Difference between revisions of "Team:NPU-China/Demonstrate"

 
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           <li class="dropdown active">
 
             <a href="#" class="dropdown-toggle" data-toggle="dropdown">Project
 
             <a href="#" class="dropdown-toggle" data-toggle="dropdown">Project
 
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           <li class="dropdown">
 
             <a href="#" class="dropdown-toggle" data-toggle="dropdown">Notebook
 
             <a href="#" class="dropdown-toggle" data-toggle="dropdown">Notebook
 
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           <ul class="nav nav-pills nav-stacked" data-spy="affix" style="width:250px; position:fixed;">
 
           <ul class="nav nav-pills nav-stacked" data-spy="affix" style="width:250px; position:fixed;">
 
             <li class="active">
 
             <li class="active">
−
               <a href="#section-1">DNA gel extraction</a>
+
               <a href="#section-1">Core-part</a>
−
            </li>
+
−
            <li>
+
−
              <a href="#section-2">Electrophoresis</a>
+
−
            </li>
+
−
            <li>
+
−
              <a href="#section-3">Gibson assenbly</a>
+
−
            </li>
+
−
            <li>
+
−
              <a href="#section-4">HPLC</a>
+
−
            </li>
+
−
            <li>
+
−
              <a href="#section-5">Knock out genes of Ecoli</a>
+
−
            </li>
+
−
            <li>
+
−
              <a href="#section-6">Crispr-cas9</a>
+
−
            </li>
+
−
            <li>
+
−
              <a href="#section-7">Plasmid preparation</a>
+
−
            </li>
+
−
            <li>
+
−
              <a href="#section-8">Plasmid transformation</a>
+
−
            </li>
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−
            <li>
+
−
              <a href="#section-9">Point mutation</a>
+
−
            </li>
+
−
            <li>
+
−
              <a href="#section-10">Reagents</a>
+
 
             </li>
 
             </li>
 
             <li>
 
             <li>
−
               <a href="#section-11">Whole cell catalysis</a>
+
               <a href="#section-2">System</a>
 
             </li>
 
             </li>
 
             <li>
 
             <li>
−
               <a href="#section-12">the LiAc SS carrier DNA PEG method</a>
+
               <a href="#section-3">Pathway</a>
 
             </li>
 
             </li>
 
             <li>
 
             <li>
−
               <a href="#section-14">Measure protein concentration</a>
+
               <a href="#section-4">Product</a>
 
             </li>
 
             </li>
 
             <li>
 
             <li>
−
               <a href="#section-13">References</a>
+
               <a href="#section-5">Conclusion</a>
 
             </li>
 
             </li>
 
           </ul>
 
           </ul>
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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;">DNA gel extraction</h2>
+
 
           <h4>
 
           <h4>
−
            1. Excise the agarose gel slice, transfer the gel slice into a 1.5ml microfuge tube.
+
          Acrylic acid is a byproduct of CEAS2 enzyme, the catalytic effect of wild type
−
             <br /> 2. Add a 3X sample volume of Buffer DE-A. the weight of gel is equivalent to a 100ul volume.
+
             ceaS2 enzyme is very weak.  
−
            <br /> 3. Resuspend the gel in Buffer DE-A by vortexing. Heat at 75℃ until the gel is completely dissolved(no more
+
<br/>
−
            than 10 min)
+
We used the AEMD platform to analyze the ceaS2
−
            <br /> 4. Add 0.5X Buffer DE-A volume of Buffer DE-B, mix.
+
            enzyme and screened the 38 mutants in the range of 5 Å around the active site
−
            <br /> 5. Place a miniprep colume into a 2ml microfuge tube. Transfer the solubilized agarose from step 4 into the
+
            to carry out molecular cloning of point mutation, and then tested the acrylic
−
            column. Centrifuge at 12,000xg for 1 min.
+
             acid yield by HPLC after whole cell catalysis. Because there are a large number
−
            <br /> 6. Discard the filtrate. Add 500ul of Buffer W1. Centrifuge at 12,000xg for 30s.
+
            of mutants, we divided them into five batches to carry out the reaction, the  
−
            <br /> 7. Discard the filtrate. Add 700ul of Buffer W2. Centrifuge at 12,000xg for 30s.
+
             results are as follows:
−
             <br /> 8. Repeat wash with W2. Centrifuge at 12,000xg for 1min.
+
−
            <br /> 9. Discard the filtrate. Centrifuge at 12,000xg for 1min.
+
−
            <br /> 10. Transfer the miniprep column into a clean 1.5ml microfuge tube. Add 25-30ul of Eluent or deionized water
+
−
            to the center of the membrane. Let it stand for 1min at room temperature. Centrifuge at 12,000xg for 1min. (pre-warming
+
−
             the Eluent at 65℃)
+
 
