.</strong> | |
− | <p class=" | + | </p> |
− | <div class="table_div"> | + | <p class="caption">Sequencing results from all the parts.</p> |
− | <table class="table_content" id="table3"> | + | <div class="table_div" style="overflow-x: auto;" id="table3"> |
+ | <table class="table_content" id="table3" style="overflow-x: auto;"> | ||
<thead> | <thead> | ||
<tr> | <tr> | ||
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</table> | </table> | ||
</div> | </div> | ||
− | + | <button type="button" class="collapsible"> | |
− | + | <h4>Conclusion</h4> | |
+ | <i class="fas fa-plus" id="plus"></i> | ||
+ | <i class="fas fa-minus" id="minus"></i> | ||
+ | </button> | ||
+ | <div class="collapsible_content"> | ||
+ | <p>Generally, the results were quite successful and no major mutations were observed. However, two | ||
+ | clear missense mutations were identified in alpha-amylases from <em>B. amyloliquefaciens</em> | ||
+ | and <em>B. licheniformis</em> that may have an effect on the protein functionality. It would | ||
+ | have been nice if we had the time to restart the cloning from the beginning, unfortunately, the | ||
+ | cassette plasmids were already assembled and the time was very limited. In Part 3b full coverage | ||
+ | of the gene couldn’t be achieved, repeating the sequencing was planned but sadly we | ||
+ | didn’t have the time.</p> | ||
+ | <p>All in all, the mutations found will be taken into account for further results of those enzymes. | ||
+ | </p> | ||
+ | </div> | ||
+ | <h3>Constructs digestion</h3> | ||
+ | <p>We used the restriction enzyme BsmBI to check for the correct ligation of the different constructs of | ||
+ | the library. There are two sites for the enzyme BsmBI in the vector employed (pRS246) for our | ||
+ | constructs, the sites are in part 1 and part 5, on both sides of the insert of interest | ||
+ | (<strong>Figure 4</strong>). The use of BsmBI allows us to check for the size (<strong>Table | ||
+ | 4</strong>) of the construct and assess the success of the assembly. The results of the | ||
+ | digestion can be found in <strong>Figure 5</strong>.</p> | ||
+ | <figure> | ||
+ | <img src="https://static.igem.org/mediawiki/2021/1/17/T--Groningen--Results_Figure4_2.png" width="50%" alt=""> | ||
+ | <figcaption>Scheme of one of the constructs from the library, SP001, where the sites for the enzyme | ||
+ | BsmBI are displayed.</figcaption> | ||
+ | </figure> | ||
+ | <p class="caption">Expected sizes of the different constructs of the library. The elements of each | ||
+ | sample can be found in the pages of the Parts plasmids.</p> | ||
+ | <div class="table_div" style="overflow-x: auto;" id="table4"> | ||
+ | <table class="table_content"> | ||
+ | <thead> | ||
+ | <tr> | ||
+ | <th>Sample</th> | ||
+ | <th>Expected sizes A (bp)</th> | ||
+ | <th>Expected sizes B (bp)</th> | ||
+ | <th>Sample</th> | ||
+ | <th>Expected sizes A (bp)</th> | ||
+ | <th>Expected sizes B (bp)</th> | ||
+ | </tr> | ||
+ | </thead> | ||
+ | <tbody> | ||
+ | <tr> | ||
+ | <td>SP001</td> | ||
+ | <td>3729</td> | ||
+ | <td>3387</td> | ||
+ | <td>SP034</td> | ||
+ | <td>3729</td> | ||
+ | <td>2983</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>SP002</td> | ||
+ | <td>3729</td> | ||
+ | <td>2869</td> | ||
+ | <td>SP035</td> | ||
+ | <td>3729</td> | ||
+ | <td>2970</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>SP005</td> | ||
+ | <td>3729</td> | ||
+ | <td>3387</td> | ||
+ | <td>SP037</td> | ||
+ | <td>3729</td> | ||
+ | <td>3387</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>SP006</td> | ||
+ | <td>3729</td> | ||
+ | <td>2953</td> | ||
+ | <td>SP038</td> | ||
+ | <td>3729</td> | ||
+ | <td>3387</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>SP008</td> | ||
+ | <td>3729</td> | ||
+ | <td>3237</td> | ||
+ | <td>SP039</td> | ||
+ | <td>3729</td> | ||
+ | <td>2982</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>SP009</td> | ||
+ | <td>3729</td> | ||
+ | <td>3237</td> | ||
+ | <td>SP040</td> | ||
+ | <td>3729</td> | ||
+ | <td>2970</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>SP010</td> | ||
+ | <td>3729</td> | ||
+ | <td>3387</td> | ||
+ | <td>SP041</td> | ||
+ | <td>3729</td> | ||
+ | <td>2802</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>SP011</td> | ||
+ | <td>3729</td> | ||
+ | <td>2776</td> | ||
+ | <td>SP042</td> | ||
+ | <td>3729</td> | ||
+ | <td>2982</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>SP012</td> | ||
+ | <td>3729</td> | ||
+ | <td>2755</td> | ||
+ | <td>SP043</td> | ||
+ | <td>3729</td> | ||
+ | <td>2953</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>SP013</td> | ||
+ | <td>3729</td> | ||
+ | <td>3381</td> | ||
+ | <td>SP044</td> | ||
+ | <td>3729</td> | ||
+ | <td>2886</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>SP014</td> | ||
+ | <td>3729</td> | ||
+ | <td>2982</td> | ||
+ | <td>SP045</td> | ||
+ | <td>3729</td> | ||
+ | <td>2982</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>SP015</td> | ||
+ | <td>3729</td> | ||
+ | <td>2869</td> | ||
+ | <td>SP046</td> | ||
+ | <td>3729</td> | ||
+ | <td>2866</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>SP016</td> | ||
+ | <td>3729</td> | ||
+ | <td>2790</td> | ||
+ | <td>SP047</td> | ||
+ | <td>3729</td> | ||
+ | <td>2755</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>SP017</td> | ||
+ | <td>3729</td> | ||
+ | <td>2869</td> | ||
+ | <td>SP048</td> | ||
+ | <td>3729</td> | ||
+ | <td>2970</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>SP018</td> | ||
+ | <td>3729</td> | ||
+ | <td>2880</td> | ||
+ | <td>SP049</td> | ||
+ | <td>3729</td> | ||
+ | <td>2749</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>SP019</td> | ||
+ | <td>3729</td> | ||
+ | <td>2796</td> | ||
+ | <td>SP050</td> | ||
+ | <td>3729</td> | ||
+ | <td>2970</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>SP020</td> | ||
+ | <td>3729</td> | ||
+ | <td>2886</td> | ||
+ | <td>SP051</td> | ||
+ | <td>3729</td> | ||
+ | <td>2970</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>SP021</td> | ||
+ | <td>3729</td> | ||
+ | <td>3387</td> | ||
+ | <td>SP052</td> | ||
+ | <td>3729</td> | ||
+ | <td>2982</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>SP022</td> | ||
+ | <td>3729</td> | ||
+ | <td>2982</td> | ||
+ | <td>SP053</td> | ||
+ | <td>3729</td> | ||
+ | <td>2755</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>SP023</td> | ||
+ | <td>3729</td> | ||
+ | <td>2970</td> | ||
+ | <td>SP054</td> | ||
+ | <td>3729</td> | ||
+ | <td>3291</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>SP024</td> | ||
+ | <td>3729</td> | ||
+ | <td>2886</td> | ||
+ | <td>SP055</td> | ||
+ | <td>3729</td> | ||
+ | <td>2982</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>SP025</td> | ||
+ | <td>3729</td> | ||
+ | <td>2802</td> | ||
+ | <td>SP056</td> | ||
+ | <td>3729</td> | ||
+ | <td>2796</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>SP026</td> | ||
+ | <td>3729</td> | ||
+ | <td>2982</td> | ||
+ | <td>SP057</td> | ||
+ | <td>3729</td> | ||
+ | <td>2970</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>SP027</td> | ||
+ | <td>3729</td> | ||
+ | <td>3237</td> | ||
+ | <td>SP058</td> | ||
+ | <td>3729</td> | ||
+ | <td>2755</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>SP028</td> | ||
+ | <td>3729</td> | ||
+ | <td>2982</td> | ||
+ | <td>SP059</td> | ||
+ | <td>3729</td> | ||
+ | <td>2886</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>SP029</td> | ||
+ | <td>3729</td> | ||
+ | <td>2982</td> | ||
+ | <td>SP060</td> | ||
+ | <td>3729</td> | ||
+ | <td>3237</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>SP030</td> | ||
+ | <td>3729</td> | ||
+ | <td>2866</td> | ||
+ | <td>SP061</td> | ||
+ | <td>3729</td> | ||
+ | <td>2970</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>SP031</td> | ||
+ | <td>3729</td> | ||
+ | <td>2886</td> | ||
+ | <td>SP062</td> | ||
+ | <td>3729</td> | ||
+ | <td>2976</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>SP032</td> | ||
+ | <td>3729</td> | ||
+ | <td>2970</td> | ||
+ | <td>SP063</td> | ||
+ | <td>3729</td> | ||
+ | <td>2982</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>SP033</td> | ||
+ | <td>3729</td> | ||
+ | <td>3387</td> | ||
+ | <td>SP064</td> | ||
+ | <td>3729</td> | ||
+ | <td>2869</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>pRS426-GFP</td> | ||
+ | <td>3729</td> | ||
+ | <td>1338</td> | ||
+ | <td></td> | ||
+ | <td></td> | ||
+ | <td></td> | ||
+ | </tr> | ||
+ | </tbody> | ||
+ | </table> | ||
+ | </div> | ||
+ | <figure> | ||
+ | <img src="https://static.igem.org/mediawiki/2021/4/47/T--Groningen--Results_Figure5.png"> | ||
+ | <figcaption>Digestion of the library of constructs with the enzyme BsmBI.</figcaption> | ||
+ | </figure> | ||
+ | <button type="button" class="collapsible"> | ||
+ | <h4>Conclusion</h4> | ||
+ | <i class="fas fa-plus" id="plus"></i> | ||
+ | <i class="fas fa-minus" id="minus"></i> | ||
+ | </button> | ||
+ | <div class="collapsible_content"> | ||
+ | <p>After the digestion of all the constructs it can be seen that the expected sizes of the | ||
+ | engineered plasmids match the sizes of the bands shown in the gel. The efficiency of the Golden | ||
+ | Gate assembly is high. There are two samples in which a band of the size of the GFP-drop out is | ||
+ | observed, SP012 and SP058, besides the expected size. This result may be due to contamination | ||
+ | while pipetting. Nevertheless, the expected band can be observed, the samples were used in follow | ||
+ | up experiments, consideration on alpha amylase activity will be taken into account. | ||
+ | Additionally, some samples (SP006, SP011, SP022, SP023, SP044, SP046, SP047, SP052, SP053, | ||
+ | SP055) show faint bands, likely evidencing that the concentration of DNA was low in the first | ||
+ | place. The faint bands have the expected size, and considering the high efficiency in the rest | ||
+ | of the digests we conclude that our Golden Gate assemblies and further screening was highly | ||
+ | successful. No samples were excluded after digestion.</p> | ||
+ | </div> | ||
</section> | </section> | ||
<section id="aAmylase"> | <section id="aAmylase"> | ||
<div class="section-header"> | <div class="section-header"> | ||
<h2 class="headline">Alpha amylase production</h2> | <h2 class="headline">Alpha amylase production</h2> | ||
+ | </div> | ||
+ | <p>For our third proof of concept, we wanted to show that our GMO does indeed show alpha-amylase | ||
+ | activity after taking up our construct. We aimed to measure the alpha-amylase production of 64 | ||
+ | samples listed at our <a href="https://2021.igem.org/Team:Groningen/Engineering#prod">Engineering | ||
+ | page</a>. This was tested with two main experiments. The principal one being an alpha amylase | ||
+ | kit assay used to perform <strong>high throughput</strong> experiments and obtain | ||
+ | <strong>quantitative </strong>data, needed for our model (see <a | ||
+ | href="https://2021.igem.org/Team:Groningen/Model">Model page</a>). Additionally, a | ||
+ | <strong>qualitative assay</strong> was employed to confirm the production of alpha-amylase in some | ||
+ | of the samples. To understand more how this assay works, see the <a | ||
+ | href="https://2021.igem.org/Team:Groningen/Experiments">Experiments page</a>. | ||
