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results are as follows: | results are as follows: | ||
<br /> | <br /> | ||
− | <img src="https://static.igem.org/mediawiki/2017/2/2a/Sxt_%281%29.png" style="max-width:60%;"> | + | <img src="https://static.igem.org/mediawiki/2017/2/2a/Sxt_%281%29.png" style="max-width:60%;"><br /> |
− | <img src="https://static.igem.org/mediawiki/2017/2/2a/Sxt_%281%29.png" style="max-width:60%;"> | + | <img src="https://static.igem.org/mediawiki/2017/2/2a/Sxt_%281%29.png" style="max-width:60%;"><br /> |
− | <img src="https://static.igem.org/mediawiki/2017/2/2a/Sxt_%281%29.png" style="max-width:60%;"> | + | <img src="https://static.igem.org/mediawiki/2017/2/2a/Sxt_%281%29.png" style="max-width:60%;"><br /> |
− | <img src="https://static.igem.org/mediawiki/2017/2/2a/Sxt_%281%29.png" style="max-width:60%;"> | + | <img src="https://static.igem.org/mediawiki/2017/2/2a/Sxt_%281%29.png" style="max-width:60%;"><br /> |
− | <img src="https://static.igem.org/mediawiki/2017/2/2a/Sxt_%281%29.png" style="max-width:60%;"> | + | <img src="https://static.igem.org/mediawiki/2017/2/2a/Sxt_%281%29.png" style="max-width:60%;"><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) | ||
Line 219: | Line 219: | ||
bacteria OD changes were monitored.The results are as follows: | bacteria OD changes were monitored.The results are as follows: | ||
<br /> | <br /> | ||
− | <img src="https://static.igem.org/mediawiki/2017/2/2a/Sxt_%281%29.png" style="max-width:60%;"> | + | <img src="https://static.igem.org/mediawiki/2017/2/2a/Sxt_%281%29.png" style="max-width:60%;"><br /> |
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 | ||
<br /> | <br /> | ||
− | <img src="https://static.igem.org/mediawiki/2017/2/2a/Sxt_%281%29.png" style="max-width:60%;"> | + | <img src="https://static.igem.org/mediawiki/2017/2/2a/Sxt_%281%29.png" style="max-width:60%;"><br /> |
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 | ||
<br /> | <br /> | ||
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: | + | 500mg / L and 1000mg / L two kinds of acrylic acid concentration to analyze:<br /> |
− | <img src="https://static.igem.org/mediawiki/2017/2/2a/Sxt_%281%29.png" style="max-width:60%;"> | + | <img src="https://static.igem.org/mediawiki/2017/2/2a/Sxt_%281%29.png" style="max-width:60%;"><br /> |
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 | ||
<br /> | <br /> | ||
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indicator | indicator | ||
</br> | </br> | ||
− | <img src="https://static.igem.org/mediawiki/2017/2/2a/Sxt_%281%29.png" style="max-width:60%;"> | + | <img src="https://static.igem.org/mediawiki/2017/2/2a/Sxt_%281%29.png" style="max-width:60%;"><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. | ||
Line 285: | Line 285: | ||
cerevisiae. | cerevisiae. | ||
</br> | </br> | ||
− | <img src="https://static.igem.org/mediawiki/2017/2/2a/Sxt_%281%29.png" style="max-width:60%;"> | + | <img src="https://static.igem.org/mediawiki/2017/2/2a/Sxt_%281%29.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/2017/2/2a/Sxt_%281%29.png" style="max-width:60%;"> | + | <img src="https://static.igem.org/mediawiki/2017/2/2a/Sxt_%281%29.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. | ||
Line 299: | Line 299: | ||
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 | ||
− | indicator | + | indicator<br /> |
− | <img src="https://static.igem.org/mediawiki/2017/2/2a/Sxt_%281%29.png" style="max-width:60%;"> | + | <img src="https://static.igem.org/mediawiki/2017/2/2a/Sxt_%281%29.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 | ||
also found in the process of the experiment that after knocking out the 9 genes, S. | also found in the process of the experiment that after knocking out the 9 genes, S. | ||
cerevisiae colony growth became very slow, indicating that a more tender method | cerevisiae colony growth became very slow, indicating that a more tender method | ||
− | should be adopted, such as RNAi, to inhibit the bypass pathway. | + | should be adopted, such as RNAi, to inhibit the bypass pathway.<br /> |
</a> | </a> | ||
<h4> </h4> | <h4> </h4> | ||
− | <img src="https://static.igem.org/mediawiki/2017/3/3b/Sxt_%282%29.png" style="max-width:60%;"> | + | <img src="https://static.igem.org/mediawiki/2017/3/3b/Sxt_%282%29.png" style="max-width:60%;"><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/2017/1/16/Sxt_%283%29.png" style="max-width:60%;"> | + | <img src="https://static.igem.org/mediawiki/2017/1/16/Sxt_%283%29.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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Reaction time: 16h | Reaction time: 16h | ||
Glucose concentration: 4g/L | Glucose concentration: 4g/L | ||
− | Glycerol concentration: 1% | + | Glycerol concentration: 1%<br /> |
− | <img src="https://static.igem.org/mediawiki/2017/1/16/Sxt_%283%29.png" style="max-width:60%;"> | + | <img src="https://static.igem.org/mediawiki/2017/1/16/Sxt_%283%29.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 | ||
Line 338: | Line 338: | ||
4.3 The effects of different pH on the amount of acrylic acid were investigated. | 4.3 The effects of different pH on the amount of acrylic acid were investigated. | ||
The results are as follows: | The results are as follows: | ||
− | Reaction conditions: 12h reaction time, 1% concentration of substrate glycerol | + | Reaction conditions: 12h reaction time, 1% concentration of substrate glycerol <br /> |
− | <img src="https://static.igem.org/mediawiki/2017/1/16/Sxt_%283%29.png" style="max-width:60%;"> | + | <img src="https://static.igem.org/mediawiki/2017/1/16/Sxt_%283%29.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 | ||
Line 346: | Line 346: | ||
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/2017/1/16/Sxt_%283%29.png" style="max-width:60%;"> | + | <img src="https://static.igem.org/mediawiki/2017/1/16/Sxt_%283%29.png" style="max-width:60%;"><br /> |
− | <img src="https://static.igem.org/mediawiki/2017/1/16/Sxt_%283%29.png" style="max-width:60%;"> | + | <img src="https://static.igem.org/mediawiki/2017/1/16/Sxt_%283%29.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 | + | 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/2017/1/16/Sxt_%283%29.png" style="max-width:60%;"> | + | <img src="https://static.igem.org/mediawiki/2017/1/16/Sxt_%283%29.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. | ||
Line 363: | Line 363: | ||
GAACF1.0. | GAACF1.0. | ||
<br /> | <br /> | ||
− | <img src="https://static.igem.org/mediawiki/2017/1/16/Sxt_%283%29.png" style="max-width:60%;"> | + | <img src="https://static.igem.org/mediawiki/2017/1/16/Sxt_%283%29.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: | ||
Line 377: | Line 377: | ||
high-throughput screening. And then, we will transform them into the existing chassis | high-throughput screening. And then, we will transform them into the existing chassis | ||
organism. We believe that we will create a new technology for acrylic acid production | organism. We believe that we will create a new technology for acrylic acid production | ||
− | which has more industrialization prospect! | + | which has more industrialization prospect!<br /> |
<br /> | <br /> | ||
</h4> | </h4> |
Revision as of 17:27, 1 November 2017