             <br />
 
             <br />
 +
          <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/d/dc/NPU-image02.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/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
 +
          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
 +
          the data in order to facilitate the analysis of the catalytic effect of each mutation point
 +
          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:
 +
          <br />
 +
          <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
 +
          vertical axis is the multiple of the acrylic acid yield of each mutational site compared
 +
          to each corresponding batch of the wild type. It can be seen that there were 11
 +
          mutational sites, whose yields were higher than the wild type ceaS2, in the 38 mutant
 +
          programs, and the F438M mutant had the highest yield of 11 times the wild type. The
 +
          effect was significant.
 +
          <br />
 
           </h4>
 
           </h4>
−
           <h2 id="section-2" style="padding-top: 100px; margin-top: -50px;">Electrophoresis</h2>
+
           <h2 id="section-2" style="padding-top: 100px; margin-top: -50px;">2.System：S. cerevisiae is more suitable for chassis cells than E. coli</h2>
−
           <h4>Agarose-Electrophoresis is used in order to see if the PCR product is correct and seperate DNA by the number of
+
           <h4>
−
            base pairs.
+
          Acrylic acid has strong chemical reactivity and is very destructive to cell
−
            <br /> ●marker was the 2 kb Plus DNA Ladder
+
          membrane. Therefore, the chassis cells’ tolerance to acrylic acid is a "roof" factor
−
            <br /> ●fill pockets with 5 µl DNA ladder or 10 µl sample volume with 6x loading buffer
+
          that restricts high yield of acrylic acid.<br>
−
            <br /> ●running conditions: 130 V for 30-40 minutes
+
          We chose E. coli and S. cerevisiae, the two most convenient model chassis
−
            <br />
+
          organisms in prokaryotic and eukaryotic organisms. In order to investigatethe
 +
          chassis cells’ tolerance to acrylic acid, we set up a cytotoxicity test where the two
 +
          chassis cells grew in different concentrations of acrylic acid medium, and the  
 +
          bacteria OD changes were monitored.The results are as follows:
 +
          <br />
 +
          <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
 +
          <br />
 +
          <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
 +
          <br />
 +
<br>
 +
          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 />
 +
<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
 +
          <br />
 +
<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
 +
          <br />
 +
<br>
 +
          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.
 +
          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.<br><br>
 +
          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
 +
          pathway further confirm this conclusion.
 +
          <br />
 
           </h4>
 
           </h4>
−
           <h2 id="section-3" style="padding-top: 100px; margin-top: -50px;">Gibson assembly</h2>
+
           <h2 id="section-3" style="padding-top: 100px; margin-top: -50px;">3.Pathway：Successfully build a new acrylic acid synthesis pathway and increase acrylic acid production</h2>
 
