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<img src="https://static.igem.org/mediawiki/2017/e/e6/NPU-image03.png" style="max-width:60%;"><br /> | <img src="https://static.igem.org/mediawiki/2017/e/e6/NPU-image03.png" style="max-width:60%;"><br /> | ||
<img src="https://static.igem.org/mediawiki/2017/a/a4/NPU-image04.png" style="max-width:60%;"><br /> | <img src="https://static.igem.org/mediawiki/2017/a/a4/NPU-image04.png" style="max-width:60%;"><br /> | ||
− | <img src="https://static.igem.org/mediawiki/2017/b/b2/NPU-image05.png" style="max-width:60%;"><br /> | + | <img src="https://static.igem.org/mediawiki/2017/b/b2/NPU-image05.png" style="max-width:60%;"><br /><br> |
In the figure, the horizontal axis stands for each different point mutation. We selected | In the figure, the horizontal axis stands for each different point mutation. We selected | ||
− | two reaction times 21h and 42h, the vertical axis is acrylic acid production (mg / L) | + | two reaction times 21h and 42h, the vertical axis is acrylic acid production (mg / L)<br> |
Due to the differences in wild type between different batches, we will normalize all | Due to the differences in wild type between different batches, we will normalize all | ||
the data in order to facilitate the analysis of the catalytic effect of each mutation point | the data in order to facilitate the analysis of the catalytic effect of each mutation point | ||
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Acrylic acid has strong chemical reactivity and is very destructive to cell | Acrylic acid has strong chemical reactivity and is very destructive to cell | ||
membrane. Therefore, the chassis cells’ tolerance to acrylic acid is a "roof" factor | membrane. Therefore, the chassis cells’ tolerance to acrylic acid is a "roof" factor | ||
− | that restricts high yield of acrylic acid. | + | that restricts high yield of acrylic acid.<br> |
We chose E. coli and S. cerevisiae, the two most convenient model chassis | We chose E. coli and S. cerevisiae, the two most convenient model chassis | ||
organisms in prokaryotic and eukaryotic organisms. In order to investigatethe | organisms in prokaryotic and eukaryotic organisms. In order to investigatethe | ||
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500mg / L, E. coli bacterial growth was inhibited or even declined while S. | 500mg / L, E. coli bacterial growth was inhibited or even declined while S. | ||
cerevisiae normally grew and entered a stable period. And when the concentration of | cerevisiae normally grew and entered a stable period. And when the concentration of | ||
− | acrylic acid reached 1000 mg / L, the growth of S. cerevisiae was then inhibited. | + | acrylic acid reached 1000 mg / L, the growth of S. cerevisiae was then inhibited.<br><br> |
Conclusion: S. cerevisiae has a better tolerance to acrylic acid toxicity than E. | Conclusion: S. cerevisiae has a better tolerance to acrylic acid toxicity than E. | ||
coli, and may be more suitable for use as chassis cells, and our results of the | coli, and may be more suitable for use as chassis cells, and our results of the | ||
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As for S. cerevisiae, since S. cerevisiae itself has a higher activity of hydrogen | As for S. cerevisiae, since S. cerevisiae itself has a higher activity of hydrogen | ||
peroxide reductase, the reducing power module onlyhas NOX enzyme. Theacrylic | peroxide reductase, the reducing power module onlyhas NOX enzyme. Theacrylic | ||
− | acid yields ofapplying new and old synthetic pathways are as follows: | + | acid yields ofapplying new and old synthetic pathways are as follows:<br> |
Conditions: reaction time 72h, PH8.0, glycerol concentration 2% | Conditions: reaction time 72h, PH8.0, glycerol concentration 2% | ||
Normalized the results based on the acrylic acid yield of BY4741-ceas2 as the | Normalized the results based on the acrylic acid yield of BY4741-ceas2 as the | ||
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<br> | <br> | ||
It can be seen that, similar to the results of E. coli, the introduction of new | It can be seen that, similar to the results of E. coli, the introduction of new | ||
− | pathways does improve the ability of S. cerevisiae synthesizing acrylic acid. | + | pathways does improve the ability of S. cerevisiae synthesizing acrylic acid. <br> |
Compared the old pathway introduced only ceaS2 enzyme, acrylic acid | Compared the old pathway introduced only ceaS2 enzyme, acrylic acid | ||
production was increased by 3 times after introduction of GlyDH enzymes and | production was increased by 3 times after introduction of GlyDH enzymes and | ||
DAK enzymes. And the yield of acrylic acid was increased by 5 times compared | DAK enzymes. And the yield of acrylic acid was increased by 5 times compared | ||
− | to the old pathway after the addition of the reducing power module. | + | to the old pathway after the addition of the reducing power module.<br> |
We also used CRISPR-CAS9 to optimize the bypass metabolic pathway of the S. | We also used CRISPR-CAS9 to optimize the bypass metabolic pathway of the S. | ||
cerevisiae. | cerevisiae. | ||
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WT is the corresponding nucleic acid stripe of wild-type S.C BY4741; M is a | WT is the corresponding nucleic acid stripe of wild-type S.C BY4741; M is a | ||
GeneRuler 1 kb DNA ladder; lanes 1, 2, 3 are three selected nucleic acid stripes of | GeneRuler 1 kb DNA ladder; lanes 1, 2, 3 are three selected nucleic acid stripes of | ||
− | monoclonal colonies. | + | monoclonal colonies.<br> |
We also tested the acrylic acid synthesis ability of the transformed strain. The results | We also tested the acrylic acid synthesis ability of the transformed strain. The results | ||
are as follows: | are as follows: |
Revision as of 21:26, 1 November 2017