+ | </p> | ||
+ | <h3>Alpha-amylase starch breakdown</h3> | ||
+ | <p>Functionality of alpha-amylase can <strong>qualitatively</strong> be proved by observing the breakdown | ||
+ | of starch. The ability to break down starch is only available when amylase is produced, which | ||
+ | in the case of <em>S. cerevisiae</em> and <em>S. paradoxus</em> doesn’t happen natively | ||
+ | (<strong>Figure 6e</strong>). However, the assembled constructs should be able to break down starch. | ||
+ | In order to confirm this, selective media plates without uracil (specific for the assembled | ||
+ | constructs) were created with the addition of 1% starch. Several samples were cultured ON and | ||
+ | treated before being plated on the starch plates, as described in the <a | ||
+ | href="https://2021.igem.org/Team:Groningen/Notebook">notebook</a> (wk 39), after which pictures | ||
+ | were taken (<strong>Figure 6</strong>) of the with/without iodine treated starch plates. Samples | ||
+ | tested with this assay can be found in <strong>Table 5</strong>.</p> | ||
+ | <p class="caption">Composition of samples that were tested by the Halo assay.</p> | ||
+ | <div class="table_div" style="overflow-x: auto;" id="table5"> | ||
+ | <table class="table_content"> | ||
+ | <thead> | ||
+ | <tr> | ||
+ | <th>Sample</th> | ||
+ | <th>Yeast Strain</th> | ||
+ | <th>Promoter</th> | ||
+ | <th>Gene</th> | ||
+ | <th>Terminator</th> | ||
+ | </tr> | ||
+ | </thead> | ||
+ | <tbody> | ||
+ | <tr> | ||
+ | <td>SP001</td> | ||
+ | <td>ySB77</td> | ||
+ | <td>pPGK1</td> | ||
+ | <td>αMF + BS</td> | ||
+ | <td>tTDH1</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>SP010</td> | ||
+ | <td>ySB77</td> | ||
+ | <td>pHHF1</td> | ||
+ | <td>αMF + BS</td> | ||
+ | <td>tTDH1</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>SP021</td> | ||
+ | <td>ySB77</td> | ||
+ | <td>pPGK1</td> | ||
+ | <td>αMFΔ + BA</td> | ||
+ | <td>tTDH1</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>SP026</td> | ||
+ | <td>ySB76</td> | ||
+ | <td>pHHF1</td> | ||
+ | <td>αMF + BL</td> | ||
+ | <td>tTDH1</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>SP027</td> | ||
+ | <td>ySB76</td> | ||
+ | <td>pRNR1</td> | ||
+ | <td>BS</td> | ||
+ | <td>tTDH1</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>SP034</td> | ||
+ | <td>ySB76</td> | ||
+ | <td>pTEF1</td> | ||
+ | <td>αMF + BL</td> | ||
+ | <td>tTDH1</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>SP035</td> | ||
+ | <td>ySB76</td> | ||
+ | <td>pPAB1</td> | ||
+ | <td>αMF_no_EAEA + BL</td> | ||
+ | <td>tTDH1</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>SP051</td> | ||
+ | <td>ySB76</td> | ||
+ | <td>pRNR1</td> | ||
+ | <td>αMF_no_EAEA + BL</td> | ||
+ | <td>tTDH1</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>SP063</td> | ||
+ | <td>ySB76</td> | ||
+ | <td>pRNR2</td> | ||
+ | <td>αMF + BL</td> | ||
+ | <td>tTDH1</td> | ||
+ | </tr> | ||
+ | </tbody> | ||
+ | </table> | ||
+ | </div> | ||
+ | <figure> | ||
+ | <img src="https://static.igem.org/mediawiki/2021/4/4c/T--Groningen--Results_Figure6.png"> | ||
+ | <figcaption>Halo assay for 9 constructs and non engineered <em>Saccharomyces spp.</em> in triplets, | ||
+ | each plate contains 3 samples, SP001, SP010 and SP021 in plate 1 (left one), SP026, SP027, SP034 | ||
+ | in plate 2 (middle) and SP035, SP061 and SP063 in plate 3 (right). Cells were pretreated | ||
+ | differently to check for intra- or extracellular alpha amylase activity. A) Pure cells, B) | ||
+ | Supernatant of overnight culture, C) Lysate of cells by bead beating 10s, D) Lysate of cells by | ||
+ | bead beating during 10s + 30s.</figcaption> | ||
+ | </figure> | ||
+ | <button type="button" class="collapsible"> | ||
+ | <h4>Conclusion</h4> | ||
+ | <i class="fas fa-plus" id="plus"></i> | ||
+ | <i class="fas fa-minus" id="minus"></i> | ||
+ | </button> | ||
+ | <div class="collapsible_content"> | ||
+ | <p>As can be seen in <b>Figure 6</b>, a larger halo was observed in the cells’ lysate samples. Halos were | ||
+ | also visible in most of the supernatant samples. This implies that alpha-amylase is present | ||
+ | <strong>extra- and intracellular</strong> after cell lysis. We were able to confirm that this | ||
+ | activity was only produced in the strains that had taken up the created constructs, as the non | ||
+ | engineered strains didn’t show any halo at all. Overall, we see a difference in the alpha-amylase activity when samples were treated differently, suggesting that the secretion of the | ||
+ | enzyme varies between constructs. More specifically to our project, we planned to isolate the | ||
+ | protein from the supernatant, this way the processing of the cells is minimal and lysating is | ||
+ | not directly necessary. Therefore, it was interesting to find that lysating some of the samples | ||
+ | did produce a bigger halo, since this could change our proposed way of extracting alpha-amylase. | ||
+ | Only few samples of the library were tested through this essay, therefore general conclusions | ||
+ | can not be drawn, but confirmation of alpha amylase-activity could be assessed. | ||
+ | </p> | ||
+ | </div> | ||
+ | <h3>Alpha-amylase assay kit</h3> | ||
+ | <p>We used a colorimetric assay to obtain <strong>quantitative data</strong> of the activity of the | ||
+ | alpha amylase produced by the engineered yeast, allowing for comparison between samples and for | ||
+ | quantitative data to use in our model.</p> | ||
+ | <figure> | ||
+ | <img src="https://static.igem.org/mediawiki/2021/8/8c/T--Groningen--Results_Figure7_2.png"> | ||
+ | <figcaption>Activity of alpha amylase in a random set of samples from the library. The sample | ||
+ | number refers to the SP0 (being #3 the same as SP003). Activity is measured as the absolute | ||
+ | change in absorbance due to the appearance of p-nitrophenol after 3h.</figcaption> | ||
+ | </figure> | ||
+ | <p>The assay is based on the capacity of alpha amylase to cleavage ethylidene-pNP-G7, resulting in | ||
+ | <strong>p-nitrophenol</strong>, a product that can be read at a wavelength of 405 nm, more | ||
+ | details on the <a href="https://2021.igem.org/Team:Groningen/Experiments">Experiments page</a>. | ||
+ | A testing round was first performed (<strong>Figure 7</strong>) in duplicates per sample to | ||
+ | check how the experiment was done and identify possible steps that may need to be optimized. | ||
+ | This round also helped to exclude samples SP003, SP004 and SP036 for further analysis as we were | ||
+ | tight on reagents and the results weren’t promising. | ||
+ | </p> | ||
+ | <p>During the high throughput experiments, we tested the capacity to produce <strong>functional | ||
+ | alpha-amylase</strong> of all the samples which were not excluded from the subset of the | ||
+ | combinatorial library (the excluded samples are listed on the <a | ||
+ | href="https://2021.igem.org/Team:Groningen/Model#art1">Model page</a>, section Accounting for | ||
+ | lost samples). Activity of alpha-amylase in triplicates of each sample can be found in | ||
+ | <strong>Figure 8</strong>. Supernatants from overnight cultures were incubated with | ||
+ | ethylidene-pNP-G7 in 96 well plates and the appearance of p-nitrophenol was measured every 5 min | ||
+ | during the first hour, then once after 2h and 3h. If alpha-amylase was in the supernatant, an | ||
+ | <strong>increase of absorbance</strong> was expected due to the appearance of p-nitrophenol, an | ||
+ | indirect measurement of alpha-amylase activity. Therefore, activity (y-axis of <strong>Figure 8</strong>) | ||
+ | was measured as the increase of absorbance (quantity of p-nitrophenol) that appears | ||
+ | per time unit (d[p-nitrophenol]/dt). | ||
+ | </p> | ||
+ | <figure> | ||
+ | <img src="https://static.igem.org/mediawiki/2021/b/b5/T--Groningen--Results_Figure8.png"> | ||
+ | <figcaption>High throughput experiment on alpha amylase activity. We tested the activity of | ||
+ | alpha amylase in triplicates in 60 samples. The different graphs account for 3 different | ||
+ | 96-well plates a) Samples measured together in a 96-well plate on October 2nd, b) Samples | ||
+ | measured together in a 96-well plate on October 5th and c) Samples measured together in a | ||
+ | 96-well plate on October 5th.</figcaption> | ||
+ | </figure> | ||
+ | <p>After having excluded the samples in which the variance between replicates deviated too much from | ||
+ | the average variance, a table summarizing the mean activity of each sample can be found below. | ||
+ | The different combinations from the subset library are also displayed in <strong>Table | ||
+ | 6</strong>.</p> | ||
+ | <p class="caption">Alpha-amylase activity of the construct from the subset of combinations. The | ||
+ | different components from each sample are also displayed.</p> | ||
+ | <div class="table_div" style="overflow-x: auto;" id="table6"> | ||
+ | <table class="table_content" style="overflow-x: auto;"> | ||
+ | <thead> | ||
+ | <tr> | ||
+ | <th>Sample</th> | ||
+ | <th>Strain</th> | ||
+ | <th>Promoter</th> | ||
+ | <th>Secretion signal</th> | ||
+ | <th>Gene</th> | ||
+ | <th>Activity values (d[p-nitrophenol]/dt)</th> | ||
+ | </tr> | ||
+ | </thead> | ||
+ | <tbody> | ||
+ | <tr> | ||
+ | <td>SP001</td> | ||
+ | <td>ySB77</td> | ||
+ | <td>pPGK1</td> | ||
+ | <td>αMF</td> | ||
+ | <td><em>B.subtilis</em></td> | ||
+ | <td>0,005476099</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>SP002</td> | ||
+ | <td>ySB85</td> | ||
+ | <td>pRNR1</td> | ||
+ | <td>αMFΔ</td> | ||
+ | <td><em>A.oryzae</em></td> | ||
+ | <td>3,4293E-05</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>SP005</td> | ||
+ | <td>ySB78</td> | ||
+ | <td>pHHF1</td> | ||
+ | <td>αMF</td> | ||
+ | <td><em>B.subtilis</em></td> | ||
+ | <td>0,000260468</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>SP008</td> | ||
+ | <td>ySB78</td> | ||
+ | <td>pHHF1</td> | ||
+ | <td>Native</td> | ||
+ | <td><em>B.subtilis</em></td> | ||
+ | <td>0,000192798</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>SP009</td> | ||
+ | <td>ySB85</td> | ||
+ | <td>pRNR1</td> | ||
+ | <td>Native</td> | ||
+ | <td><em>B.subtilis</em></td> | ||
+ | <td>0,00052307</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>SP010</td> | ||
+ | <td>ySB77</td> | ||
+ | <td>pHHF1</td> | ||
+ | <td>αMF</td> | ||
+ | <td><em>B.subtilis</em></td> | ||
+ | <td>0,001100158</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>SP012</td> | ||
+ | <td>ySB78</td> | ||
+ | <td>pRNR1</td> | ||
+ | <td>Native</td> | ||
+ | <td><em>A.oryzae</em></td> | ||
+ | <td>9,26803E-05</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>SP013</td> | ||
+ | <td>ySB85</td> | ||
+ | <td>pRAD27</td> | ||
+ | <td>αMF</td> | ||
+ | <td><em>B.subtilis</em></td> | ||
+ | <td>0,000945715</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>SP014</td> | ||
+ | <td>ySB76</td> | ||
+ | <td>pREV1</td> | ||
+ | <td>αMF</td> | ||
+ | <td><em>B.amyloliquefaciens</em></td> | ||
+ | <td>0,00001</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>SP015</td> | ||
+ | <td>ySB76</td> | ||
+ | <td>pPGK1</td> | ||
+ | <td>αMFΔ</td> | ||
+ | <td><em>A.oryzae</em></td> | ||
+ | <td>1,44142E-05</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>SP016</td> | ||