           <h4>
 
           <h4>
−
            1. Set up the reaction.
+
          In order to increase the ability of the chassis cells convert ing glycerol to DHAP or
−
            <br /> V(ul)=(0.02*bp)/concentration(ng/ul)
+
          4G3P, we designed a new GlyDH-DAK glycerol metabolic pathway. To maintain
−
            <br /> 2. Add the fragments into Gibson system.
+
          the supply of the reducing power of GlyDH enzymes, the NOX-CAT reducing
−
             <br /> 3. Incubate samples in a thermocycler at 50 °C for 1h.
+
          power module was also introduced, which eventually forms the acrylic synthesis
−
            <br /> 4. Purify the product using DNA purification kit.
+
          pathway — GDNCC Pathways.
−
            <br /> 5. Transform the product into the competent cells of E.coli BL21 , following the transformation protocol.
+
<br>
−
            <br />
+
          First, we introduced new pathways into two chassis cells through two or three
−
            <br /> </h4>
+
          plasmid vectors.
−
           <img src="https://static.igem.org/mediawiki/2017/2/2a/Sxt_%281%29.png" style="max-width:60%;">
+
  <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
 +
<br><br>
 +
          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:
 +
<br>
 +
          Conditions: reaction time 42h, PH8.0, glycerol concentration 1%
 +
<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
 +
          of the GlyDH enzyme and the DAK enzyme compared to the introduction of only
 +
          the ceaS2 enzyme in old pathway. And the acrylic acid yield is increased by 8
 +
          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.
 +
<br>
 +
<br>
 +
          As for S. cerevisiae, since S. cerevisiae itself has a higher activity of hydrogen
 +
          peroxide reductase, the reducing power module onlyhas NOX enzyme. Theacrylic
 +
          acid yields ofapplying new and old synthetic pathways are as follows:<br>
 +
          Conditions: reaction time 72h, PH8.0, glycerol concentration 2%
 +
          Normalized the results based on the acrylic acid yield of BY4741-ceas2 as the
 +
          indicator.
 +
          </br>
 +
<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
 +
          pathways does improve the ability of S. cerevisiae synthesizing acrylic acid. <br>
 +
          Compared the old pathway introduced only ceaS2 enzyme, acrylic acid
 +
          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
 +
          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.
 +
          cerevisiae.
 +
          </br>
 +
          <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.
 +
          cerevisiae's DLD genes:
 +
          </br>
 +
           <img src="https://static.igem.org/mediawiki/2017/e/e6/NPU-image16.png" style="max-width:60%;"><br />
 +
          Fig 6 S.C BY4741DLD1gene Agarose gel figure of colonies verification after CRISPR
 +
          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
 +
          monoclonal colonies.<br>
 +
          We also tested the acrylic acid synthesis ability of the transformed strain. The results
 +
          are as follows:
 +
          Conditions: reaction time 72h, PH8.0, glycerol concentration 2%
 +
          Normalized the results based on the acrylic acid yield of BY4741-ceas2 as the
 +
          indicator.<br />
 +
          <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
 +
          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.
 +
          cerevisiae colony growth became very slow, indicating that a more tender method
 +
          should be adopted, such as RNAi, to inhibit the bypass pathway.<br />
 
           </a>
 
           </a>
−
          <h4>&nbsp;</h4>
+
        <br />
−
 
+
−
          <img src="https://static.igem.org/mediawiki/2017/3/3b/Sxt_%282%29.png" style="max-width:60%;">
+
 
           </a>
 
           </a>
−
           <h4>&nbsp;</h4>
+
           <h4>&#160;</h4>
−
           <h4>&nbsp;</h4>
+
           <h4>&#160;</h4>
−
           <h2 id="section-4" style="padding-top: 100px; margin-top: -50px;">HPLC</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>
−
            for acrylic acid
+
          There are several important conditions for whole cell reaction: enzyme induction
 +
          temperature, carbon source, Buffer, PH, reaction time. We set different control
 +
          experiments with E.coli BL21 (DE3) as the chassis cells. The results are as follows:
 +
          4.1 The effects of different induction temperatures on the amount of acrylic acid were
 +
          investigated. The results are as follows:
 +
          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/c/c3/NPU-image18.png" style="max-width:60%;"><br />
−
          </a>
+
 
           <h4>
 
           <h4>
−
            <br /> Samples have to be centrifuged at 12,000xg for 5 min in order to remove solids (cells/precipitates).
+
          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>
−
            <br /> All instruments we used is ultra-high performance liquid chromatography mass spectrometry instrument ABSciex
+
−
            5600 TripleTOF.
+
−
            <br /> a) HPLC:
+
−
            <br /> Colum: Waters SunFire C18(4.6×250 mm)
+
−
            <br /> Detector wavelength: 210 nm
+
−
            <br /> Flowing phase: 90% 10mM ammonium acetate/ 10% methanol
+
−
            <br /> Colum temperature: 35℃
+
−
            <br /> Flow rate: 1mL/min
+
−
            <br /> b) MS detecting condition:
+
−
            <br /> Anion mode
+
−
            <br /> Ion Source Gas1: 55 psi
+
−
            <br /> Ion Source Gas2: 55 psi
+
−
            <br /> Curtain Gas: 35 psi
+
−
            <br /> IonSpray Voltage Floating: 4500 v
+
−
            <br /> Interface Heater Temperature: 550℃
+
−
            <br />
+
−
            <br />
+
−
          </h4>
+
−
          <h2 id="section-5" style="padding-top: 100px; margin-top: -50px;">Knock out genes of E ▪ coli (MG1655)</h2>
+
−
          <h4>1. Pre-chill 1.5ml and 2ml microcentrifuge tubes, deionized water, 10% glycerol. Add 1ml LB to 2ml microcentrifuge
+
−
            tubes.
+
−
            <br /> 2. When OD600=0.6, incubate competent cells on ice for 20 min.
+
−
            <br /> 3. Transfer the competent cells to 50 mL pre-chilled centrifuge tube. Centrifuge at 5,500r/min, 4 ℃ for 5 min.
+
−
            <br /> 4. Discard the filtrate. Add 30ml of pre-chilled deionized water, resuspend the cells gently.
+
−
            <br /> 5. Centrifuge at 5,500r/min, 4 ℃ for 5 min. Discard the filtrate. Repeat wash with deionized water.
+
−
            <br /> 6. Discard the filtrate. Add 30ml of pre-chilled 10% glycerol, resuspend the cells gently.
+
−
            <br /> 7. Centrifuge at 6,500r/min, 4 ℃ for 5 min. Discard the filtrate. Repeat wash with 10% glycerol.
+
−
            <br /> 8. Discard the filtrate and leave over 1ml 10% glycerol to resuspend the competent cells, pipet 80ul into each
+
−
            1.5ml EP tube, add 5ul of DNA and carefully mix with the competent cells. Let it stand for 2min.
+
−
            <br /> 9. Add electrocompetent to DNA on ice. Move the mixture to the cuvette. Dry and shock the cells(2500V). Add
+
−
            1ml of LB medium. Incubate at 30℃ for 4 hours shaking at 220rpm, pipet 100ul from each tube onto the appropriate
+
−
            plate, and spread the mixture evenly across the plate. Incubate at 30℃ overnight. Position the plates with the
+
−
            agar side at the top, and the lid at the bottom.
+
−
            <br />
+
−
          </h4>
+
−
           <h2 id="section-6" style="padding-top: 100px; margin-top: -50px;">Knock out the genes of Saccharomyces cerevisiae with Crispr-Cas9</h2>
+
−
 
