Difference between revisions of "Team:H14Z1 Hangzhou/Demonstrate"

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                 <h1 class="content_title">Demonstrate</h1>
 
                 <h1 class="content_title">Demonstrate</h1>
 
                 <div class="content_conts">
 
                 <div class="content_conts">
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                     <!------------------------HPLC sample------------------------------ -->
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                     <!------------------------Part 1------------------------------ -->
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                     <h3 class="content_subtitle">HPLC sample</h3>
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                     <h3 class="content_subtitle">Functional validation of GSH module and SAM module by HPLC</h3>
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                     <p class="mt8 content_context">
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                     <p class="content_context">
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                         <span style="width:40px">Fig1.</span><span style="width:500px">GSH standard products peak at
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                         To further validate the GSH module and SAM module, we tested the GSH and SAM content in final
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                            7.5min</span>
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                        engineered strain L. lactis/pNZ-GMcA.
 
                     </p>
 
                     </p>
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                     <p class="mt8 content_context">
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                     <p class="content_context">
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                         <span style="width:40px">Fig2.</span><span style="width:500px">NZ9000 intracellular extracts
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                         It is known that L. lactis NZ9000 cannot form GSH and can form little SAM by itself. As shown
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                            from no-load cells</span>
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                        in Figure.1, no GSH was detected in L. lactis NZ9000 as expected. And GSH was obviously
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                        appeared during the fermentation, illustrating that GSH module was effective. And Figure.2
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                        showed that more SAM were accumulated in strain L. lactis/pNZ-GMcA than wild-type, illustrating
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                        the good function of SAM module.
 
                     </p>
 
                     </p>
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                     <p class="mt8 content_context">
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                     <div>
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                         <span style="width:40px">Fig3.</span><span style="width:500px">intracellular extracts
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                        <p><img style="width: 50%;" src="https://static.igem.org/mediawiki/2018/d/d0/T--H14Z1_Hangzhou--project_demonstrate_fig1.png"></p>
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                             of.NZ9000/pNZ-gshF</span>
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                        <p class="content_context" style="text-align:center; font-size:8px">
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                            Figure. 1 HPLC analysis of GSH samples
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                         </p>
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                    </div>
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                    <div>
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                        <p><img style="width: 50%;" src="https://static.igem.org/mediawiki/2018/e/e5/T--H14Z1_Hangzhou--project_demonstrate_fig2.png"></p>
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                        <p class="content_context" style="text-align:center; font-size:8px">
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                             Figure.2 HPLC analysis of SAM samples
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                        </p>
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                    </div>
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                    <!------------------------Part 2------------------------------ -->
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                    <h3 class="content_subtitle">Functional validation of adhesion factor module by self-aggregation
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                        value assay</h3>
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                    <p class="content_context">
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                        To validate adhesion factor module, we carried out self-aggregation assay which could reflect
 +
                        the adhesivity of strains. As shown in Figure. 3, by introducing plasmid pNZ-GMcA, L. lactis
 +
                        could have obviously improvement in self-aggregation value, illustrating that adhesion module
 +
                        was expressed successfully and worked well.
 
                     </p>
 
                     </p>
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                     <p><img style="width: 60%;" src="https://static.igem.org/mediawiki/2018/4/44/T--H14Z1_Hangzhou--results_demonstrate_fig1.PNG"></p>
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                     <p><img style="width: 50%;" src="https://static.igem.org/mediawiki/2018/3/31/T--H14Z1_Hangzhou--project_demonstrate_fig3.png"></p>
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                     <p class="content_context" style="text-indent:2em; text-align:justify;">
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                     <p class="content_context" style="text-align:center; font-size:8px">
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                         According to the area calculation, the product was determined by internal standard
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                         Figure. 3 Comparison of self-aggregation value between L.lactis NZ9000 and L. lactis/pNZ-GMcA
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                        method. Sample size and standard volume are mixed for testing.
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                        The results show that the sealing area is not much different at about 7.45 min, but the area of
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                        7.75 min is half of the original. It is verified that the peak is the product peak of GSH.
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                     </p>
 
                     </p>
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                     <p><img style="width: 60%;" src="https://static.igem.org/mediawiki/2018/c/c1/T--H14Z1_Hangzhou--results_demonstrate_fig2.PNG"></p>
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                     <p class="mt8 content_context" style="text-align:center">
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                     <!------------------------Part 3------------------------------ -->
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                         <span style="width:80px">Fig4.</span><span>GSH concentration / cell dry
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                    <h3 class="content_subtitle">Functional comparison among different composite modules</h3>
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                            weight</span>
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                     <p class="content_context">
 +
                         After constructing plasmids containing different combination of the three modules, they were
 +
                        introduced to L. lactis NZ9000 by electroporation, separately, as shown in Figure. 4. Then, the
 +
                        function of these engineered strains were validated by detected the GSH and SAM content and
 +
                        self-aggregation value. As depicted in Figure. 5, while adding the modules to the strain, the
 +
                        function of this module was obtained no matter single expression or combinational expression.
 