+ | <td>ySB77</td> | ||
+ | <td>pRAD27</td> | ||
+ | <td>Native</td> | ||
+ | <td><em>B.licheniformis</em></td> | ||
+ | <td>1,2205E-05</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>SP018</td> | ||
+ | <td>ySB76</td> | ||
+ | <td>pRAD27</td> | ||
+ | <td>αMFΔ</td> | ||
+ | <td><em>B.amyloliquefaciens</em></td> | ||
+ | <td>0,00001</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>SP019</td> | ||
+ | <td>ySB78</td> | ||
+ | <td>pTEF1</td> | ||
+ | <td>Native</td> | ||
+ | <td><em>B.licheniformis</em></td> | ||
+ | <td>1,08206E-05</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>SP022</td> | ||
+ | <td>ySB77</td> | ||
+ | <td>pREV1</td> | ||
+ | <td>αMF</td> | ||
+ | <td><em>B.amyloliquefaciens</em></td> | ||
+ | <td>1,14565E-05</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>SP023</td> | ||
+ | <td>ySB77</td> | ||
+ | <td>pRNR2</td> | ||
+ | <td>αMF_no_EAEA</td> | ||
+ | <td><em>B.licheniformis</em></td> | ||
+ | <td>0,00001</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>SP024</td> | ||
+ | <td>ySB77</td> | ||
+ | <td>pTEF1</td> | ||
+ | <td>αMFΔ</td> | ||
+ | <td><em>B.licheniformis</em></td> | ||
+ | <td>0,000341834</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>SP025</td> | ||
+ | <td>ySB78</td> | ||
+ | <td>pPAB1</td> | ||
+ | <td>Native</td> | ||
+ | <td><em>B.amyloliquefaciens</em></td> | ||
+ | <td>1,15319E-05</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>SP026</td> | ||
+ | <td>ySB76</td> | ||
+ | <td>pHHF1</td> | ||
+ | <td>αMF</td> | ||
+ | <td><em>B.licheniformis</em></td> | ||
+ | <td>0,000200209</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>SP027</td> | ||
+ | <td>ySB76</td> | ||
+ | <td>pRNR1</td> | ||
+ | <td>Native</td> | ||
+ | <td><em>B.subtilis</em></td> | ||
+ | <td>0,001931731</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>SP028</td> | ||
+ | <td>ySB77</td> | ||
+ | <td>pTEF1</td> | ||
+ | <td>αMF</td> | ||
+ | <td><em>B.amyloliquefaciens</em></td> | ||
+ | <td>1,20322E-05</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>SP029</td> | ||
+ | <td>ySB85</td> | ||
+ | <td>pRPL18B</td> | ||
+ | <td>αMF</td> | ||
+ | <td><em>B.licheniformis</em></td> | ||
+ | <td>5,32401E-05</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>SP030</td> | ||
+ | <td>ySB85</td> | ||
+ | <td>pTDH3</td> | ||
+ | <td>αMFΔ</td> | ||
+ | <td><em>B.amyloliquefaciens</em></td> | ||
+ | <td>0,00001</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>SP031</td> | ||
+ | <td>ySB77</td> | ||
+ | <td>pPGK1</td> | ||
+ | <td>αMFΔ</td> | ||
+ | <td><em>B.amyloliquefaciens</em></td> | ||
+ | <td>2,44344E-05</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>SP032</td> | ||
+ | <td>ySB78</td> | ||
+ | <td>pRPL18B</td> | ||
+ | <td>αMF_no_EAEA</td> | ||
+ | <td><em>B.amyloliquefaciens</em></td> | ||
+ | <td>1,85894E-05</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>SP033</td> | ||
+ | <td>ySB85</td> | ||
+ | <td>pREV1</td> | ||
+ | <td>αMF</td> | ||
+ | <td><em>B.subtilis</em></td> | ||
+ | <td>0,000845811</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>SP034</td> | ||
+ | <td>ySB76</td> | ||
+ | <td>pTEF1</td> | ||
+ | <td>αMF</td> | ||
+ | <td><em>B.licheniformis</em></td> | ||
+ | <td>0,000211464</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>SP035</td> | ||
+ | <td>ySB76</td> | ||
+ | <td>pPAB1</td> | ||
+ | <td>αMF_no_EAEA</td> | ||
+ | <td><em>B.licheniformis</em></td> | ||
+ | <td>0,00021031</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>SP037</td> | ||
+ | <td>ySB85</td> | ||
+ | <td>pRPL18B</td> | ||
+ | <td>αMF</td> | ||
+ | <td><em>B.subtilis</em></td> | ||
+ | <td>0,00113086</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>SP038</td> | ||
+ | <td>ySB78</td> | ||
+ | <td>pRNR2</td> | ||
+ | <td>αMF</td> | ||
+ | <td><em>B.subtilis</em></td> | ||
+ | <td>0,002933777</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>SP039</td> | ||
+ | <td>ySB85</td> | ||
+ | <td>pTEF1</td> | ||
+ | <td>αMF</td> | ||
+ | <td><em>B.licheniformis</em></td> | ||
+ | <td>1,41915E-05</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>SP041</td> | ||
+ | <td>ySB85</td> | ||
+ | <td>pHHF1</td> | ||
+ | <td>Native</td> | ||
+ | <td><em>B.amyloliquefaciens</em></td> | ||
+ | <td>1,17257E-05</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>SP044</td> | ||
+ | <td>ySB77</td> | ||
+ | <td>pRNR2</td> | ||
+ | <td>αMFΔ</td> | ||
+ | <td><em>B.licheniformis</em></td> | ||
+ | <td>4,16163E-05</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>SP045</td> | ||
+ | <td>ySB78</td> | ||
+ | <td>pPAB1</td> | ||
+ | <td>αMF</td> | ||
+ | <td><em>B.licheniformis</em></td> | ||
+ | <td>9,44327E-05</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>SP046</td> | ||
+ | <td>ySB77</td> | ||
+ | <td>pTDH3</td> | ||
+ | <td>αMFΔ</td> | ||
+ | <td><em>B.licheniformis</em></td> | ||
+ | <td>1,15677E-05</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>SP047</td> | ||
+ | <td>ySB77</td> | ||
+ | <td>pPGK1</td> | ||
+ | <td>Native</td> | ||
+ | <td><em>A.oryzae</em></td> | ||
+ | <td>0,00019171</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>SP049</td> | ||
+ | <td>ySB85</td> | ||
+ | <td>pRAD27</td> | ||
+ | <td>Native</td> | ||
+ | <td><em>A.oryzae</em></td> | ||
+ | <td>0,00029359</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>SP050</td> | ||
+ | <td>ySB77</td> | ||
+ | <td>pPGK1</td> | ||
+ | <td>αMF_no_EAEA</td> | ||
+ | <td><em>B.amyloliquefaciens</em></td> | ||
+ | <td>1,39692E-05</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>SP051</td> | ||
+ | <td>ySB76</td> | ||
+ | <td>pRNR1</td> | ||
+ | <td>αMF_no_EAEA</td> | ||
+ | <td><em>B.licheniformis</em></td> | ||
+ | <td>0,000290985</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>SP052</td> | ||
+ | <td>ySB77</td> | ||
+ | <td>pPAB1</td> | ||
+ | <td>αMF</td> | ||
+ | <td><em>B.amyloliquefaciens</em></td> | ||
+ | <td>1,01931E-05</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>SP053</td> | ||
+ | <td>ySB85</td> | ||
+ | <td>pHHF1</td> | ||
+ | <td>Native</td> | ||
+ | <td><em>A.oryzae</em></td> | ||
+ | <td>1,35179E-05</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>SP054</td> | ||
+ | <td>ySB85</td> | ||
+ | <td>pRPL18B</td> | ||
+ | <td>αMFΔ</td> | ||
+ | <td><em>B.subtilis</em></td> | ||
+ | <td>0,000154906</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>SP055</td> | ||
+ | <td>ySB77</td> | ||
+ | <td>pRPL18B</td> | ||
+ | <td>αMF</td> | ||
+ | <td><em>B.licheniformis</em></td> | ||
+ | <td>0,00001</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>SP056</td> | ||
+ | <td>ySB78</td> | ||
+ | <td>pPAB1</td> | ||
+ | <td>Native</td> | ||
+ | <td><em>B.licheniformis</em></td> | ||
+ | <td>1,94392E-05</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>SP058</td> | ||
+ | <td>ySB78</td> | ||
+ | <td>pRNR2</td> | ||
+ | <td>Native</td> | ||
+ | <td><em>A.oryzae</em></td> | ||
+ | <td>6,79755E-05</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>SP060</td> | ||
+ | <td>ySB85</td> | ||
+ | <td>pREV1</td> | ||
+ | <td>Native</td> | ||
+ | <td><em>B.subtilis</em></td> | ||
+ | <td>0,000223757</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>SP061</td> | ||
+ | <td>ySB85</td> | ||
+ | <td>pREV1</td> | ||
+ | <td>αMF_no_EAEA</td> | ||
+ | <td><em>B.amyloliquefaciens</em></td> | ||
+ | <td>1,40926E-05</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>SP062</td> | ||
+ | <td>ySB85</td> | ||
+ | <td>pRAD27</td> | ||
+ | <td>αMF</td> | ||
+ | <td><em>B.amyloliquefaciens</em></td> | ||
+ | <td>1,38021E-05</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>SP063</td> | ||
+ | <td>ySB76</td> | ||
+ | <td>pRNR2</td> | ||
+ | <td>αMF</td> | ||
+ | <td><em>B.licheniformis</em></td> | ||
+ | <td>0,000596797</td> | ||
+ | </tr> | ||
+ | </tbody> | ||
+ | </table> | ||
+ | </div> | ||
+ | <button type="button" class="collapsible"> | ||
+ | <h4>Conclusion</h4> | ||
+ | <i class="fas fa-plus" id="plus"></i> | ||
+ | <i class="fas fa-minus" id="minus"></i> | ||
+ | </button> | ||
+ | <div class="collapsible_content"> | ||
+ | <p>Experiment were performed in triplicate to account for stochasticity, samples SP006, SP011, | ||
+ | SP020, SP021, SP042 and SP059 were excluded, as the variance between replicates deviated from | ||
+ | the mean variance, evidencing that the signal was more likely an artefact than actual alpha | ||
+ | amylase activity. For the rest of the samples displayed in the graphs it can be concluded that | ||
+ | there is a high variability in terms of how active alpha amylase is. Some constructs | ||
+ | aren’t successful in expressing active alpha amylase in the supernatant (any sample with | ||
+ | an activity value below 0,00001 was considered non active). However, other samples produce | ||
+ | bigger amounts of p-nitrophenol and thus have more alpha-amylase activity in the supernatant. | ||
+ | </p> | ||
+ | <p>Some <strong>general trends</strong> can be observed by looking at the graphs and the table, for | ||
+ | instance, a relatively high alpha amylase activity is observed in samples that contain the alpha | ||
+ | amylase gene from the <strong>host </strong><strong><em>B. subtilis</em></strong>. The | ||
+ | interpretation of such a big set of samples and identification of the best performers is an | ||
+ | arduous task. AI technology could help getting deeper insights on our library and finding a | ||
+ | better combination from the combinatorial library, more on this can be found in the <a | ||
+ | href="https://2021.igem.org/Team:Groningen/Model">model page</a>.</p> | ||
</div> | </div> | ||
</section> | </section> | ||
Line 581: | Line 1,523: | ||
<h2 class="headline">Improving alpha amylase production</h2> | <h2 class="headline">Improving alpha amylase production</h2> | ||
</div> | </div> | ||
+ | <p>We used the Automated Recommendation Tool (ART) to model which samples from the combinatorial library | ||
+ | discussed on the <a href="https://2021.igem.org/Team:Groningen/Engineering">engineering page</a> | ||
+ | would yield a high alpha-amylase activity in the supernatant (exploitative recommendations). | ||
+ | Furthermore, the ART also provides recommendations on which samples should be tested in a future | ||
+ | DBTL-cycle in order to improve the performance of the model (exploratory recommendations). On this | ||
+ | page, only the final results of the ART are discussed. How these results are generated and how to | ||
+ | analyze them is discussed on the <a href="https://2021.igem.org/Team:Groningen/Model#art">modeling | ||
+ | page</a>.</p> | ||
+ | <h3>Exploitative recommendations</h3> | ||
+ | <p class="caption">Exploitative recommendation given by the ART.</p> | ||
+ | <div class="table_div" style="overflow-x: auto;" id="table7"> | ||
+ | <table class="table_content"> | ||
+ | <thead> | ||
+ | <tr> | ||
+ | <th>Rank</th> | ||
+ | <th>strain</th> | ||
+ | <th>promoter</th> | ||
+ | <th>secretion</th> | ||
+ | <th>gene</th> | ||
+ | <th>Predicted ln(activity)</th> | ||
+ | <th>Predicted activity</th> | ||
+ | </tr> | ||
+ | </thead> | ||
+ | <tbody> | ||
+ | <tr> | ||
+ | <td>0</td> | ||
+ | <td>ySB78</td> | ||
+ | <td>pPGK1</td> | ||
+ | <td>αMF</td> | ||
+ | <td><em>B.subtilis</em></td> | ||
+ | <td>-6.241298</td> | ||