+
−
          <img src="https://static.igem.org/mediawiki/2017/c/c6/Sxt_%284%29.png" style="max-width:60%;">
+
−
          </a>
+
−
          <h4>&nbsp;</h4>
+
−
 
+
−
          <img src="https://static.igem.org/mediawiki/2017/8/8d/Sxt_%285%29.png" style="max-width:60%;">
+
−
          </a>
+
 
           <h4>
 
           <h4>
−
            <br /> 2. Purify the PCR product with a DNA purification kit.
+
          4.2 the results of production of acrylic acid with different carbon sources
−
            <br /> 3. Add the appropriate amount of DMT enzyme, hold for one hour at 37 ° C.
+
          Condition: PH7.4  
−
            <br /> 4. transform the DNA into competent cells.
+
          Reaction time: 16h
−
            <br /> 50ul competent cell + 15ul purified DNA，incubate on ice for 30min，heat shock 45s，incubate on ice for 2min，add
+
          Glucose concentration: 4g/L
−
            LB medium and incubate for 1h.
+
          Glycerol concentration: 1%<br /></h4>
−
            <br /> 5. Pipet 100ul from each tube onto the plate with resistance, and spread the mixture evenly across the plate.
+
           <img src="https://static.igem.org/mediawiki/2017/8/80/NPU-image19.png" style="max-width:60%;"><br />
−
            Incubate for 12h. Position the plates with the agar side at the top, and the lid at the bottom.
+
−
            <br /> 6. use a sterile pipet tip to pick Saccharomyces cerevisiae from plates，throw the tip into the tubes of 5 ml
+
−
            of LB + antibiotics，incubate in a rotary shaker. Prepare plasmid with kit for sequencing.
+
−
            <br /> 7. Transfer plasmid and fragment into Saccharomyces cerevisiae using the LiAc SS carrier DNA PEG method.</h4>
+
−
           <img src="https://static.igem.org/mediawiki/2017/e/e0/Sxt_%286%29.png" style="max-width:60%;">
+
−
          </a>
+
 
           <h4>
 
           <h4>
−
            <br /> 8. Prepare the template: use a sterile toothpick to pick Saccharomyces cerevisiae from plates，put the toothpick
+
          It can be seen that the yield of acrylic acid was higher when the glycerol was used
−
            into 100ul 20mMNaOH and mix，99° C boiling for 30min. </h4>
+
           as the carbon source, because the carbon flow rate of the glycerol metabolic
−
           <img src="https://static.igem.org/mediawiki/2017/8/84/Sxt_%287%29.png" style="max-width:60%;">
+
           pathway was more concentrated, thus turning more carbon source into acrylic
−
           </a>
+
           acid. Plus, the glycerol itself owning a higher reducing powermay also be one of
−
           <h4>&nbsp;</h4>
+
           the reasons.
−
 
+
           4.3  The effects of different pH on the amount of acrylic acid were investigated.
−
          <img src="https://static.igem.org/mediawiki/2017/a/a8/Sxt_%288%29.png" style="max-width:60%;">
+
           The results are as follows:
−
           </a>
+
           Reaction conditions: 12h reaction time, 1% concentration of substrate glycerol </h4><br />
−
 