                     </p>
 
                     </p>
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                     <p class="content_context" style="text-indent:2em; text-align:justify;">
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                    <p><img style="width: 50%;" src="https://static.igem.org/mediawiki/2018/3/30/T--H14Z1_Hangzhou--project_demonstrate_fig4.png"></p>
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                         Comparing the GSH yields of the two groups, the first group was lower than the second group in
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                     <p class="content_context" style="text-align:center; font-size:8px">
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                        general, and both groups had the highest GSH yields after 6 hours of induction. Up to 8.62mg/g.
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                         Figure. 4 Schematic diagram of transferring plasmids to L. lactis by electroporation
 
                     </p>
 
                     </p>
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                     <!------------------------HPLC detection of SAM------------------------------ -->
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                     <p><img style="width: 50%;" src="https://static.igem.org/mediawiki/2018/0/02/T--H14Z1_Hangzhou--project_demonstrate_fig5.png"></p>
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                    <h3 class="content_subtitle">HPLC detection of SAM</h3>
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                     <p class="content_context" style="text-align:center; font-size:8px">
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                     <p class="content_context" style="text-indent:2em; text-align:justify;">
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                         Figure. 5 Functional comparison among different composite modules in L. lactis.
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                         Fermentation conditions: when OD600=0.4 was added with different concentration of inducer, the
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                        final concentration was 20ng/ml, 50ng/ml, 100ng/ml nisin was induced and methionine at the
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                        final concentration of 1g/l was added
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                        as substrate.
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                     </p>
 
                     </p>
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                     <p>
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                        <span class="content_context">a：20ng/ml</span>
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                     <!------------------------Part 4------------------------------ -->
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                        <span class="content_context">b：50ng/ml</span>
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                    <h3 class="content_subtitle">Process demonstrate of smart yogurts with three target modules</h3>
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                         <span class="content_context">c：100ng/ml</span>
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                    <p class="content_context">
 +
                        After validation of the function of the final engineered strain L. lactis/pNZ-GMcA, we applied
 +
                        it to produce smart yogurts. As depicted below, we produced three kinds of smart yogurts. One
 +
                        was produced by using wild-type strain L.lactis NZ9000 adding GSH and SAM in the process.
 +
                        Another one was produced by using engineered L. lactis/pNZ-GMcA and the last one using
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                         wild-type strain L.bulgaricus and engineered L. lactis/pNZ-GMcA without adding GSH and SAM.
 
                     </p>
 
                     </p>
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                     <p><img style="width: 60%;" src="https://static.igem.org/mediawiki/2018/e/ed/T--H14Z1_Hangzhou--results_demonstrate_fig3.PNG"></p>
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                     <p><img style="width: 50%;" src="https://static.igem.org/mediawiki/2018/9/9d/T--H14Z1_Hangzhou--project_demonstrate_fig6.png"></p>
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                     <p class="mt8 content_context" style="text-align:center">
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                     <p class="content_context" style="text-align:center; font-size:8px">
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                         <span style="width:80px">Fig5.</span><span>SAM liquid phase detection chart</span>
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                         Figure. 6 Schematic diagram of producing smart yogurts
 
                     </p>
 
                     </p>
  
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                     <p><img style="width: 60%;" src="https://static.igem.org/mediawiki/2018/8/89/T--H14Z1_Hangzhou--results_demonstrate_fig4.PNG"></p>
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                    <!------------------------Part 5------------------------------ -->
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                     <p class="mt8 content_context" style="text-align:center">
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                    <h3 class="content_subtitle">The content of GSH and SAM in the smart yogurts</h3>
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                         <span style="width:80px">Fig6.</span><span>SAM2 production in
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                    <p class="content_context">
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                            NZ9000/pNZ-metk cells</span>
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                        The GSH and SAM content of the smart yogurts in the fermentation at 6 and 12 hours were
 +
                        detected. As shown in Figure. 7, the smart yogurt made by using engineered L. lactis contained
 +
                        obvious more GSH and SAM. The content increased with the increase of cell numbers.
 +
                    </p>
 +
                     <p><img style="width: 50%;" src="https://static.igem.org/mediawiki/2018/c/cf/T--H14Z1_Hangzhou--project_demonstrate_fig7.png"></p>
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                     <p class="content_context" style="text-align:center; font-size:8px">
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                         Figure. 7 GSH and SAM content in the smart yogurts at 6 and 12 hours. Asterisk represented not
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                        detected.
 