+ | <td>0.001947</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>1</td> | ||
+ | <td>ySB78</td> | ||
+ | <td>pTDH3</td> | ||
+ | <td>αMF</td> | ||
+ | <td><em>B.subtilis</em></td> | ||
+ | <td>-6.323765</td> | ||
+ | <td>0.001793</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>2</td> | ||
+ | <td>ySB76</td> | ||
+ | <td>pRNR2</td> | ||
+ | <td>αMF</td> | ||
+ | <td><em>B.subtilis</em></td> | ||
+ | <td>-6.363736</td> | ||
+ | <td>0.001723</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>3</td> | ||
+ | <td>ySB85</td> | ||
+ | <td>pPGK1</td> | ||
+ | <td>αMF</td> | ||
+ | <td><em>B.subtilis</em></td> | ||
+ | <td>-6.388392</td> | ||
+ | <td>0.001681</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>4</td> | ||
+ | <td>ySB76</td> | ||
+ | <td>pPGK1</td> | ||
+ | <td>αMF</td> | ||
+ | <td><em>B.subtilis</em></td> | ||
+ | <td>-6.435489</td> | ||
+ | <td>0.001604</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>5</td> | ||
+ | <td>ySB77</td> | ||
+ | <td>pRNR2</td> | ||
+ | <td>αMF</td> | ||
+ | <td><em>B.subtilis</em></td> | ||
+ | <td>-6.521911</td> | ||
+ | <td>0.001471</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>6</td> | ||
+ | <td>ySB77</td> | ||
+ | <td>pTDH3</td> | ||
+ | <td>αMF</td> | ||
+ | <td><em>B.subtilis</em></td> | ||
+ | <td>-6.566358</td> | ||
+ | <td>0.001407</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>7</td> | ||
+ | <td>ySB76</td> | ||
+ | <td>pHHF1</td> | ||
+ | <td>αMF</td> | ||
+ | <td><em>B.subtilis</em></td> | ||
+ | <td>-6.573551</td> | ||
+ | <td>0.001397</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>8</td> | ||
+ | <td>ySB76</td> | ||
+ | <td>pRNR1</td> | ||
+ | <td>αMF</td> | ||
+ | <td><em>B.subtilis</em></td> | ||
+ | <td>-6.666158</td> | ||
+ | <td>0.001273</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>9</td> | ||
+ | <td>ySB76</td> | ||
+ | <td>pTDH3</td> | ||
+ | <td>αMF</td> | ||
+ | <td><em>B.subtilis</em></td> | ||
+ | <td>-6.676021</td> | ||
+ | <td>0.001261</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>10</td> | ||
+ | <td>ySB85</td> | ||
+ | <td>pRNR1</td> | ||
+ | <td>αMF</td> | ||
+ | <td><em>B.subtilis</em></td> | ||
+ | <td>-6.689628</td> | ||
+ | <td>0.001244</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>11</td> | ||
+ | <td>ySB77</td> | ||
+ | <td>pRNR1</td> | ||
+ | <td>αMF</td> | ||
+ | <td><em>B.subtilis</em></td> | ||
+ | <td>-6.707219</td> | ||
+ | <td>0.001222</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>12</td> | ||
+ | <td>ySB76</td> | ||
+ | <td>pPAB1</td> | ||
+ | <td>αMF</td> | ||
+ | <td><em>B.subtilis</em></td> | ||
+ | <td>-6.75746</td> | ||
+ | <td>0.001162</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>13</td> | ||
+ | <td>ySB77</td> | ||
+ | <td>pREV1</td> | ||
+ | <td>αMF</td> | ||
+ | <td><em>B.subtilis</em></td> | ||
+ | <td>-6.892306</td> | ||
+ | <td>0.001016</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>14</td> | ||
+ | <td>ySB78</td> | ||
+ | <td>pRNR1</td> | ||
+ | <td>αMF</td> | ||
+ | <td><em>B.subtilis</em></td> | ||
+ | <td>-6.892674</td> | ||
+ | <td>0.001015</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>15</td> | ||
+ | <td>ySB85</td> | ||
+ | <td>pPAB1</td> | ||
+ | <td>αMF</td> | ||
+ | <td><em>B.subtilis</em></td> | ||
+ | <td>-6.914319</td> | ||
+ | <td>0.000993</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>16</td> | ||
+ | <td>ySB76</td> | ||
+ | <td>pTEF1</td> | ||
+ | <td>αMF</td> | ||
+ | <td><em>B.subtilis</em></td> | ||
+ | <td>-6.930234</td> | ||
+ | <td>0.000978</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>17</td> | ||
+ | <td>ySB78</td> | ||
+ | <td>pPAB1</td> | ||
+ | <td>αMF</td> | ||
+ | <td><em>B.subtilis</em></td> | ||
+ | <td>-6.938977</td> | ||
+ | <td>0.000969</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>18</td> | ||
+ | <td>ySB76</td> | ||
+ | <td>pREV1</td> | ||
+ | <td>αMF</td> | ||
+ | <td><em>B.subtilis</em></td> | ||
+ | <td>-6.942261</td> | ||
+ | <td>0.000966</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>19</td> | ||
+ | <td>ySB85</td> | ||
+ | <td>pTEF1</td> | ||
+ | <td>αMF</td> | ||
+ | <td><em>B.subtilis</em></td> | ||
+ | <td>-6.95399</td> | ||
+ | <td>0.000955</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>20</td> | ||
+ | <td>ySB77</td> | ||
+ | <td>pPAB1</td> | ||
+ | <td>αMF</td> | ||
+ | <td><em>B.subtilis</em></td> | ||
+ | <td>-6.968985</td> | ||
+ | <td>0.000941</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>21</td> | ||
+ | <td>ySB78</td> | ||
+ | <td>pRPL18B</td> | ||
+ | <td>αMF</td> | ||
+ | <td><em>B.subtilis</em></td> | ||
+ | <td>-6.980546</td> | ||
+ | <td>0.00093</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>22</td> | ||
+ | <td>ySB85</td> | ||
+ | <td>pTDH3</td> | ||
+ | <td>αMF</td> | ||
+ | <td><em>B.subtilis</em></td> | ||
+ | <td>-6.996441</td> | ||
+ | <td>0.000915</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>23</td> | ||
+ | <td>ySB77</td> | ||
+ | <td>pTEF1</td> | ||
+ | <td>αMF</td> | ||
+ | <td><em>B.subtilis</em></td> | ||
+ | <td>-7.012753</td> | ||
+ | <td>0.0009</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>24</td> | ||
+ | <td>ySB78</td> | ||
+ | <td>pREV1</td> | ||
+ | <td>αMF</td> | ||
+ | <td><em>B.subtilis</em></td> | ||
+ | <td>-7.057184</td> | ||
+ | <td>0.000861</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>25</td> | ||
+ | <td>ySB78</td> | ||
+ | <td>pRAD27</td> | ||
+ | <td>αMF</td> | ||
+ | <td><em>B.subtilis</em></td> | ||
+ | <td>-7.06477</td> | ||
+ | <td>0.000855</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>26</td> | ||
+ | <td>ySB85</td> | ||
+ | <td>pRNR2</td> | ||
+ | <td>αMF</td> | ||
+ | <td><em>B.subtilis</em></td> | ||
+ | <td>-7.082414</td> | ||
+ | <td>0.00084</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>27</td> | ||
+ | <td>ySB78</td> | ||
+ | <td>pRNR1</td> | ||
+ | <td>αMF_no_EAEA</td> | ||
+ | <td><em>B.subtilis</em></td> | ||
+ | <td>-7.096215</td> | ||
+ | <td>0.000828</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>28</td> | ||
+ | <td>ySB78</td> | ||
+ | <td>pTEF1</td> | ||
+ | <td>αMF</td> | ||
+ | <td><em>B.subtilis</em></td> | ||
+ | <td>-7.134024</td> | ||
+ | <td>0.000798</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>29</td> | ||
+ | <td>ySB76</td> | ||
+ | <td>pRAD27</td> | ||
+ | <td>αMF</td> | ||
+ | <td><em>B.subtilis</em></td> | ||
+ | <td>-7.223168</td> | ||
+ | <td>0.000729</td> | ||
+ | </tr> | ||
+ | </tbody> | ||
+ | </table> | ||
+ | </div> | ||
+ | <p><strong>Table 7</strong> shows that all exploitative recommendations contain the alpha-amylase | ||
+ | encoding genes from <em>B.subtilis</em>. Furthermore, the αMF secretion peptide occurs in 29 | ||
+ | out of 30 recommendations. It is also striking that 8 out of 10 recommendations contain either the | ||
+ | pRNR2, pTDH3 or pPGK1 promoter, with the pPGK1 promoter occurring in 3 out of the top 5 | ||
+ | recommendations. No clear pattern can be noticed in the recommendations regarding which | ||
+ | <em>Saccharomyces spp.</em> should be used as a chassis. | ||
+ | </p> | ||
+ | <h3>Exploratory recommendations</h3> | ||
+ | <p class="caption">Exploratory recommendation given by the ART</p> | ||
+ | <div class="table_div" style="overflow-x: auto;" id="table8"> | ||
+ | <table class="table_content"> | ||
+ | <thead> | ||
+ | <tr> | ||
+ | <th>Rank</th> | ||
+ | <th>strain</th> | ||
+ | <th>promoter</th> | ||
+ | <th>secretion</th> | ||
+ | <th>gene</th> | ||
+ | <th>Predicted ln(activity)</th> | ||
+ | <th>Predicted activity</th> | ||
+ | </tr> | ||
+ | </thead> | ||
+ | <tbody> | ||
+ | <tr> | ||
+ | <td><strong>0</strong></td> | ||
+ | <td>ySB78</td> | ||
+ | <td>pTDH3</td> | ||
+ | <td>αMF</td> | ||
+ | <td>B.amyloliquefaciens</td> | ||
+ | <td>-10.158696</td> | ||
+ | <td>0.000039</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td><strong>1</strong></td> | ||
+ | <td>ySB78</td> | ||
+ | <td>pTDH3</td> | ||
+ | <td>Native</td> | ||
+ | <td>B.amyloliquefaciens</td> | ||
+ | <td>-10.167739</td> | ||
+ | <td>0.000038</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td><strong>2</strong></td> | ||
+ | <td>ySB78</td> | ||
+ | <td>pRPL18B</td> | ||
+ | <td>αMF_no_EAEA</td> | ||
+ | <td>A.oryzae</td> | ||
+ | <td>-9.694006</td> | ||
+ | <td>0.000062</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td><strong>3</strong></td> | ||
+ | <td>ySB78</td> | ||
+ | <td>pPAB1</td> | ||
+ | <td>αMF_no_EAEA</td> | ||
+ | <td>B.licheniformis</td> | ||
+ | <td>-9.807968</td> | ||
+ | <td>0.000055</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td><strong>4</strong></td> | ||
+ | <td>ySB78</td> | ||
+ | <td>pTDH3</td> | ||
+ | <td>αMF</td> | ||
+ | <td>B.subtilis</td> | ||
+ | <td>-6.323765</td> | ||
+ | <td>0.001793</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td><strong>5</strong></td> | ||
+ | <td>ySB78</td> | ||
+ | <td>pPGK1</td> | ||
+ | <td>αMF</td> | ||
+ | <td>B.amyloliquefaciens</td> | ||
+ | <td>-10.157919</td> | ||
+ | <td>0.000039</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td><strong>6</strong></td> | ||
+ | <td>ySB78</td> | ||
+ | <td>pTDH3</td> | ||
+ | <td>Native</td> | ||
+ | <td>B.subtilis</td> | ||
+ | <td>-7.246373</td> | ||
+ | <td>0.000713</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td><strong>7</strong></td> | ||
+ | <td>ySB78</td> | ||
+ | <td>pRNR1</td> | ||
+ | <td>αMF_no_EAEA</td> | ||
+ | <td>B.amyloliquefaciens</td> | ||
+ | <td>-10.167606</td> | ||
+ | <td>0.000038</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td><strong>8</strong></td> | ||
+ | <td>ySB78</td> | ||
+ | <td>pRNR1</td> | ||
+ | <td>αMF_no_EAEA</td> | ||
+ | <td>A.oryzae</td> | ||
+ | <td>-9.742028</td> | ||
+ | <td>0.000059</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td><strong>9</strong></td> | ||
+ | <td>ySB78</td> | ||
+ | <td>pHHF1</td> | ||
+ | <td>αMF_no_EAEA</td> | ||
+ | <td>B.amyloliquefaciens</td> | ||
+ | <td>-10.261079</td> | ||
+ | <td>0.000035</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td><strong>10</strong></td> | ||
+ | <td>ySB78</td> | ||
+ | <td>pREV1</td> | ||
+ | <td>αMFΔ</td> | ||
+ | <td>A.oryzae</td> | ||
+ | <td>-9.40958</td> | ||
+ | <td>0.000082</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td><strong>11</strong></td> | ||
+ | <td>ySB78</td> | ||
+ | <td>pPGK1</td> | ||
+ | <td>αMF</td> | ||
+ | <td>A.oryzae</td> | ||
+ | <td>-9.517837</td> | ||
+ | <td>0.000074</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td><strong>12</strong></td> | ||
+ | <td>ySB77</td> | ||
+ | <td>pPGK1</td> | ||
+ | <td>αMF</td> | ||
+ | <td>A.oryzae</td> | ||
+ | <td>-9.564904</td> | ||
+ | <td>0.00007</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td><strong>13</strong></td> | ||
+ | <td>ySB77</td> | ||
+ | <td>pPGK1</td> | ||
+ | <td>αMF</td> | ||
+ | <td>B.licheniformis</td> | ||
+ | <td>-9.353338</td> | ||
+ | <td>0.000087</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td><strong>14</strong></td> | ||
+ | <td>ySB78</td> | ||
+ | <td>pRAD27</td> | ||
+ | <td>αMF_no_EAEA</td> | ||
+ | <td>A.oryzae</td> | ||
+ | <td>-9.827786</td> | ||
+ | <td>0.000054</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td><strong>15</strong></td> | ||
+ | <td>ySB78</td> | ||
+ | <td>pRNR1</td> | ||
+ | <td>αMF_no_EAEA</td> | ||