+
           <img src="https://static.igem.org/mediawiki/2017/d/d7/NPU-image20.png" style="max-width:60%;"><br />
−
           <h4>&nbsp;</h4>
+
−
          <h2 id="section-7" style="padding-top: 100px; margin-top: -50px;">plasmid preparation</h2>
+
−
          <h4>Tiangen mini plasmid kit
+
−
            <br />
+
−
          </h4>
+
−
 
+
−
          <h2 id="section-8" style="padding-top: 100px; margin-top: -50px;">Plasmid transformation</h2>
+
−
          <h4>1. Pipette 50µl of competent cells and 2µl of plasmid into 1.5ml tube
+
−
            <br /> 2. Heat shock tubes at 42°C for 30s
+
−
            <br /> 3. Incubate on ice for 2min
+
−
            <br /> 4. Pipette 250µl LB media to each transformation
+
−
            <br /> 5. Incubate at 37°C for 1h
+
−
            <br /> 6. Plating
+
−
            <br /> 7. Pick single colonies
+
−
            <br /> Reference: http://parts.igem.org/Help:Protocols/Transformation
+
−
            <br />
+
−
           </h4>
+
−
 
+
−
          <h2 id="section-9" style="padding-top: 100px; margin-top: -50px;">Point mutation</h2>
+
−
 
+
−
 
+
−
           <img src="https://static.igem.org/mediawiki/2017/0/0f/Sxt_%289%29.png" style="max-width:60%;">
+
−
          </a>
+
−
          <h4>&nbsp;</h4>
+
−
 
+
−
           <img src="https://static.igem.org/mediawiki/2017/6/6c/Sxt_%2810%29.png" style="max-width:60%;">
+
−
          </a>
+
 
           <h4>
 
           <h4>
−
            <br /> 2. Purify the PCR product with a DNA purification kit.
+
          It can be drawn that PH8.0 was most suitable for acrylic acid production; the  
−
            <br /> 3. Add the appropriate amount of DMT enzyme, hold for one hour at 37 ° C.
+
          reason may be that alkaline environment made E.coli more resistant to acrylic
−
            <br /> 4. Transform 5μl digested DNA into competent cells DH5α, incubate on ice for 30min.
+
          acid.
−
            <br /> 42° C heat shock, 45s. Incubate on ice for 2min.
+
          4.4 The effect of different Buffer on the amount of acrylic acid were investigated.  
−
            <br /> add 200μl of LB. incubate at 37 °C for 1 h, 220rpm/min.
+
          The results are as follows:</h4>
−
            <br /> 5. pipet 200μl from each tube onto the plate with appropriate resistance, and spread the mixture evenly across
+
          <img src="https://static.igem.org/mediawiki/2017/8/86/NPU-image21.png" style="max-width:60%;"><br />
−
            the plate. Incubate at 37℃ overnight. Position the plates with the agar side at the top, and the lid at the bottom.
+
          <img src="https://static.igem.org/mediawiki/2017/e/ef/NPU-image22.png" style="max-width:60%;"><br /> <h4>
−
            <br /> 6. Select single colonies for sequencing.
+
          It can be seen that the DHa or G3P activity of the two substrates of ceaS2 enzyme was
 +
          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 </h4><br />
 +
          <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
 +
          cell catalytic reaction endured for 16h. The sampling point should be set after 16h.
 
             <br />
 
             <br />
−
          </h4>
 
−
 
−
          <h2 id="section-10" style="padding-top: 100px; margin-top: -50px;">Reagents</h2>
 
−
          <h4>1. LB medium（lysogeny broth）
 
−
            <br /> The recipe for 1l LB media is as follows:
 
−
            <br /> Tryptone 10g/L
 
−
            <br /> Yeast extract 5g/L
 
−
            <br /> NaCl 10g/L
 
−
            <br /> 2. 0.1mM Kanamycin
 
−
            <br /> MW of Kanamycin：582.58
 
−
            <br /> Store at -20℃
 
−
            <br /> 3. LB plate
 
−
            <br /> The recipe for 1l LB plate is as follows:
 
−
            <br /> Tryptone 10g/L
 
−
            <br /> Yeast extract 5g/L
 
−
            <br /> NaCl 10g/L
 
−
            <br /> 15g Agar
 
−
            <br /> Add appropriate amount of resistance.
 