                     </p>
 
                     </p>
  
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                     <p class="content_context" style="text-indent:2em; text-align:justify;">
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                    <!------------------------Part 6------------------------------ -->
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                         Fig6. shows that although there is no significant metK protein on SDS-PAGE, the intracellular
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                    <h3 class="content_subtitle">Application of patent for the production of smart yogurts</h3>
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                        SAM production is increased in varying degrees at different induction concentrations. The
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                     <p class="content_context">
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                        highest yield of 50 ng/ml is 15.2 mg/g.
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                        At last, we have applied a Chinese patent for producing smart yogurts using the engineered
 +
                        strain containing three modules. And it was registered by National Patent Office of China.
 +
                    </p>
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                    <p><img style="width: 50%;" src="https://static.igem.org/mediawiki/2018/b/bf/T--H14Z1_Hangzhou--project_demonstrate_fig8.png"></p>
 +
                    <p class="content_context" style="text-align:center; font-size:8px">
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                         Figure. 8 The patent application for producing smart yogurts
 
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Revision as of 21:58, 17 October 2018


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Demonstrate

Functional validation of GSH module and SAM module by HPLC

To further validate the GSH module and SAM module, we tested the GSH and SAM content in final engineered strain L. lactis/pNZ-GMcA.

It is known that L. lactis NZ9000 cannot form GSH and can form little SAM by itself. As shown in Figure.1, no GSH was detected in L. lactis NZ9000 as expected. And GSH was obviously appeared during the fermentation, illustrating that GSH module was effective. And Figure.2 showed that more SAM were accumulated in strain L. lactis/pNZ-GMcA than wild-type, illustrating the good function of SAM module.

Figure. 1 HPLC analysis of GSH samples

Figure.2 HPLC analysis of SAM samples

Functional validation of adhesion factor module by self-aggregation value assay

To validate adhesion factor module, we carried out self-aggregation assay which could reflect the adhesivity of strains. As shown in Figure. 3, by introducing plasmid pNZ-GMcA, L. lactis could have obviously improvement in self-aggregation value, illustrating that adhesion module was expressed successfully and worked well.

Figure. 3 Comparison of self-aggregation value between L.lactis NZ9000 and L. lactis/pNZ-GMcA

Functional comparison among different composite modules

After constructing plasmids containing different combination of the three modules, they were introduced to L. lactis NZ9000 by electroporation, separately, as shown in Figure. 4. Then, the function of these engineered strains were validated by detected the GSH and SAM content and self-aggregation value. As depicted in Figure. 5, while adding the modules to the strain, the function of this module was obtained no matter single expression or combinational expression.

Figure. 4 Schematic diagram of transferring plasmids to L. lactis by electroporation

Figure. 5 Functional comparison among different composite modules in L. lactis.

Process demonstrate of smart yogurts with three target modules

After validation of the function of the final engineered strain L. lactis/pNZ-GMcA, we applied it to produce smart yogurts. As depicted below, we produced three kinds of smart yogurts. One was produced by using wild-type strain L.lactis NZ9000 adding GSH and SAM in the process. Another one was produced by using engineered L. lactis/pNZ-GMcA and the last one using wild-type strain L.bulgaricus and engineered L. lactis/pNZ-GMcA without adding GSH and SAM.

Figure. 6 Schematic diagram of producing smart yogurts

The content of GSH and SAM in the smart yogurts

The GSH and SAM content of the smart yogurts in the fermentation at 6 and 12 hours were detected. As shown in Figure. 7, the smart yogurt made by using engineered L. lactis contained obvious more GSH and SAM. The content increased with the increase of cell numbers.

Figure. 7 GSH and SAM content in the smart yogurts at 6 and 12 hours. Asterisk represented not detected.

Application of patent for the production of smart yogurts

At last, we have applied a Chinese patent for producing smart yogurts using the engineered strain containing three modules. And it was registered by National Patent Office of China.

Figure. 8 The patent application for producing smart yogurts