Team:NCTU Formosa/CSP data

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<br><br>
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<div id="blueBox"><p> Carotenoid Pathway­</p></div>
<div id="blueBox"><p> Carotenoid Pathway­</p></div>
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<div id="Box"><h2>Data</h2>
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<h2>Introduction</h2> 
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<p>This pathway explain how Fernesyl Diphosphate is catalyzed to Zeathaxine.There are three color products in the pathway:red Lycopene, orange beta-Carotene and yellow Zeathaxine.</p>
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<div><img src = "https://static.igem.org/mediawiki/2011/thumb/a/ac/Nctu_cp_1.PNG/800px-Nctu_cp_1.PNG" width="700"></div>
 
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<br><b>Figure 1. </b> The overview of the Carotenoid pathway <br><br>
 
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<font color="red">Lycopene</font><br>Lycopene is a bright red carotenoid pigment but has no Vit A activity .  Lycopene is also an important intermediate in the biosynthesis of many carotenoids.
 
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<font color="orange">Bata-Carotene</font><br>Bata-Carotene is a strong red-orange pigment abundant in plants and fruits. It’s a member of carotenes and it can be distinguished by two beta-rings at both ends of the molecule. beta–Carotene is the most well-known pre-Vit A among all the carotenoids.
 
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<font color="yellow">Zeaxanthin</font><br>Zeaxanthin is one of the two primary xanthophyll carotenoids contained within the retina of the eye.  Zeaxanthin provides the primary yellow pigment.
 