−
            <br /> 4. 2YT medium
 
−
            <br /> The recipe for 1l LB plate is as follows:
 
−
            <br /> Tryptone 16g/L
 
−
            <br /> Yeast extract 10g/L
 
−
            <br /> NaCl 5g/L
 
−
            <br /> 5. 0.5mM IPTG
 
−
            <br /> MW of IPTG：238.30
 
−
            <br /> Store at -20℃
 
−
            <br /> 6. 50mM PBS buffer,PH8.0
 
−
            <br /> A：0.05mol/L Na2HPO4
 
−
            <br /> B：0.05mol/L KH2P04
 
−
            <br /> 137mMNaCl，2.7mMKCl，10mMNa2HPO4,2mMKH2PO4 for 1L.
 
 
             <br />
 
             <br />
−
 
 
           </h4>
 
           </h4>
−
 
+
           <h2 id="section-5" style="padding-top: 100px; margin-top: -50px;">5.Conclusion </h2> <h4>
−
           <h2 id="section-11" style="padding-top: 100px; margin-top: -50px;">Whole-cell catalysis</h2>
+
          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
 +
          acrylic acid that we have acquired is 211.655 mg / L, which is 200 times than that of
 +
          GAACF1.0. </h4>
 +
          <br />
 +
          <img src="https://static.igem.org/mediawiki/2017/a/a1/NPU-25.png" style="max-width:60%;"><br />
 
           <h4>
 
           <h4>
−
            Whole cell catalysis means using complete biological organisms (ie, whole cells, tissues or even individuals) as a catalyst.
+
          Fig7. Yeast strain: BY4741-ceaS2-gld-DAK; Condition of whole cell catalysis: PH:
−
            The essence is using enzymes in cells for catalysis. The method is a kind of biocatalytic technology between
+
          7.4; Concentration of the substrate glycerol: 2%.  
−
            fermentation and extract enzyme for catalysis. The advantage of whole cell catalysis is that the intracellular
+
<br>
−
            complete multi-enzyme system can achieve the cascade reaction of enzyme, so as to make up the deficiency of cascade
+
<br>
−
            reaction in reaction which only use pure enzyme and improve the catalytic efficiency. While eliminating the complex
+
The chromatogam of the sample by
−
            process in enzyme purification, it is easier to carry out the reaction and lower production costs. <br>
+
          HPLC shows the yield is up to 211.655 mg / L according to the standard curve.
−
            1. Prepare
+
          211.655mg/L , currently this is the highest yield of acrylic acid biosynthesis, where
−
            sterile tubes of 5 ml of 2YT+antibiotics. Use a sterile pipet tip to pick bacteria from plates. Throw the tip
+
          glycerol serves as the carbon source.
−
            into the tubes. Incubate in a rotary shaker at37℃ for 3-4h.
+
          As an undergraduate team, in just a few months, we have tried our best to create an  
−
            <br /> 2. Transfer 120µl of bacteria from a slant culture into an Erlenmeyer flask containing 60 mL LB medium with
+
          efficient acrylic cell factory. We were surprised by the huge increase in GAACF 2.0  
−
            appropriate resistance, incubate at 37 °C.
+
          production, which is only the production of wild-type ceaS2. Because it is a
−
            <br /> 3. When the OD600 reaches 0.6-0.8, the induction of IPTG (0.5 mM) should be carried out. Incubate at 30 °C on
+
          continuing project, we are planning to screen for more active mutants on the basis of
−
            a rotary shaker incubator at 220 rpm for 14 h.
+
          several productive mutations using HTS for point saturation mutations and  
−
            <br /> 4. Harvest the bacteria(6000rpm/min 7min). Wash with 30ml PBS.
+
          high-throughput screening. And then, we will transform them into the existing chassis
−
            <br /> 5. The biocatalytic reaction mixture contained 10% glycerol, E▪ coli and 50mM PBS. Reaction time gradient: 8h,
+
          organism. We believe that we will create a new technology for acrylic acid production
−
            16h, 32h.
+
          which has more industrialization prospect!<br />
−
            <br /> 6. Use HPLC for further analysis.
+
 
             <br />
 
             <br />
−
            <br /> Reference: Li N, He Y, Chen Y, et al. Production of cyclic adenosine-3′,5′-monophosphate by whole cell catalysis
+
       
−
            using recombinant Escherichia coli, overexpressing adenylate cyclase[J]. Korean Journal of Chemical Engineering,
+
−
            2013, 30(4):913-917.
+
−
            <br />
+
−
          </h4>
+
−
 
