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<h2 id="section-1" style="padding-top: 100px; margin-top: -50px;">1. Core-part:the activity of rate limiting enzyme ceaS2 has been improved</h2> | <h2 id="section-1" style="padding-top: 100px; margin-top: -50px;">1. Core-part:the activity of rate limiting enzyme ceaS2 has been improved</h2> | ||
<h4> | <h4> | ||
Acrylic acid is a byproduct of CEAS2 enzyme, the catalytic effect of wild type | Acrylic acid is a byproduct of CEAS2 enzyme, the catalytic effect of wild type | ||
− | ceaS2 enzyme is very weak. We used the AEMD platform to analyze the ceaS2 | + | ceaS2 enzyme is very weak. |
+ | <br/> | ||
+ | We used the AEMD platform to analyze the ceaS2 | ||
enzyme and screened the 38 mutants in the range of 5 Å around the active site | enzyme and screened the 38 mutants in the range of 5 Å around the active site | ||
to carry out molecular cloning of point mutation, and then tested the acrylic | to carry out molecular cloning of point mutation, and then tested the acrylic | ||
Line 187: | Line 179: | ||
results are as follows: | results are as follows: | ||
<br /> | <br /> | ||
− | <img src="https://static.igem.org/mediawiki/2017/ | + | <img src="https://static.igem.org/mediawiki/2017/8/8b/NPU-image01.png" style="max-width:60%;"><br /> |
− | <img src="https://static.igem.org/mediawiki/2017/ | + | <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/ | + | <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/ | + | <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/ | + | <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 | ||
− | compared to the respective | + | compared to the respective WT, that is, to compare each mutation point to The batch WT |
yield multiple is a new indicator, the result is as follows: | yield multiple is a new indicator, the result is as follows: | ||
<br /> | <br /> | ||
− | <img src="https://static.igem.org/mediawiki/2017/ | + | <br /> |
+ | <img src="https://static.igem.org/mediawiki/2017/e/e4/NPU-26.png" style="max-width:110%;"><br /> | ||
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 | ||
Line 212: | Line 205: | ||
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 | ||
Line 219: | Line 212: | ||
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/ | + | <img src="https://static.igem.org/mediawiki/2017/a/a8/NPU-image07.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/ | + | <img src="https://static.igem.org/mediawiki/2017/8/82/NPU-image08.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 /> | ||
+ | <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:<br /> | 500mg / L and 1000mg / L two kinds of acrylic acid concentration to analyze:<br /> | ||
− | <img src="https://static.igem.org/mediawiki/2017/ | + | <br> |
+ | <img src="https://static.igem.org/mediawiki/2017/1/13/NPU-image09.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 /> | ||
− | <img src="https://static.igem.org/mediawiki/2017/ | + | <br> |
+ | <img src="https://static.igem.org/mediawiki/2017/4/46/NPU-image10.png" style="max-width:60%;"><br /> | ||
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 | ||
<br /> | <br /> | ||
+ | <br> | ||
As can be seen from the results, when the concentration of acrylic acid reached | As can be seen from the results, when the concentration of acrylic acid reached | ||
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 | ||
Line 249: | Line 246: | ||
power module was also introduced, which eventually forms the acrylic synthesis | power module was also introduced, which eventually forms the acrylic synthesis | ||
pathway — GDNCC Pathways. | pathway — GDNCC Pathways. | ||
+ | <br> | ||
First, we introduced new pathways into two chassis cells through two or three | First, we introduced new pathways into two chassis cells through two or three | ||
plasmid vectors. | plasmid vectors. | ||
− | pET-28a-ceaS2 | + | <br> |
− | + | <br>pET-28a-ceaS2; pCDFDuet-gld-DAK; pETDuet-NOX-CAT; YCplac33-LEU-ceaS2; YCplac33-LEU-ceaS2-NOX; YCplac33-URA-gld-DAK | |
− | + | <br> | |
− | + | <br> | |
− | + | <img src="https://static.igem.org/mediawiki/2017/c/c7/NPU-image11.png" style="max-width:60%;"> | |
− | + | <img src="https://static.igem.org/mediawiki/2017/0/00/NPU-image12.png" style="max-width:60%;"> | |
− | + | <br> | |
+ | Fig5 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: | ||
+ | <br> | ||
Conditions: reaction time 42h, PH8.0, glycerol concentration 1% | Conditions: reaction time 42h, PH8.0, glycerol concentration 1% | ||
− | <img src="https://static.igem.org/mediawiki/2017/ | + | <br> |
+ | <img src="https://static.igem.org/mediawiki/2017/b/b5/NPU-image13.png" style="max-width:60%;"><br /> | ||
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 | ||
Line 268: | Line 270: | ||
times compared to the old one after the addition of the reducing power module. The | times compared to the old one after the addition of the reducing power module. The | ||
new pathway does enhance the ability of E. colisynthesizing acrylic acid. | new pathway does enhance the ability of E. colisynthesizing acrylic acid. | ||
+ | <br> | ||
+ | <br> | ||
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 | ||
