Shuimoliuyun (Talk | contribs) |
Shuimoliuyun (Talk | contribs) |
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1:E.gld+DAK;2:S-ceaS2;3,E.NOX-CAT;4.S.NOX-ceaS2;5:DAK;6:NOX;7,ceaS2; | 1:E.gld+DAK;2:S-ceaS2;3,E.NOX-CAT;4.S.NOX-ceaS2;5:DAK;6:NOX;7,ceaS2; | ||
8:gld;9:s.gld-DAK;10:CAT | 8:gld;9:s.gld-DAK;10:CAT | ||
+ | <br><br> | ||
We also used the whole cell catalytic reaction and HPLC determination method to determine the amount of acrylic acid produced. | We also used the whole cell catalytic reaction and HPLC determination method to determine the amount of acrylic acid produced. | ||
For E. coli, yields of using new and old synthetic pathways of acrylic acid are as follows: | For E. coli, yields of using new and old synthetic pathways of acrylic acid are as follows: | ||
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cerevisiae. | cerevisiae. | ||
</br> | </br> | ||
− | <img src="https://static.igem.org/mediawiki/2017/b/ | + | <img src="https://static.igem.org/mediawiki/2017/b/b0/%E9%85%B5%E6%AF%8D%E8%B7%AF%E5%BE%84%E5%9B%BE.png" style="max-width:60%;"><br /> |
Colonial verification results show that we have successfully knocked out the S. | Colonial verification results show that we have successfully knocked out the S. | ||
cerevisiae's DLD genes: | cerevisiae's DLD genes: | ||
</br> | </br> | ||
− | <img src="https://static.igem.org/mediawiki/2017/ | + | <img src="https://static.igem.org/mediawiki/2017/e/e6/NPU-image16.png" style="max-width:60%;"><br /> |
Fig XX S.C BY4741DLD1gene Agarose gel figure of colonies verification after CRISPR | Fig XX S.C BY4741DLD1gene Agarose gel figure of colonies verification after CRISPR | ||
knockout. | knockout. | ||
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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 | ||
indicator<br /> | indicator<br /> | ||
− | <img src="https://static.igem.org/mediawiki/2017/ | + | <img src="https://static.igem.org/mediawiki/2017/2/2e/NPU-image17.png" style="max-width:60%;"><br /> |
It can be seen that the optimization of bypass metabolic flux is conducive to the | It can be seen that the optimization of bypass metabolic flux is conducive to the | ||
concentration of metabolic flux and improving the yield of acrylic acid. Of coursewe | concentration of metabolic flux and improving the yield of acrylic acid. Of coursewe | ||
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<h4> </h4> | <h4> </h4> | ||
− | + | <br /> | |
</a> | </a> | ||
<h4> </h4> | <h4> </h4> | ||
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Induction time: 14h | Induction time: 14h | ||
</h4> | </h4> | ||
− | <img src="https://static.igem.org/mediawiki/2017/ | + | <img src="https://static.igem.org/mediawiki/2017/c/c3/NPU-image18.png" style="max-width:60%;"><br /> |
It can be seen that when the induction temperature was 30 ℃, the enzyme expression and activity were the highest, and the yield of acrylic acid was the best. | It can be seen that when the induction temperature was 30 ℃, the enzyme expression and activity were the highest, and the yield of acrylic acid was the best. | ||
</br> | </br> | ||
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Glucose concentration: 4g/L | Glucose concentration: 4g/L | ||
Glycerol concentration: 1%<br /> | Glycerol concentration: 1%<br /> | ||
− | <img src="https://static.igem.org/mediawiki/2017/ | + | <img src="https://static.igem.org/mediawiki/2017/8/80/NPU-image19.png" style="max-width:60%;"><br /> |
It can be seen that the yield of acrylic acid was higher when the glycerol was used | It can be seen that the yield of acrylic acid was higher when the glycerol was used | ||
as the carbon source, because the carbon flow rate of the glycerol metabolic | as the carbon source, because the carbon flow rate of the glycerol metabolic | ||
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The results are as follows: | The results are as follows: | ||
Reaction conditions: 12h reaction time, 1% concentration of substrate glycerol <br /> | Reaction conditions: 12h reaction time, 1% concentration of substrate glycerol <br /> | ||
− | <img src="https://static.igem.org/mediawiki/2017/ | + | <img src="https://static.igem.org/mediawiki/2017/d/d7/NPU-image20.png" style="max-width:60%;"><br /> |
It can be drawn that PH8.0 was most suitable for acrylic acid production; the | It can be drawn that PH8.0 was most suitable for acrylic acid production; the | ||
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4.4 The effect of different Buffer on the amount of acrylic acid were investigated. | 4.4 The effect of different Buffer on the amount of acrylic acid were investigated. | ||
The results are as follows: | The results are as follows: | ||
− | |||
<img src="https://static.igem.org/mediawiki/2017/8/86/NPU-image21.png" style="max-width:60%;"><br /> | <img src="https://static.igem.org/mediawiki/2017/8/86/NPU-image21.png" style="max-width:60%;"><br /> | ||
+ | <img src="https://static.igem.org/mediawiki/2017/e/ef/NPU-image22.png" style="max-width:60%;"><br /> | ||
It can be seen that the DHa or G3P activity of the two substrates of ceaS2 enzyme was | It can be seen that the DHa or G3P activity of the two substrates of ceaS2 enzyme was | ||
higher under PBS buffer condition. | higher under PBS buffer condition. | ||
4.5 The effects of different reaction time on the amount of acrylic acid were investigated. The results are shown as follows<br /> | 4.5 The effects of different reaction time on the amount of acrylic acid were investigated. The results are shown as follows<br /> | ||
− | <img src="https://static.igem.org/mediawiki/2017/ | + | <img src="https://static.igem.org/mediawiki/2017/c/c2/NPU-image23.png" style="max-width:60%;"><br /> |
It can be drawn that the yield of acrylic acid reached a higher level after the whole | It can be drawn that the yield of acrylic acid reached a higher level after the whole | ||
cell catalytic reaction endured for 16h. The sampling point should be set after 16h. | cell catalytic reaction endured for 16h. The sampling point should be set after 16h. | ||
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GAACF1.0. | GAACF1.0. | ||
<br /> | <br /> | ||
− | <img src="https://static.igem.org/mediawiki/2017/ | + | <img src="https://static.igem.org/mediawiki/2017/d/dc/NPU-image24.png" style="max-width:60%;"><br /> |
<h4> | <h4> | ||
FigXX. Yeast strain: BY4741-ceaS2-gld-DAK; Condition of whole cell catalysis: PH: | FigXX. Yeast strain: BY4741-ceaS2-gld-DAK; Condition of whole cell catalysis: PH: |
Revision as of 18:56, 1 November 2017