+
−
          <h2 id="section-12" style="padding-top: 100px; margin-top: -50px;">the LiAc SS carrier DNA PEG method</h2>
+
−
          <h4>
+
−
            1.use a sterile pipet tip to pick Saccharomyces cerevisiae from plates，throw the tip into the tubes of appropriate medium，incubate
+
−
            in a rotary shaker for 12h.
+
−
            <br /> 2.measure OD600, transfer x(x=(50×0.2)/(OD600×dilution ratio)) ml Saccharomyces cerevisiae into 50ml YPAD.
+
−
            <br /> 3.incubate for 4-5h to make OD600 reaches 0.8-0.9.
+
−
            <br /> 4.boil ssDNA.
+
−
            <br /> 5.Centrifuge at 3000g for 5 min. Discard the filtrate. Repeat washes with 25ml deionized water twice.
+
−
            <br /> 6.Transfer the cells to 1.5 mL centrifuge tube. Add 1ml of deionized water, resuspend the cells gently.
+
−
            <br /> 7.Centrifuge at 13000rpm for 30s. Discard the filtrate.
+
−
            <br /> 8.Add 1ml of deionized water, resuspend the cells. Pipet 100ul into each 1.5 mL centrifuge tube.
+
−
            <br /> 9.Centrifuge using a Mini Centrifuge. Discard the filtrate.
+
−
            <br /> 10.System for transformation:</h4>
+
−
          <img src="https://static.igem.org/mediawiki/2017/7/70/Sxt_%2811%29.png" style="max-width:60%;">
+
−
          </a>
+
−
          <h4>
+
−
            <br /> 11.Incubate at 30℃ for 20min.
+
−
            <br /> 12.42℃ heat shock for 40min. pipet 100ul from each tube onto the appropriate plate, and spread the mixture evenly
+
−
            across the plate. Incubate at 30℃ for 2-3 days. Position the plates with the agar side at the top, and the lid
+
−
            at the bottom.
+
−
            <br /> 13.Prepare plasmid for sequencing.
+
−
            <br />
+
−
 
+
−
          </h4>
+
−
 
+
−
          <h2 id="section-14" style="padding-top: 100px; margin-top: -50px;">Measure protein concentration</h2>
+
−
          <h4>
+
−
            We used Pierce BCA Protein Assay Kit (Thermo Fisher Scientific) to measure protein concentration.
+
−
            <br> 1. Measure OD at 280 nm to get rough protein concentration, and then diluted the protein to 0.5-1 mg / mL.
+
−
            <br> 2. Prepare the reaction solution: reagent A and B in the BCA Protein Assay Kit are mixed in a 50: 1 ratio.
+
−
            <br> 3. Pipette 200 uL of reaction solution in the coated wells
+
−
            <br> 4. Pipette 25 uL of diluted protein, mixed it with the reaction solution. Hold at 37 ℃ for 30 min.
+
−
            <br> 5. Measure OD at 562 nm. Protein concentration is measured according to the protein standard curve.
+
−
            <br>
+
−
 
+
−
          </h4>
+
−
 
+
−
 
+
−
          <h2 id="section-13" style="padding-top: 100px; margin-top: -50px;">References</h2>
+
−
          <h4>
+
−
            http://parts.igem.org/Help:Protocols/Transformation
+
−
            <br /> https://2015.igem.org/Team:Aachen/Project/Overview
+
−
            <br /> http://www.zymoresearch.com/category/all-products
+
−
            <br /> http://www.corning.com/worldwide/en/products/life-sciences/resources/brands/axygen-brand-products.html
+
−
            <br /> http://www.tsingke.net/shop/
+
−
            <br /> http://www.cwbiotech.com/
+
−
            <br />
+
−
          </h4>
+
−
 
+
−
 
+
−
 
+
 
         </div>
 
         </div>
 
       </div>
 
       </div>

Latest revision as of 02:22, 2 November 2017

1. Core-part：the activity of rate limiting enzyme ceaS2 has been improved

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 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 acid yield by HPLC after whole cell catalysis. Because there are a large number of mutants, we divided them into five batches to carry out the reaction, the results are as follows:






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).
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 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:


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 to each corresponding batch of the wild type. It can be seen that there were 11 mutational sites, whose yields were higher than the wild type ceaS2, in the 38 mutant programs, and the F438M mutant had the highest yield of 11 times the wild type. The effect was significant.