+ | <td>B.licheniformis</td> | ||
+ | <td>-9.966545</td> | ||
+ | <td>0.000047</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td><strong>16</strong></td> | ||
+ | <td>ySB76</td> | ||
+ | <td>pTDH3</td> | ||
+ | <td>αMFΔ</td> | ||
+ | <td>B.licheniformis</td> | ||
+ | <td>-9.201508</td> | ||
+ | <td>0.000101</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td><strong>17</strong></td> | ||
+ | <td>ySB78</td> | ||
+ | <td>pREV1</td> | ||
+ | <td>αMF_no_EAEA</td> | ||
+ | <td>A.oryzae</td> | ||
+ | <td>-9.898024</td> | ||
+ | <td>0.00005</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td><strong>18</strong></td> | ||
+ | <td>ySB78</td> | ||
+ | <td>pHHF1</td> | ||
+ | <td>αMF_no_EAEA</td> | ||
+ | <td>B.subtilis</td> | ||
+ | <td>-7.240897</td> | ||
+ | <td>0.000717</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td><strong>19</strong></td> | ||
+ | <td>ySB77</td> | ||
+ | <td>pREV1</td> | ||
+ | <td>αMF</td> | ||
+ | <td>A.oryzae</td> | ||
+ | <td>-9.653788</td> | ||
+ | <td>0.000064</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td><strong>20</strong></td> | ||
+ | <td>ySB77</td> | ||
+ | <td>pTDH3</td> | ||
+ | <td>αMF</td> | ||
+ | <td>B.subtilis</td> | ||
+ | <td>-6.566358</td> | ||
+ | <td>0.001407</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td><strong>21</strong></td> | ||
+ | <td>ySB85</td> | ||
+ | <td>pRAD27</td> | ||
+ | <td>αMF_no_EAEA</td> | ||
+ | <td>A.oryzae</td> | ||
+ | <td>-9.909267</td> | ||
+ | <td>0.00005</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td><strong>22</strong></td> | ||
+ | <td>ySB77</td> | ||
+ | <td>pTDH3</td> | ||
+ | <td>Native</td> | ||
+ | <td>B.subtilis</td> | ||
+ | <td>-7.454713</td> | ||
+ | <td>0.000579</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td><strong>23</strong></td> | ||
+ | <td>ySB85</td> | ||
+ | <td>pRAD27</td> | ||
+ | <td>αMF</td> | ||
+ | <td>A.oryzae</td> | ||
+ | <td>-9.6954</td> | ||
+ | <td>0.000062</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td><strong>24</strong></td> | ||
+ | <td>ySB78</td> | ||
+ | <td>pRNR2</td> | ||
+ | <td>αMF</td> | ||
+ | <td>A.oryzae</td> | ||
+ | <td>-9.705825</td> | ||
+ | <td>0.000061</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td><strong>25</strong></td> | ||
+ | <td>ySB78</td> | ||
+ | <td>pHHF1</td> | ||
+ | <td>αMF_no_EAEA</td> | ||
+ | <td>B.licheniformis</td> | ||
+ | <td>-10.126075</td> | ||
+ | <td>0.00004</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td><strong>26</strong></td> | ||
+ | <td>ySB77</td> | ||
+ | <td>pHHF1</td> | ||
+ | <td>αMF</td> | ||
+ | <td>A.oryzae</td> | ||
+ | <td>-9.733338</td> | ||
+ | <td>0.000059</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td><strong>27</strong></td> | ||
+ | <td>ySB78</td> | ||
+ | <td>pPGK1</td> | ||
+ | <td>αMF_no_EAEA</td> | ||
+ | <td>B.licheniformis</td> | ||
+ | <td>-10.143406</td> | ||
+ | <td>0.000039</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td><strong>28</strong></td> | ||
+ | <td>ySB78</td> | ||
+ | <td>pTDH3</td> | ||
+ | <td>αMF_no_EAEA</td> | ||
+ | <td>B.amyloliquefaciens</td> | ||
+ | <td>-10.471749</td> | ||
+ | <td>0.000028</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td><strong>29</strong></td> | ||
+ | <td>ySB78</td> | ||
+ | <td>pPGK1</td> | ||
+ | <td>αMF</td> | ||
+ | <td>B.licheniformis</td> | ||
+ | <td>-9.498398</td> | ||
+ | <td>0.000075</td> | ||
+ | </tr> | ||
+ | </tbody> | ||
+ | </table> | ||
+ | </div> | ||
+ | <p>The exploratory results of the ART are shown in <strong>Table 8</strong>. While these patterns might | ||
+ | seem mostly random at first sight, there are some striking patterns that reflect the frequency and | ||
+ | variance distributions shown in <b>figures 7 and 8</b> discussed in the “Step 1: Creating a | ||
+ | well-balanced subset of the combinatorial library” section on the <a | ||
+ | href="https://2021.igem.org/Team:Groningen/Model#art1">modeling page</a>. For instance the pTDH3 | ||
+ | promoter which was shown to be underrepresented in the training data, is part of 8 out the 30 | ||
+ | exploratorium recommendations. If all promoters were equally distributed across the recommendations, | ||
+ | it should have occurred around 3 times. Also the αMF_no_EAEA secretion signal occurs more | ||
+ | often (13 out of 30) than expected (around 7.5 out of 30). However, the ySB76 occurs a lot less (1 | ||
+ | out of 30) than expected (around 7.5 out of 30) even though figure 8 on the <a | ||
+ | href="https://2021.igem.org/Team:Groningen/Model#art1">modeling page</a> shows it contributes | ||
+ | less to the overall variance in the dataset than the other strains.</p> | ||
+ | <button type="button" class="collapsible"> | ||
+ | <h4>Conclusion</h4> | ||
+ | <i class="fas fa-plus" id="plus"></i> | ||
+ | <i class="fas fa-minus" id="minus"></i> | ||
+ | </button> | ||
+ | <div class="collapsible_content"> | ||
+ | <p>Based on the results from the ART, it is expected that a device containing the alpha-amylase | ||
+ | encoding gene from <em>B.subtilis</em> and the αMF secretion signal will yield a high | ||
+ | alpha-amylase activity in the supernatant. Furthermore, the exploratory recommendations show | ||
+ | some correlations with the frequency and variance distributions of the part-variants shown on | ||
+ | the <a href="https://2021.igem.org/Team:Groningen/Model#art">modeling page</a>.</p> | ||
+ | </div> | ||
+ | </section> | ||
+ | <section id="discussion"> | ||
+ | <div class="section-header"> | ||
+ | <h2 class="headline">Final discussion, finding the most active alpha amylase and future perspectives | ||
+ | </h2> | ||
+ | </div> | ||
+ | <p>Overall, we were able to successfully clone and functionally express alpha-amylase in | ||
+ | <em>Saccharomyces spp</em>. using Golden Gate Assembly. However, the level of alpha-amylase activity | ||
+ | did differ based on the way of extraction (lysising or not) and the combination of construct and | ||
+ | chassis. Some results by themselves may not appear to be very<strong> conclusive</strong>, but | ||
+ | bringing all of them together helped us to get a better insight on how to find the most active alpha-amylase. | ||
+ | </p> | ||
+ | <p>When checking the activity of alpha-amylase the samples that holded the native sequence (Parts 3 and | ||
+ | Parts 3b) from <em>B. amyloliquefaciens</em> and <em>licheniformis, </em>a <strong>general | ||
+ | trend</strong> was observed, the samples didn’t perform very well (in terms of alpha-amylase | ||
+ | activity). Combining these results together with the sequencing results, it turns out that | ||
+ | those two samples had clear <strong>missense mutations</strong> that may affect the performance of | ||
+ | them. Two possible scenarios could explain the lack of activity, 1. the deficient production of the | ||
+ | enzyme or 2. the lack of functionality. Considering the experiments performed it is hard to tell | ||
+ | what holds true in this case. Performing direct measurements like SDS gel electrophoresis to | ||
+ | identify the presence of the protein would have given us more insights into where the problem was. | ||
+ | <strong>Further experiments</strong> would be needed to check for the consequences of the mutations. | ||
+ | Ideally, we would have avoided unknown mutations by an early detection of them, unfortunately, at the | ||
+ | time we found them all the constructs of the library were already assembled and we didn’t have | ||
+ | time to restart again. </p> | ||
+ | <p>Besides the engineering strategy, a very important part when expressing heterologous proteins is | ||
+ | measuring the <strong>successful expression and activity</strong> of the protein. Using different | ||
+ | assays as we did is a safe and insightful manner of testing the performance of your device. Far from | ||
+ | being redundant, we draw <strong>different but complementary conclusions</strong> from both | ||
+ | experiments. The halo test helped us understand the importance of the secretion of the enzyme. On the other hand, | ||
+ | the high throughput experiment enabled for more precise comparison between samples and proved to be | ||
+ | an efficient way of getting quantitative data from your devices. </p> | ||
+ | <p>In our attempt to screen as many combinations as possible to find the one that would achieve the | ||
+ | <strong>highest yield of functional alpha-amylase</strong>, we combined <em>in vitro</em> high | ||
+ | throughput experiments with a machine learning tool (ART). By doing so we expanded the search space, | ||
+ | by not being just restricted to the actual combinations that were tested in the lab. When merging | ||
+ | these two elements (experiments and machine learning), we encountered some aspects that needed to be | ||
+ | taken into consideration. In our case, the subset of samples from the combinatorial library that are | ||
+ | going to be evaluated experimentally needed to be thoroughly selected, trying to find an | ||
+ | <strong>even</strong> and<strong> representative </strong>distribution of all the elements inside | ||
+ | each category (as explained in the <a href="https://2021.igem.org/Team:Groningen/Model#art">Model | ||
+ | page</a>). Another important aspect is the selection of a performance measure that is | ||
+ | representative of what would constitute a “good” device. We selected the activity of | ||
+ | alpha-amylase in the supernatant of the machine to be this performance measure as it represents what | ||
+ | we aimed to achieve (an efficient production of alpha-amylase) and initial results showed that it | ||
+ | can be accurately measured.</p> | ||
+ | <p>There are some improvements that need to be made in the experiments pipeline (see section | ||
+ | Improvements in the <a href="https://2021.igem.org/Team:Groningen/Model#art">Model page</a>). In the | ||
+ | next DBTL cycle those changes would need to be implemented in order to get more accurate | ||
+ | results. </p> | ||
+ | <p>In general, we learnt that when expressing a new gene in a chassis, it is rather unpredictable how | ||
+ | the different elements in the device would interact between each other<strong>.</strong> From our | ||
+ | experience, we believe that by using this kind of technology more knowledge about the performance of | ||
+ | your device can be acquired and the optimal results can presumably be obtained in fewer DBTL cycles. | ||
+ | </p> | ||
</section> | </section> | ||
− | <section> | + | <section id="ref"> |
<div class="section-header"> | <div class="section-header"> | ||
<h2 class="headline">References</h2> | <h2 class="headline">References</h2> | ||
</div> | </div> | ||
+ | <ol class="references"> | ||
+ | <li>Declerck N, Machius M, Wiegand G, Huber R, Gaillardin C. Probing structural determinants specifying high thermostability in Bacillus licheniformis alpha-amylase. J Mol Biol. 2000 Aug 25;301(4):1041-57. doi: 10.1006/jmbi.2000.4025. PMID: 10966804. | ||
+ | </li> | ||
+ | </ol> | ||
</section> | </section> | ||
</div> | </div> |
Latest revision as of 01:22, 22 October 2021
Results
Introduction
On this page the main findings and interpretations of the results of our project can be found. In our project we aimed to achieve four different goals. These four proof of concepts are summarized in Figure 1.
