Shuimoliuyun (Talk | contribs) |
Shuimoliuyun (Talk | contribs) |
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results are as follows: | results are as follows: | ||
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<img src="https://static.igem.org/mediawiki/2017/d/dc/NPU-image02.png" style="max-width:60%;"><br /> | <img src="https://static.igem.org/mediawiki/2017/d/dc/NPU-image02.png" style="max-width:60%;"><br /> | ||
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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) | ||
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yield multiple is a new indicator, the result is as follows: | yield multiple is a new indicator, the result is as follows: | ||
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The horizontal axis in the figure is the position of each mutational site, and the | The horizontal axis in the figure is the position of each mutational site, and the | ||
vertical axis is the multiple of the acrylic acid yield of each mutational site compared | vertical axis is the multiple of the acrylic acid yield of each mutational site compared | ||
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bacteria OD changes were monitored.The results are as follows: | bacteria OD changes were monitored.The results are as follows: | ||
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Fig1. OD of E.coli MG1655 under acrylic acid of different concentration and time | Fig1. OD of E.coli MG1655 under acrylic acid of different concentration and time | ||
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Fig2. OD of S. cerevisiaeBY4741 under acrylic acid of different concentration and time | Fig2. OD of S. cerevisiaeBY4741 under acrylic acid of different concentration and time | ||
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Two kinds of chassis cells have different tolerance to acrylic acid. Here we selected | Two kinds of chassis cells have different tolerance to acrylic acid. Here we selected | ||
500mg / L and 1000mg / L two kinds of acrylic acid concentration to analyze:<br /> | 500mg / L and 1000mg / L two kinds of acrylic acid concentration to analyze:<br /> | ||
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Fig3. A comparison of OD of BY4741 and MG1655 under 500mg/L acrylic acid | Fig3. A comparison of OD of BY4741 and MG1655 under 500mg/L acrylic acid | ||
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Fig4. A comparison of OD of BY4741 and MG1655 under 1000mg/L acrylic acid | Fig4. A comparison of OD of BY4741 and MG1655 under 1000mg/L acrylic acid | ||
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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: | ||
Conditions: reaction time 42h, PH8.0, glycerol concentration 1% | Conditions: reaction time 42h, PH8.0, glycerol concentration 1% | ||
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It can be seen that the acrylic acid yield is increased by 3 times after the introduction | It can be seen that the acrylic acid yield is increased by 3 times after the introduction | ||
of the GlyDH enzyme and the DAK enzyme compared to the introduction of only | of the GlyDH enzyme and the DAK enzyme compared to the introduction of only | ||
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indicator | indicator | ||
</br> | </br> | ||
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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. | ||
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cerevisiae. | cerevisiae. | ||
</br> | </br> | ||
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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> | ||
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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 /> | ||
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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> | ||
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</a> | </a> | ||
<h4> </h4> | <h4> </h4> | ||
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Induction time: 14h | Induction time: 14h | ||
</h4> | </h4> | ||
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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 /> | ||
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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 /> | ||
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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: | ||
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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 /> | ||
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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 /> | ||
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<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 18:39, 1 November 2017