2.System：S. cerevisiae is more suitable for chassis cells than E. coli

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 that restricts high yield of acrylic acid.
We chose E. coli and S. cerevisiae, the two most convenient model chassis organisms in prokaryotic and eukaryotic organisms. In order to investigatethe chassis cells’ tolerance to acrylic acid, we set up a cytotoxicity test where the two chassis cells grew in different concentrations of acrylic acid medium, and the bacteria OD changes were monitored.The results are as follows:

Fig1. OD of E.coli MG1655 under acrylic acid of different concentration and time

Fig2. OD of S. cerevisiaeBY4741 under acrylic acid of different concentration and time

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:


Fig3. A comparison of OD of BY4741 and MG1655 under 500mg/L acrylic acid


Fig4. A comparison of OD of BY4741 and MG1655 under 1000mg/L acrylic acid

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. 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.

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 pathway further confirm this conclusion.

3.Pathway：Successfully build a new acrylic acid synthesis pathway and increase acrylic acid production

In order to increase the ability of the chassis cells convert ing glycerol to DHAP or 4G3P, we designed a new GlyDH-DAK glycerol metabolic pathway. To maintain the supply of the reducing power of GlyDH enzymes, the NOX-CAT reducing power module was also introduced, which eventually forms the acrylic synthesis pathway — GDNCC Pathways.
First, we introduced new pathways into two chassis cells through two or three plasmid vectors.

pET-28a-ceaS2; pCDFDuet-gld-DAK; pETDuet-NOX-CAT; YCplac33-LEU-ceaS2; YCplac33-LEU-ceaS2-NOX; YCplac33-URA-gld-DAK


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

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:
Conditions: reaction time 42h, PH8.0, glycerol concentration 1%

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 the ceaS2 enzyme in old pathway. And the acrylic acid yield is increased by 8 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.

As for S. cerevisiae, since S. cerevisiae itself has a higher activity of hydrogen peroxide reductase, the reducing power module onlyhas NOX enzyme. Theacrylic acid yields ofapplying new and old synthetic pathways are as follows:
Conditions: reaction time 72h, PH8.0, glycerol concentration 2% Normalized the results based on the acrylic acid yield of BY4741-ceas2 as the indicator.



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.
Compared the old pathway introduced only ceaS2 enzyme, acrylic acid 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 to the old pathway after the addition of the reducing power module.
We also used CRISPR-CAS9 to optimize the bypass metabolic pathway of the S. cerevisiae.

Colonial verification results show that we have successfully knocked out the S. cerevisiae's DLD genes:

Fig 6 S.C BY4741DLD1gene Agarose gel figure of colonies verification after CRISPR knockout.

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 monoclonal colonies.
We also tested the acrylic acid synthesis ability of the transformed strain. The results are as follows: Conditions: reaction time 72h, PH8.0, glycerol concentration 2% Normalized the results based on the acrylic acid yield of BY4741-ceas2 as the indicator.

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 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 should be adopted, such as RNAi, to inhibit the bypass pathway.

 

 

4.Product：Multi - Conditional Optimization of Acrylic Cell Factory Catalytic Reaction Process

There are several important conditions for whole cell reaction: enzyme induction temperature, carbon source, Buffer, PH, reaction time. We set different control experiments with E.coli BL21 (DE3) as the chassis cells. The results are as follows: 4.1 The effects of different induction temperatures on the amount of acrylic acid were investigated. The results are as follows: Induction time: 14h


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.


4.2 the results of production of acrylic acid with different carbon sources Condition: PH7.4 Reaction time: 16h Glucose concentration: 4g/L Glycerol concentration: 1%


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 pathway was more concentrated, thus turning more carbon source into acrylic acid. Plus, the glycerol itself owning a higher reducing powermay also be one of the reasons. 4.3 The effects of different pH on the amount of acrylic acid were investigated. The results are as follows: Reaction conditions: 12h reaction time, 1% concentration of substrate glycerol



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 acid. 4.4 The effect of different Buffer on the amount of acrylic acid were investigated. The results are as follows:



It can be seen that the DHa or G3P activity of the two substrates of ceaS2 enzyme was 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



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.

5.Conclusion

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 acrylic acid that we have acquired is 211.655 mg / L, which is 200 times than that of GAACF1.0.



Fig7. Yeast strain: BY4741-ceaS2-gld-DAK; Condition of whole cell catalysis: PH: 7.4; Concentration of the substrate glycerol: 2%.

The chromatogam of the sample by 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. As an undergraduate team, in just a few months, we have tried our best to create an efficient acrylic cell factory. We were surprised by the huge increase in GAACF 2.0 production, which is only the production of wild-type ceaS2. Because it is a continuing project, we are planning to screen for more active mutants on the basis of several productive mutations using HTS for point saturation mutations and 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 which has more industrialization prospect!