Ammonia as the only nitrogen source
As we plan on feeding our GMO with ammonia extracted from the MOF, an essential part of our project is that our GMO should be able to grow in the presence of ammonia as its only nitrogen source. Therefore, we grew 6 different Saccharomyces strains under different ammonia concentrations to see if the presence of ammonia would affect their growth. In the end we were able to identify the best performing strains. The protocol can be found in the Experiments page.
As can be seen from Figure 2, there are 4 strains that outperformed in the experiment. These strains were chosen for further experimentation. The doubling time of the chosen strains can be seen in Table 1.
Strain | TD (min) | ||
---|---|---|---|
0.05 g/L | 5 g/L | 7.5 g/L | |
ySB76 | 51,71 | 70,71 | 69,3 |
ySB77 | 130,75 | 90 | 60,78 |
ySB78 | 101,91 | 94,93 | 88,85 |
ySB85 | 103,43 | 91,18 | 108,28 |
We learned that two of the strains that we were planning to use didn’t perform well when ammonia was the only source of nitrogen. This experiment helped us to exclude them for further tests as they weren’t suitable for our purpose (see Description page). Four strains proved to grow in different concentrations of ammonia, with strain ySB76 (S. cerevisiae) being the fastest grower overall. We could not observe a clear tendency that would suggest the optimal ammonia concentration for growing the cells. This result is relevant for our project, since the concentration of this nutrient doesn't necessarily need to be kept constant, which allows for more flexibility in ammonia concentration of the cell culture and thus more flexibility in how much ammonia the MOF should be able to capture. As we wanted to test different chassis in the optimization process, after this experiment we decided to express alpha amylase in yYS76, yYB77, ySB78 and ySB85, which correspond with 3 S. cerevisiae strains and 1 S. paradoxus strain.
Engineering success
For our second proof of concept, we wanted to demonstrate that it is possible to use Golden Gate Assembly to clone heterologous genes for alpha-amylase in Saccharomyces spp. Going from the gene of interest to the cassette plasmid expressed in Saccharomyces spp. is a long process. Throughout it there were some checkpoints that we used to confirm the success of the cloning: GFP screening; DNA concentration measurements with a Nanodrop; basic parts sequencing; and digestion of the final cassette plasmids.
GFP screening
The empty plasmid (pRS426__ConLS'-GFPdropout-ConRE'-URA3-2micron-Kan) used in the Golden Gate cassette plasmid assemblies contains a GFP dropout. This way, when the assembled plasmid is inserted correctly, the GFP insert will dropout and white colonies will be formed. Whenever the plasmid was not inserted correctly, the GFP dropout will still be present and colonies will appear green when examined under UV-light (Figure 3). With this simple technique we make sure that the chosen colonies have uptaken the assembled plasmid without the need of performing sequencing or digestion experiments prior to plasmid isolation. This GFP screening was used both for assembling our part plasmids and cassette plasmids.
GFP screening smoothened the process, considering the amount of samples that we needed to assemble. Generally, all the transformed E.coli with cassette plasmids (64 plates) showed a high efficiency of the Golden Gate assembly; a few countable green colonies were observed in contrast to hundreds of white colonies. One sample (SP007) didn’t show any white colonies, the sample was excluded for future experiments.
DNA concentrations
After purification of cassette plasmids amplified in E. coli, DNA concentrations were measured in order to assure a successful purification prior to Saccharomyces spp. transformations, results can be found in Table 2. These measurements were performed using a nanodrop spectrophotometer, measuring the DNA concentration at a spectrum of wavelengths.
Sample number | ng/ul | A260/280 ratio | Sample number | ng/ul | A260/280 ratio |
---|---|---|---|---|---|
SP001 | 22,6 | 1,823 | SP034 | 80 | 0,001 |
SP002 | 15,6 | 1,803 | SP035 | 35,5 | 1,529 |
SP003 | 29 | 1,598 | SP036 | 15 | 1,523 |
SP004 | 19,7 | 1,662 | SP037 | 29,4 | 1,699 |
SP005 | 54,3 | 2,027 | SP038 | 48,5 | 1,960 |
SP006 | 53,5 | 1,508 | SP039 | 14,6 | 2,021 |
SP008 | 69,5 | 1,853 | SP040 | 18,3 | 1,649 |
SP009 | 16,1 | 2,414 | SP041 | 25,1 | 1,668 |
SP010 | 12,1 | 1,898 | SP042 | 45,1 | 1,488 |
SP011 | 13,3 | 1,430 | SP043 | 15 | 2,150 |
SP012 | 58,9 | 1,812 | SP044 | 29,9 | 1,472 |
SP013 | 19,5 | 2,058 | SP045 | 78,4 | 1,708 |
SP014 | 29,1 | 1,473 | SP046 | 33 | 1,451 |
SP015 | 28,3 | 1,461 | SP047 | 62,3 | 1,489 |
SP016 | 52,3 | 1,458 | SP048 | 97,6 | 0,002 |
SP017 | 72,1 | 1,553 | SP049 | 13,4 | 1,949 |
SP018 | 34,7 | 1,627 | SP050 | 119 | 0,002 |
SP019 | 79,8 | 1,788 | SP051 | 21,2 | 1,612 |
SP020 | 15,7 | 1,627 | SP052 | 56,5 | 1,471 |
SP021 | 17,9 | 1,729 | SP053 | 6,3 | 2,117 |
SP022 | 71 | 1,514 | SP054 | 39,2 | 1,798 |
SP023 | 13,2 | 1,688 | SP055 | 25,7 | 1,490 |
SP024 | 10,6 | 2,078 | SP056 | 50,7 | 2,069 |
SP025 | 82,7 | 0,002 | SP057 | 73,3 | 1,806 |
SP026 | 9,5 | 1,602 | SP058 | 51,7 | 1,987 |
SP027 | 47,2 | 1,512 | SP059 | 30,6 | 1,363 |
SP028 | 64,2 | 1,544 | SP060 | 61,6 | 1,936 |
SP029 | 10,5 | 2,297 | SP061 | 62,4 | 1,848 |
SP030 | 16,6 | 1,892 | SP062 | 82,4 | 1,741 |
SP031 | 51,3 | 1,507 | SP063 | 66 | 1,547 |
SP032 | 45 | 2,067 | SP064 | 58,8 | 1,718 |
SP033 | 48,7 | 1,617 |
The A260/280 ratio represents the purity of the DNA, an ideal sample would have a value of approximately 1.8. Due to large variation in DNA concentration and A260/280 ratio, 20 μl of each cassette plasmid was used in the Saccharomyces spp. transformation instead of 5 μl. Overall, the concentration of DNA in the samples after transformation and plasmid isolation were good and ensured the continuation with Saccharomyces spp. transformations.
DNA sequencing
To ensure that our ordered G-blocks for part 3 and part 3B we made were correct, we sequenced the basic parts (4 full alpha amylases and 4 alpha amylases without the native secretion peptide) assembled in the entry vector (more on this on the Engineering page). pYTK001 (entry vector) with Part 3 and 3b was sequenced using two primers flanking the gene, the vector is used to compose the cassettes plasmids in further experiments. All sequencing results are summarized in Table 3.
Part | Alpha amylase Gene | Mutations | Comments |
---|---|---|---|
Part 3 | From A. oryzae | - | Good quality sequence, 100% gene coverage. |
From B. amyloliquefaciens | c.301C>Tp.T101I | The amino acid is not in the substrate binding site of the protein. 100 % gene coverage. | |
From B. licheniformis | c.690G>Tp.D231Y | Mutations in the flanking aspartate have shown a decrease in thermostability [1]. No data about our mutation. 100% gene coverage. | |
From B. subtilis | - | Good quality sequence, 100% gene coverage. | |
Part 3b | From A. oryzae | - | Good forward sequencing, 63% gene coverage. |
From B. amyloliquefaciens | - | Good forward sequencing, 52% | |
From B. licheniformis | - | Good forward sequencing, 66% gene coverage. | |
From B. subtilis | - | Good forward sequencing, 50% gene coverage. |
Generally, the results were quite successful and no major mutations were observed. However, two clear missense mutations were identified in alpha-amylases from B. amyloliquefaciens and B. licheniformis that may have an effect on the protein functionality. It would have been nice if we had the time to restart the cloning from the beginning, unfortunately, the cassette plasmids were already assembled and the time was very limited. In Part 3b full coverage of the gene couldn’t be achieved, repeating the sequencing was planned but sadly we didn’t have the time.
All in all, the mutations found will be taken into account for further results of those enzymes.
Constructs digestion
We used the restriction enzyme BsmBI to check for the correct ligation of the different constructs of the library. There are two sites for the enzyme BsmBI in the vector employed (pRS246) for our constructs, the sites are in part 1 and part 5, on both sides of the insert of interest (Figure 4). The use of BsmBI allows us to check for the size (Table 4) of the construct and assess the success of the assembly. The results of the digestion can be found in Figure 5.
