Difference between revisions of "Team:H14Z1 Hangzhou/Demonstrate"
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<p class="content_context" style="text-indent:2em; text-align:justify"> | <p class="content_context" style="text-indent:2em; text-align:justify"> | ||
To further validate the GSH module and SAM module, we tested the GSH and SAM content in final | To further validate the GSH module and SAM module, we tested the GSH and SAM content in final | ||
| − | engineered strain L. lactis/pNZ-GMcA. | + | engineered strain <i>L. lactis</i>/pNZ-GMcA. |
</p> | </p> | ||
<p class="content_context" style="text-indent:2em; text-align:justify"> | <p class="content_context" style="text-indent:2em; text-align:justify"> | ||
| − | It is known that L. lactis NZ9000 cannot form GSH and can form little SAM by itself. As shown | + | It is known that <i>L. lactis</i> 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 | + | in Figure.1, no GSH was detected in <i>L. lactis</i> NZ9000 as expected. And GSH was obviously |
appeared during the fermentation, illustrating that GSH module was effective. And Figure.2 | 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 | + | showed that more SAM were accumulated in strain <i>L. lactis</i>/pNZ-GMcA than wild-type, illustrating |
the good function of SAM module. | the good function of SAM module. | ||
</p> | </p> | ||
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<p class="content_context" style="text-indent:2em; text-align:justify"> | <p class="content_context" style="text-indent:2em; text-align:justify"> | ||
To validate adhesion factor module, we carried out self-aggregation assay which could reflect | 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 | + | the adhesivity of strains. As shown in Figure. 3, by introducing plasmid pNZ-GMcA, <i>L. lactis</i> |
could have obviously improvement in self-aggregation value, illustrating that adhesion module | could have obviously improvement in self-aggregation value, illustrating that adhesion module | ||
was expressed successfully and worked well. | was expressed successfully and worked well. | ||
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<p><img style="width: 45%; margin-top: 12px;" src="https://static.igem.org/mediawiki/2018/3/31/T--H14Z1_Hangzhou--project_demonstrate_fig3.png"></p> | <p><img style="width: 45%; margin-top: 12px;" src="https://static.igem.org/mediawiki/2018/3/31/T--H14Z1_Hangzhou--project_demonstrate_fig3.png"></p> | ||
<p class="content_context" style="text-align:center; font-size:18px"> | <p class="content_context" style="text-align:center; font-size:18px"> | ||
| − | Figure. 3 Comparison of self-aggregation value between L.lactis NZ9000 and L. lactis/pNZ-GMcA | + | Figure. 3 Comparison of self-aggregation value between L.lactis NZ9000 and <i>L. lactis</i>/pNZ-GMcA |
</p> | </p> | ||
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<p class="content_context" style="text-indent:2em; text-align:justify"> | <p class="content_context" style="text-indent:2em; text-align:justify"> | ||
After constructing plasmids containing different combination of the three modules, they were | After constructing plasmids containing different combination of the three modules, they were | ||
| − | introduced to L. lactis NZ9000 by electroporation, separately. Then, the function of these | + | introduced to <i>L. lactis</i> NZ9000 by electroporation, separately. Then, the function of these |
engineered strains were validated by detected the GSH and SAM content and self-aggregation | engineered strains were validated by detected the GSH and SAM content and self-aggregation | ||
value. | value. | ||
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<p><img style="width: 80%; margin-top: 12px; margin-left: 3em" src="https://static.igem.org/mediawiki/2018/9/94/T--H14Z1_Hangzhou--demonstrate_fig11.png"></p> | <p><img style="width: 80%; margin-top: 12px; margin-left: 3em" src="https://static.igem.org/mediawiki/2018/9/94/T--H14Z1_Hangzhou--demonstrate_fig11.png"></p> | ||
<p class="content_context" style="text-align:center; font-size:18px"> | <p class="content_context" style="text-align:center; font-size:18px"> | ||
| − | Figure. 4 Functional comparison among different composite modules in L. lactis. | + | Figure. 4 Functional comparison among different composite modules in <i>L. lactis</i>. |
</p> | </p> | ||
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<h3 class="content_subtitle">Process demonstrate of smart yogurts with three target modules</h3> | <h3 class="content_subtitle">Process demonstrate of smart yogurts with three target modules</h3> | ||
<p class="content_context" style="text-indent:2em; text-align:justify"> | <p class="content_context" style="text-indent:2em; text-align:justify"> | ||
| − | After validation of the function of the final engineered strain L. lactis/pNZ-GMcA, we applied | + | After validation of the function of the final engineered strain <i>L. lactis</i>/pNZ-GMcA, we applied |
it to produce smart yogurts. As depicted below, we produced three kinds of smart yogurts. One | 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. | 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 | + | Another one was produced by using engineered <i>L. lactis</i>/pNZ-GMcA and the last one using |
| − | wild-type strain L.bulgaricus and engineered L. lactis/pNZ-GMcA without adding GSH and SAM. | + | wild-type strain L.bulgaricus and engineered <i>L. lactis</i>/pNZ-GMcA without adding GSH and SAM. |
</p> | </p> | ||
<p><img style="width: 65%; " src="https://static.igem.org/mediawiki/2018/9/9d/T--H14Z1_Hangzhou--project_demonstrate_fig6.png"></p> | <p><img style="width: 65%; " src="https://static.igem.org/mediawiki/2018/9/9d/T--H14Z1_Hangzhou--project_demonstrate_fig6.png"></p> | ||
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<p class="content_context" style="text-indent:2em; text-align:justify"> | <p class="content_context" style="text-indent:2em; text-align:justify"> | ||
The GSH and SAM content of the smart yogurts in the fermentation at 6 and 12 hours were | The GSH and SAM content of the smart yogurts in the fermentation at 6 and 12 hours were | ||
| − | detected. As shown in Figure. 6, the smart yogurt made by using engineered L. lactis contained | + | detected. As shown in Figure. 6, the smart yogurt made by using engineered <i>L. lactis</i> contained |
obvious more GSH and SAM. The content increased with the increase of cell numbers. | obvious more GSH and SAM. The content increased with the increase of cell numbers. | ||
</p> | </p> | ||
Latest revision as of 01:21, 18 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. Then, the function of these engineered strains were validated by detected the GSH and SAM content and self-aggregation value.
Figure. 4 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. 5 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. 6, 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. 6 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. 7 The patent application for producing smart yogurts