− | indicator | + | indicator. |
</br> | </br> | ||
− | <img src="https://static.igem.org/mediawiki/2017/ | + | <br> |
+ | <img src="https://static.igem.org/mediawiki/2017/5/5e/NPU-image14.png" style="max-width:60%;"><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. | ||
</br> | </br> | ||
− | <img src="https://static.igem.org/mediawiki/2017/ | + | <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 | + | Fig 6 S.C BY4741DLD1gene Agarose gel figure of colonies verification after CRISPR |
− | knockout. | + | knockout.<br> |
− | + | <br> | |
+ | 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: | ||
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<br /> | + | indicator.<br /> |
− | <img src="https://static.igem.org/mediawiki/2017/2/ | + | <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 | ||
Line 307: | Line 314: | ||
should be adopted, such as RNAi, to inhibit the bypass pathway.<br /> | should be adopted, such as RNAi, to inhibit the bypass pathway.<br /> | ||
</a> | </a> | ||
− | + | <br /> | |
− | + | ||
− | + | ||
</a> | </a> | ||
− | <h4>& | + | <h4> </h4> |
− | <h4>& | + | <h4> </h4> |
<h2 id="section-4" style="padding-top: 100px; margin-top: -50px;">4.Product:Multi - Conditional Optimization of Acrylic Cell Factory Catalytic Reaction Process</h2> | <h2 id="section-4" style="padding-top: 100px; margin-top: -50px;">4.Product:Multi - Conditional Optimization of Acrylic Cell Factory Catalytic Reaction Process</h2> | ||
<h4> | <h4> | ||
Line 322: | Line 327: | ||
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. | + | <h4> |
+ | 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.</h4> | ||
</br> | </br> | ||
+ | <h4> | ||
4.2 the results of production of acrylic acid with different carbon sources | 4.2 the results of production of acrylic acid with different carbon sources | ||
Condition: PH7.4 | Condition: PH7.4 | ||
Reaction time: 16h | Reaction time: 16h | ||
Glucose concentration: 4g/L | Glucose concentration: 4g/L | ||
− | Glycerol concentration: 1%<br /> | + | Glycerol concentration: 1%<br /></h4> |
− | <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 /> |
+ | <h4> | ||
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 346: | ||
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 <br /> | + | Reaction conditions: 12h reaction time, 1% concentration of substrate glycerol </h4><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 /> |
− | + | <h4> | |
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 | ||
reason may be that alkaline environment made E.coli more resistant to acrylic | reason may be that alkaline environment made E.coli more resistant to acrylic | ||
acid. | acid. | ||
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:</h4> |
− | <img src="https://static.igem.org/mediawiki/2017/ | + | <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/ | + | <img src="https://static.igem.org/mediawiki/2017/e/ef/NPU-image22.png" style="max-width:60%;"><br /> <h4> |
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 </h4><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 /> <h4> |
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 357: | Line 365: | ||
<br /> | <br /> | ||
</h4> | </h4> | ||
− | <h2 id="section-5" style="padding-top: 100px; margin-top: -50px;">5.Conclusion </h2> | + | <h2 id="section-5" style="padding-top: 100px; margin-top: -50px;">5.Conclusion </h2> <h4> |
Due to the time limit of the experiment, we did not have enough time to replace the | Due to the time limit of the experiment, we did not have enough time to replace the | ||
optimal mutation site into the existing cell factory. At present, the highest yield of | optimal mutation site into the existing cell factory. At present, the highest yield of | ||
acrylic acid that we have acquired is 211.655 mg / L, which is 200 times than that of | acrylic acid that we have acquired is 211.655 mg / L, which is 200 times than that of | ||
− | GAACF1.0. | + | GAACF1.0. </h4> |
<br /> | <br /> | ||
− | <img src="https://static.igem.org/mediawiki/2017/ | + | <img src="https://static.igem.org/mediawiki/2017/a/a1/NPU-25.png" style="max-width:60%;"><br /> |
<h4> | <h4> | ||
− | + | Fig7. Yeast strain: BY4741-ceaS2-gld-DAK; Condition of whole cell catalysis: PH: | |
− | 7.4; Concentration of the substrate glycerol: 2%. The | + | 7.4; Concentration of the substrate glycerol: 2%. |
+ | <br> | ||
+ | <br> | ||
+ | The chromatogam of the sample by | ||
HPLC shows the yield is up to 211.655 mg / L according to the standard curve. | HPLC shows the yield is up to 211.655 mg / L according to the standard curve. | ||
− | + | 211.655mg/L , currently this is the highest yield of acrylic acid biosynthesis, where | |
glycerol serves as the carbon source. | glycerol serves as the carbon source. | ||
As an undergraduate team, in just a few months, we have tried our best to create an | As an undergraduate team, in just a few months, we have tried our best to create an | ||
Line 379: | Line 390: | ||
which has more industrialization prospect!<br /> | which has more industrialization prospect!<br /> | ||
<br /> | <br /> | ||
− | + | ||
</div> | </div> | ||
</div> | </div> |
Latest revision as of 02:22, 2 November 2017