Sample | Expected sizes A (bp) | Expected sizes B (bp) | Sample | Expected sizes A (bp) | Expected sizes B (bp) |
---|---|---|---|---|---|
SP001 | 3729 | 3387 | SP034 | 3729 | 2983 |
SP002 | 3729 | 2869 | SP035 | 3729 | 2970 |
SP005 | 3729 | 3387 | SP037 | 3729 | 3387 |
SP006 | 3729 | 2953 | SP038 | 3729 | 3387 |
SP008 | 3729 | 3237 | SP039 | 3729 | 2982 |
SP009 | 3729 | 3237 | SP040 | 3729 | 2970 |
SP010 | 3729 | 3387 | SP041 | 3729 | 2802 |
SP011 | 3729 | 2776 | SP042 | 3729 | 2982 |
SP012 | 3729 | 2755 | SP043 | 3729 | 2953 |
SP013 | 3729 | 3381 | SP044 | 3729 | 2886 |
SP014 | 3729 | 2982 | SP045 | 3729 | 2982 |
SP015 | 3729 | 2869 | SP046 | 3729 | 2866 |
SP016 | 3729 | 2790 | SP047 | 3729 | 2755 |
SP017 | 3729 | 2869 | SP048 | 3729 | 2970 |
SP018 | 3729 | 2880 | SP049 | 3729 | 2749 |
SP019 | 3729 | 2796 | SP050 | 3729 | 2970 |
SP020 | 3729 | 2886 | SP051 | 3729 | 2970 |
SP021 | 3729 | 3387 | SP052 | 3729 | 2982 |
SP022 | 3729 | 2982 | SP053 | 3729 | 2755 |
SP023 | 3729 | 2970 | SP054 | 3729 | 3291 |
SP024 | 3729 | 2886 | SP055 | 3729 | 2982 |
SP025 | 3729 | 2802 | SP056 | 3729 | 2796 |
SP026 | 3729 | 2982 | SP057 | 3729 | 2970 |
SP027 | 3729 | 3237 | SP058 | 3729 | 2755 |
SP028 | 3729 | 2982 | SP059 | 3729 | 2886 |
SP029 | 3729 | 2982 | SP060 | 3729 | 3237 |
SP030 | 3729 | 2866 | SP061 | 3729 | 2970 |
SP031 | 3729 | 2886 | SP062 | 3729 | 2976 |
SP032 | 3729 | 2970 | SP063 | 3729 | 2982 |
SP033 | 3729 | 3387 | SP064 | 3729 | 2869 |
pRS426-GFP | 3729 | 1338 |
After the digestion of all the constructs it can be seen that the expected sizes of the engineered plasmids match the sizes of the bands shown in the gel. The efficiency of the Golden Gate assembly is high. There are two samples in which a band of the size of the GFP-drop out is observed, SP012 and SP058, besides the expected size. This result may be due to contamination while pipetting. Nevertheless, the expected band can be observed, the samples were used in follow up experiments, consideration on alpha amylase activity will be taken into account. Additionally, some samples (SP006, SP011, SP022, SP023, SP044, SP046, SP047, SP052, SP053, SP055) show faint bands, likely evidencing that the concentration of DNA was low in the first place. The faint bands have the expected size, and considering the high efficiency in the rest of the digests we conclude that our Golden Gate assemblies and further screening was highly successful. No samples were excluded after digestion.
Alpha amylase production
For our third proof of concept, we wanted to show that our GMO does indeed show alpha-amylase activity after taking up our construct. We aimed to measure the alpha-amylase production of 64 samples listed at our Engineering page. This was tested with two main experiments. The principal one being an alpha amylase kit assay used to perform high throughput experiments and obtain quantitative data, needed for our model (see Model page). Additionally, a qualitative assay was employed to confirm the production of alpha-amylase in some of the samples. To understand more how this assay works, see the Experiments page.
Alpha-amylase starch breakdown
Functionality of alpha-amylase can qualitatively be proved by observing the breakdown of starch. The ability to break down starch is only available when amylase is produced, which in the case of S. cerevisiae and S. paradoxus doesn’t happen natively (Figure 6e). However, the assembled constructs should be able to break down starch. In order to confirm this, selective media plates without uracil (specific for the assembled constructs) were created with the addition of 1% starch. Several samples were cultured ON and treated before being plated on the starch plates, as described in the notebook (wk 39), after which pictures were taken (Figure 6) of the with/without iodine treated starch plates. Samples tested with this assay can be found in Table 5.
Sample | Yeast Strain | Promoter | Gene | Terminator |
---|---|---|---|---|
SP001 | ySB77 | pPGK1 | αMF + BS | tTDH1 |
SP010 | ySB77 | pHHF1 | αMF + BS | tTDH1 |
SP021 | ySB77 | pPGK1 | αMFΔ + BA | tTDH1 |
SP026 | ySB76 | pHHF1 | αMF + BL | tTDH1 |
SP027 | ySB76 | pRNR1 | BS | tTDH1 |
SP034 | ySB76 | pTEF1 | αMF + BL | tTDH1 |
SP035 | ySB76 | pPAB1 | αMF_no_EAEA + BL | tTDH1 |
SP051 | ySB76 | pRNR1 | αMF_no_EAEA + BL | tTDH1 |
SP063 | ySB76 | pRNR2 | αMF + BL | tTDH1 |
As can be seen in Figure 6, a larger halo was observed in the cells’ lysate samples. Halos were also visible in most of the supernatant samples. This implies that alpha-amylase is present extra- and intracellular after cell lysis. We were able to confirm that this activity was only produced in the strains that had taken up the created constructs, as the non engineered strains didn’t show any halo at all. Overall, we see a difference in the alpha-amylase activity when samples were treated differently, suggesting that the secretion of the enzyme varies between constructs. More specifically to our project, we planned to isolate the protein from the supernatant, this way the processing of the cells is minimal and lysating is not directly necessary. Therefore, it was interesting to find that lysating some of the samples did produce a bigger halo, since this could change our proposed way of extracting alpha-amylase. Only few samples of the library were tested through this essay, therefore general conclusions can not be drawn, but confirmation of alpha amylase-activity could be assessed.
Alpha-amylase assay kit
We used a colorimetric assay to obtain quantitative data of the activity of the alpha amylase produced by the engineered yeast, allowing for comparison between samples and for quantitative data to use in our model.
The assay is based on the capacity of alpha amylase to cleavage ethylidene-pNP-G7, resulting in p-nitrophenol, a product that can be read at a wavelength of 405 nm, more details on the Experiments page. A testing round was first performed (Figure 7) in duplicates per sample to check how the experiment was done and identify possible steps that may need to be optimized. This round also helped to exclude samples SP003, SP004 and SP036 for further analysis as we were tight on reagents and the results weren’t promising.
During the high throughput experiments, we tested the capacity to produce functional alpha-amylase of all the samples which were not excluded from the subset of the combinatorial library (the excluded samples are listed on the Model page, section Accounting for lost samples). Activity of alpha-amylase in triplicates of each sample can be found in Figure 8. Supernatants from overnight cultures were incubated with ethylidene-pNP-G7 in 96 well plates and the appearance of p-nitrophenol was measured every 5 min during the first hour, then once after 2h and 3h. If alpha-amylase was in the supernatant, an increase of absorbance was expected due to the appearance of p-nitrophenol, an indirect measurement of alpha-amylase activity. Therefore, activity (y-axis of Figure 8) was measured as the increase of absorbance (quantity of p-nitrophenol) that appears per time unit (d[p-nitrophenol]/dt).
After having excluded the samples in which the variance between replicates deviated too much from the average variance, a table summarizing the mean activity of each sample can be found below. The different combinations from the subset library are also displayed in Table 6.
Sample | Strain | Promoter | Secretion signal | Gene | Activity values (d[p-nitrophenol]/dt) |
---|---|---|---|---|---|
SP001 | ySB77 | pPGK1 | αMF | B.subtilis | 0,005476099 |
SP002 | ySB85 | pRNR1 | αMFΔ | A.oryzae | 3,4293E-05 |
SP005 | ySB78 | pHHF1 | αMF | B.subtilis | 0,000260468 |
SP008 | ySB78 | pHHF1 | Native | B.subtilis | 0,000192798 |
SP009 | ySB85 | pRNR1 | Native | B.subtilis | 0,00052307 |
SP010 | ySB77 | pHHF1 | αMF | B.subtilis | 0,001100158 |
SP012 | ySB78 | pRNR1 | Native | A.oryzae | 9,26803E-05 |
SP013 | ySB85 | pRAD27 | αMF | B.subtilis | 0,000945715 |
SP014 | ySB76 | pREV1 | αMF | B.amyloliquefaciens | 0,00001 |
SP015 | ySB76 | pPGK1 | αMFΔ | A.oryzae | 1,44142E-05 |
SP016 | ySB77 | pRAD27 | Native | B.licheniformis | 1,2205E-05 |
SP018 | ySB76 | pRAD27 | αMFΔ | B.amyloliquefaciens | 0,00001 |
SP019 | ySB78 | pTEF1 | Native | B.licheniformis | 1,08206E-05 |
SP022 | ySB77 | pREV1 | αMF | B.amyloliquefaciens | 1,14565E-05 |
SP023 | ySB77 | pRNR2 | αMF_no_EAEA | B.licheniformis | 0,00001 |
SP024 | ySB77 | pTEF1 | αMFΔ | B.licheniformis | 0,000341834 |
SP025 | ySB78 | pPAB1 | Native | B.amyloliquefaciens | 1,15319E-05 |
SP026 | ySB76 | pHHF1 | αMF | B.licheniformis | 0,000200209 |
SP027 | ySB76 | pRNR1 | Native | B.subtilis | 0,001931731 |
SP028 | ySB77 | pTEF1 | αMF | B.amyloliquefaciens | 1,20322E-05 |
SP029 | ySB85 | pRPL18B | αMF | B.licheniformis | 5,32401E-05 |
SP030 | ySB85 | pTDH3 | αMFΔ | B.amyloliquefaciens | 0,00001 |
SP031 | ySB77 | pPGK1 | αMFΔ | B.amyloliquefaciens | 2,44344E-05 |
SP032 | ySB78 | pRPL18B | αMF_no_EAEA | B.amyloliquefaciens | 1,85894E-05 |
SP033 | ySB85 | pREV1 | αMF | B.subtilis | 0,000845811 |
SP034 | ySB76 | pTEF1 | αMF | B.licheniformis | 0,000211464 |
SP035 | ySB76 | pPAB1 | αMF_no_EAEA | B.licheniformis | 0,00021031 |
SP037 | ySB85 | pRPL18B | αMF | B.subtilis | 0,00113086 |
SP038 | ySB78 | pRNR2 | αMF | B.subtilis | 0,002933777 |
SP039 | ySB85 | pTEF1 | αMF | B.licheniformis | 1,41915E-05 |
SP041 | ySB85 | pHHF1 | Native | B.amyloliquefaciens | 1,17257E-05 |
SP044 | ySB77 | pRNR2 | αMFΔ | B.licheniformis | 4,16163E-05 |
SP045 | ySB78 | pPAB1 | αMF | B.licheniformis | 9,44327E-05 |
SP046 | ySB77 | pTDH3 | αMFΔ | B.licheniformis | 1,15677E-05 |
SP047 | ySB77 | pPGK1 | Native | A.oryzae | 0,00019171 |
SP049 | ySB85 | pRAD27 | Native | A.oryzae | 0,00029359 |
SP050 | ySB77 | pPGK1 | αMF_no_EAEA | B.amyloliquefaciens | 1,39692E-05 |
SP051 | ySB76 | pRNR1 | αMF_no_EAEA | B.licheniformis | 0,000290985 |
SP052 | ySB77 | pPAB1 | αMF | B.amyloliquefaciens | 1,01931E-05 |
SP053 | ySB85 | pHHF1 | Native | A.oryzae | 1,35179E-05 |
SP054 | ySB85 | pRPL18B | αMFΔ | B.subtilis | 0,000154906 |
SP055 | ySB77 | pRPL18B | αMF | B.licheniformis | 0,00001 |
SP056 | ySB78 | pPAB1 | Native | B.licheniformis | 1,94392E-05 |
SP058 | ySB78 | pRNR2 | Native | A.oryzae | 6,79755E-05 |
SP060 | ySB85 | pREV1 | Native | B.subtilis | 0,000223757 |
SP061 | ySB85 | pREV1 | αMF_no_EAEA | B.amyloliquefaciens | 1,40926E-05 |
SP062 | ySB85 | pRAD27 | αMF | B.amyloliquefaciens | 1,38021E-05 |
SP063 | ySB76 | pRNR2 | αMF | B.licheniformis | 0,000596797 |
Experiment were performed in triplicate to account for stochasticity, samples SP006, SP011, SP020, SP021, SP042 and SP059 were excluded, as the variance between replicates deviated from the mean variance, evidencing that the signal was more likely an artefact than actual alpha amylase activity. For the rest of the samples displayed in the graphs it can be concluded that there is a high variability in terms of how active alpha amylase is. Some constructs aren’t successful in expressing active alpha amylase in the supernatant (any sample with an activity value below 0,00001 was considered non active). However, other samples produce bigger amounts of p-nitrophenol and thus have more alpha-amylase activity in the supernatant.
Some general trends can be observed by looking at the graphs and the table, for instance, a relatively high alpha amylase activity is observed in samples that contain the alpha amylase gene from the host B. subtilis. The interpretation of such a big set of samples and identification of the best performers is an arduous task. AI technology could help getting deeper insights on our library and finding a better combination from the combinatorial library, more on this can be found in the model page.
Improving alpha amylase production
We used the Automated Recommendation Tool (ART) to model which samples from the combinatorial library discussed on the engineering page would yield a high alpha-amylase activity in the supernatant (exploitative recommendations). Furthermore, the ART also provides recommendations on which samples should be tested in a future DBTL-cycle in order to improve the performance of the model (exploratory recommendations). On this page, only the final results of the ART are discussed. How these results are generated and how to analyze them is discussed on the modeling page.
Exploitative recommendations
Rank | strain | promoter | secretion | gene | Predicted ln(activity) | Predicted activity |
---|---|---|---|---|---|---|
0 | ySB78 | pPGK1 | αMF | B.subtilis | -6.241298 | 0.001947 |
1 | ySB78 | pTDH3 | αMF | B.subtilis | -6.323765 | 0.001793 |
2 | ySB76 | pRNR2 | αMF | B.subtilis | -6.363736 | 0.001723 |
3 | ySB85 | pPGK1 | αMF | B.subtilis | -6.388392 | 0.001681 |
4 | ySB76 | pPGK1 | αMF | B.subtilis | -6.435489 | 0.001604 |
5 | ySB77 | pRNR2 | αMF | B.subtilis | -6.521911 | 0.001471 |
6 | ySB77 | pTDH3 | αMF | B.subtilis | -6.566358 | 0.001407 |
7 | ySB76 | pHHF1 | αMF | B.subtilis | -6.573551 | 0.001397 |
8 | ySB76 | pRNR1 | αMF | B.subtilis | -6.666158 | 0.001273 |
9 | ySB76 | pTDH3 | αMF | B.subtilis | -6.676021 | 0.001261 |
10 | ySB85 | pRNR1 | αMF | B.subtilis | -6.689628 | 0.001244 |
11 | ySB77 | pRNR1 | αMF | B.subtilis | -6.707219 | 0.001222 |
12 | ySB76 | pPAB1 | αMF | B.subtilis | -6.75746 | 0.001162 |
13 | ySB77 | pREV1 | αMF | B.subtilis | -6.892306 | 0.001016 |
14 | ySB78 | pRNR1 | αMF | B.subtilis | -6.892674 | 0.001015 |
15 | ySB85 | pPAB1 | αMF | B.subtilis | -6.914319 | 0.000993 |
16 | ySB76 | pTEF1 | αMF | B.subtilis | -6.930234 | 0.000978 |
17 | ySB78 | pPAB1 | αMF | B.subtilis | -6.938977 | 0.000969 |
18 | ySB76 | pREV1 | αMF | B.subtilis | -6.942261 | 0.000966 |
19 | ySB85 | pTEF1 | αMF | B.subtilis | -6.95399 | 0.000955 |
20 | ySB77 | pPAB1 | αMF | B.subtilis | -6.968985 | 0.000941 |
21 | ySB78 | pRPL18B | αMF | B.subtilis | -6.980546 | 0.00093 |
22 | ySB85 | pTDH3 | αMF | B.subtilis | -6.996441 | 0.000915 |
23 | ySB77 | pTEF1 | αMF | B.subtilis | -7.012753 | 0.0009 |
24 | ySB78 | pREV1 | αMF | B.subtilis | -7.057184 | 0.000861 |
25 | ySB78 | pRAD27 | αMF | B.subtilis | -7.06477 | 0.000855 |
26 | ySB85 | pRNR2 | αMF | B.subtilis | -7.082414 | 0.00084 |
27 | ySB78 | pRNR1 | αMF_no_EAEA | B.subtilis | -7.096215 | 0.000828 |
28 | ySB78 | pTEF1 | αMF | B.subtilis | -7.134024 | 0.000798 |
29 | ySB76 | pRAD27 | αMF | B.subtilis | -7.223168 | 0.000729 |
Table 7 shows that all exploitative recommendations contain the alpha-amylase encoding genes from B.subtilis. Furthermore, the αMF secretion peptide occurs in 29 out of 30 recommendations. It is also striking that 8 out of 10 recommendations contain either the pRNR2, pTDH3 or pPGK1 promoter, with the pPGK1 promoter occurring in 3 out of the top 5 recommendations. No clear pattern can be noticed in the recommendations regarding which Saccharomyces spp. should be used as a chassis.
Exploratory recommendations
Rank | strain | promoter | secretion | gene | Predicted ln(activity) | Predicted activity |
---|---|---|---|---|---|---|
0 | ySB78 | pTDH3 | αMF | B.amyloliquefaciens | -10.158696 | 0.000039 |
1 | ySB78 | pTDH3 | Native | B.amyloliquefaciens | -10.167739 | 0.000038 |
2 | ySB78 | pRPL18B | αMF_no_EAEA | A.oryzae | -9.694006 | 0.000062 |
3 | ySB78 | pPAB1 | αMF_no_EAEA | B.licheniformis | -9.807968 | 0.000055 |
4 | ySB78 | pTDH3 | αMF | B.subtilis | -6.323765 | 0.001793 |
5 | ySB78 | pPGK1 | αMF | B.amyloliquefaciens | -10.157919 | 0.000039 |
6 | ySB78 | pTDH3 | Native | B.subtilis | -7.246373 | 0.000713 |
7 | ySB78 | pRNR1 | αMF_no_EAEA | B.amyloliquefaciens | -10.167606 | 0.000038 |
8 | ySB78 | pRNR1 | αMF_no_EAEA | A.oryzae | -9.742028 | 0.000059 |
9 | ySB78 | pHHF1 | αMF_no_EAEA | B.amyloliquefaciens | -10.261079 | 0.000035 |
10 | ySB78 | pREV1 | αMFΔ | A.oryzae | -9.40958 | 0.000082 |
11 | ySB78 | pPGK1 | αMF | A.oryzae | -9.517837 | 0.000074 |
12 | ySB77 | pPGK1 | αMF | A.oryzae | -9.564904 | 0.00007 |
13 | ySB77 | pPGK1 | αMF | B.licheniformis | -9.353338 | 0.000087 |
14 | ySB78 | pRAD27 | αMF_no_EAEA | A.oryzae | -9.827786 | 0.000054 |
15 | ySB78 | pRNR1 | αMF_no_EAEA | B.licheniformis | -9.966545 | 0.000047 |
16 | ySB76 | pTDH3 | αMFΔ | B.licheniformis | -9.201508 | 0.000101 |
17 | ySB78 | pREV1 | αMF_no_EAEA | A.oryzae | -9.898024 | 0.00005 |
18 | ySB78 | pHHF1 | αMF_no_EAEA | B.subtilis | -7.240897 | 0.000717 |
19 | ySB77 | pREV1 | αMF | A.oryzae | -9.653788 | 0.000064 |
20 | ySB77 | pTDH3 | αMF | B.subtilis | -6.566358 | 0.001407 |
21 | ySB85 | pRAD27 | αMF_no_EAEA | A.oryzae | -9.909267 | 0.00005 |
22 | ySB77 | pTDH3 | Native | B.subtilis | -7.454713 | 0.000579 |
23 | ySB85 | pRAD27 | αMF | A.oryzae | -9.6954 | 0.000062 |
24 | ySB78 | pRNR2 | αMF | A.oryzae | -9.705825 | 0.000061 |
25 | ySB78 | pHHF1 | αMF_no_EAEA | B.licheniformis | -10.126075 | 0.00004 |
26 | ySB77 | pHHF1 | αMF | A.oryzae | -9.733338 | 0.000059 |
27 | ySB78 | pPGK1 | αMF_no_EAEA | B.licheniformis | -10.143406 | 0.000039 |
28 | ySB78 | pTDH3 | αMF_no_EAEA | B.amyloliquefaciens | -10.471749 | 0.000028 |
29 | ySB78 | pPGK1 | αMF | B.licheniformis | -9.498398 | 0.000075 |
The exploratory results of the ART are shown in Table 8. While these patterns might seem mostly random at first sight, there are some striking patterns that reflect the frequency and variance distributions shown in figures 7 and 8 discussed in the “Step 1: Creating a well-balanced subset of the combinatorial library” section on the modeling page. For instance the pTDH3 promoter which was shown to be underrepresented in the training data, is part of 8 out the 30 exploratorium recommendations. If all promoters were equally distributed across the recommendations, it should have occurred around 3 times. Also the αMF_no_EAEA secretion signal occurs more often (13 out of 30) than expected (around 7.5 out of 30). However, the ySB76 occurs a lot less (1 out of 30) than expected (around 7.5 out of 30) even though figure 8 on the modeling page shows it contributes less to the overall variance in the dataset than the other strains.
Based on the results from the ART, it is expected that a device containing the alpha-amylase encoding gene from B.subtilis and the αMF secretion signal will yield a high alpha-amylase activity in the supernatant. Furthermore, the exploratory recommendations show some correlations with the frequency and variance distributions of the part-variants shown on the modeling page.
Final discussion, finding the most active alpha amylase and future perspectives
Overall, we were able to successfully clone and functionally express alpha-amylase in Saccharomyces spp. using Golden Gate Assembly. However, the level of alpha-amylase activity did differ based on the way of extraction (lysising or not) and the combination of construct and chassis. Some results by themselves may not appear to be very conclusive, but bringing all of them together helped us to get a better insight on how to find the most active alpha-amylase.
When checking the activity of alpha-amylase the samples that holded the native sequence (Parts 3 and Parts 3b) from B. amyloliquefaciens and licheniformis, a general trend was observed, the samples didn’t perform very well (in terms of alpha-amylase activity). Combining these results together with the sequencing results, it turns out that those two samples had clear missense mutations that may affect the performance of them. Two possible scenarios could explain the lack of activity, 1. the deficient production of the enzyme or 2. the lack of functionality. Considering the experiments performed it is hard to tell what holds true in this case. Performing direct measurements like SDS gel electrophoresis to identify the presence of the protein would have given us more insights into where the problem was. Further experiments would be needed to check for the consequences of the mutations. Ideally, we would have avoided unknown mutations by an early detection of them, unfortunately, at the time we found them all the constructs of the library were already assembled and we didn’t have time to restart again.
Besides the engineering strategy, a very important part when expressing heterologous proteins is measuring the successful expression and activity of the protein. Using different assays as we did is a safe and insightful manner of testing the performance of your device. Far from being redundant, we draw different but complementary conclusions from both experiments. The halo test helped us understand the importance of the secretion of the enzyme. On the other hand, the high throughput experiment enabled for more precise comparison between samples and proved to be an efficient way of getting quantitative data from your devices.
In our attempt to screen as many combinations as possible to find the one that would achieve the highest yield of functional alpha-amylase, we combined in vitro high throughput experiments with a machine learning tool (ART). By doing so we expanded the search space, by not being just restricted to the actual combinations that were tested in the lab. When merging these two elements (experiments and machine learning), we encountered some aspects that needed to be taken into consideration. In our case, the subset of samples from the combinatorial library that are going to be evaluated experimentally needed to be thoroughly selected, trying to find an even and representative distribution of all the elements inside each category (as explained in the Model page). Another important aspect is the selection of a performance measure that is representative of what would constitute a “good” device. We selected the activity of alpha-amylase in the supernatant of the machine to be this performance measure as it represents what we aimed to achieve (an efficient production of alpha-amylase) and initial results showed that it can be accurately measured.
There are some improvements that need to be made in the experiments pipeline (see section Improvements in the Model page). In the next DBTL cycle those changes would need to be implemented in order to get more accurate results.
In general, we learnt that when expressing a new gene in a chassis, it is rather unpredictable how the different elements in the device would interact between each other. From our experience, we believe that by using this kind of technology more knowledge about the performance of your device can be acquired and the optimal results can presumably be obtained in fewer DBTL cycles.
References
- Declerck N, Machius M, Wiegand G, Huber R, Gaillardin C. Probing structural determinants specifying high thermostability in Bacillus licheniformis alpha-amylase. J Mol Biol. 2000 Aug 25;301(4):1041-57. doi: 10.1006/jmbi.2000.4025. PMID: 10966804.