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<h2>Design</h2>
 
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<div><img src = "https://static.igem.org/mediawiki/2011/thumb/a/ab/Nctu_cp_2.PNG/800px-Nctu_cp_2.PNG" width="700"></div>
<div><img src = "https://static.igem.org/mediawiki/2011/thumb/a/ab/Nctu_cp_2.PNG/800px-Nctu_cp_2.PNG" width="700"></div>
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<br><b>Figure 2. </b The Carotenoid pathway circuit design. Cotransform <a href ="http://partsregistry.org/Part:BBa_K539151 ">BBa_K539151</a> on psb3T5 and <a href ="http://partsregistry.org/Part:BBa_K539281 ">BBa_K539281</a> on psb4A5 into host cell DH5 α.
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<br><b> Figure 3.</b> The compositions of the Carotenoid pathway circuit. The upper strand is BBa_K539151 and the down strand is BBa_K539281.
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<p>
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<p>The upper strand of the Carotenoid pathway circuit BBa_K539151 is composed of (1) crtEBI BBa_K346090 that can produce Lycopene, (2) 37℃induced RBS BBa_K115002, (3) crtY BBa_K118008 that can produce beta-Carotene, and (4) Terminator BBa_J61048. When we assembled the upper strand, we found a point mutation of BBa_K118008 (iGEM08_Edinburgh) at SpeI site and we corrected it successfully.(ACCTAGT->ACTAGT). This corrected part is numbered as BBa_K539119.
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The main concept of the Pathway Commander is to control the flux through the metabolic pathway. Therefore, we choose the Carotenoid synthesis Pathway that can easily display different steps in the pathway by different colors. </p>
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The down stand BBa_K539281 is composed of (1) Heat sensitive cI promoter with repressor BBa_K098995, (2) crtZ BBa_I742158 that can produce Zeaxanthin, and (3) Terminator BBa_J61048.</p>
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<p>After both of the strands were constructed, we transferred the assembled insert BBa_K539151 from originally backbone(psb1k3) to psb3T5. The reason why we change the backbone is that the psb3T5 with low-copy-numbers is better for E.coli to express proteins. For the same reason, we transferred the complete insert BBa_K539281 from originally backbone (psb1k3) to psb4A5. (We chose different antibiotic backbone from the upper strand in order to cotransform.) Finally, we cotransformed DH5αwith BBa_K539151 on psb3T5 and BBa_K539281 on psb4A5.
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To test the circuit whether it can work or not, we incubate the modified E.coli in different temperatures and extract the pigments. </p>
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<ol type="disc">
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<li>30℃: It is supposed to express only CrtEBI and produce Lycopene. Show red color. <img src = "https://static.igem.org/mediawiki/2011/5/52/Nctu_cp_6.PNG" width="10">
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</li>
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<li>37℃: It is supposed to express CrtEBI and CrtY. Produce beta-Carotene and show orange color. <img src = "https://static.igem.org/mediawiki/2011/0/01/Nctu_cp_7.PNG" width="10">
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</li>
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<li>42℃: It is supposed to express CrtEBI, CrtY and CrtZ. Produce Zeaxanthin and show yellow color. <img src = "https://static.igem.org/mediawiki/2011/3/30/Nctu_cp_8.PNG" width="10"></li>
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�<h2>
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Experiment methods</h2>
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<li>
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The cells were cultured in LB over night.</li>
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<li>Transferred 200ul LB to fresh 200ml LB and incubated at 37°C. </li>
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<li>After 8 hrs, when OD ( optical density) reaches 0.1, then switch the temperature to 30°C, 37°C, 42°C respectively and incubate for 24hrs. </li>
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<li>Centrifuged at 4°C, 6000rpm. The pellets were added with 1 ml ddH20 and vortexed. </li>
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<li>Moved into 1.5ml eppendorf. Centrifuged for 20minutes at 4°C, 14000rpm. </li>
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<li>Added with 500ul acetone, and vortexed for 1hr to extract the pigments. <li>
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<div><img src = "https://static.igem.org/mediawiki/2011/thumb/c/ce/IMG_2261.JPG/800px-IMG_2261.JPG" width="5
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<br><b>Figure 4. </b> Here shows the result of our circuit. The colored pellets represent the achievement after incubate in 30°C(left two), 37°C(middle two), 42°C(right two) for 24 hours respectively.
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<div><img src = "https://static.igem.org/mediawiki/2011/0/03/Natu_cp_11.jpg" width="2
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<img src = "https://static.igem.org/mediawiki/2011/0/01/Natu_cp_12.jpg" width="2
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<img src = "https://static.igem.org/mediawiki/2011/6/6e/Natu_cp_13.jpg" width="2
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<br><b>Figure 5.</b>
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a. A close-up of 30°C incubated E.coli .
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Expressing crtE, crtB and crtI and so to produce Lycopene. <br>
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b. In addition to express crtE,crtB and crtI,37°C cultured E.coli expresses crtY so that beta-Carotene is present. <br>
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c. Furthermore, E.coli that cultured in 42°C expresses crtE, crtB , crtI,crtY and the high-temperature regulated gene , crtZ . Zeaxanthin is prodused.
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<div><img src = "https://static.igem.org/mediawiki/2011/thumb/6/62/Nctu_cp_3.PNG/800px-Nctu_cp_3.PNG" width="700"></div><br>
 
<p>
<p>
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If E.coli is incubating below 37℃, mRNAs cannot be translated by ribosomes, because temperature-sensitive RBS(ribosome biding site) <a href ="http://partsregistry.org/Part:BBa_K115002 ">BBa_K115002</a> forms the hairpin. A ribosome cannot bind to the RBS so that it cannot translate CrtY <a href ="http://partsregistry.org/Part:BBa_K118008">BBa_K118008</a>. As a result, the E.coli only can produce CrtE, CrtB and CrtI <a href ="http://partsregistry.org/Part:BBa_K346090 ">BBa_K346090</a> which convert colorless Farnesyl pyrophosphate to red Lycopene.
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In order to decrease the interruption caused by the color of E.coli itself, we should extract the pigment with organic solvent since the three pigments are hydrophobic.   So Acetone is added and vortexed to form a homogeneous colored solution.
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<div><img src = "http://partsregistry.org/wiki/images/d/d5/RNA_data_2-2.jpghttps://static.igem.org/mediawiki/2011/0/00/Natu_cp_14.jpg" width="5
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<div><img src = "https://static.igem.org/mediawiki/2011/d/d1/Natu_cp_15.jpg" width="5
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<div><img src = "https://static.igem.org/mediawiki/2011/8/89/Natu_cp_16.jpg" width="5
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<br><b>Figure 6.</b>
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Here shows color of 30°C cultured E.coli , and corresponds to the pigment, Lycopene.<br>
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b. Here shows color of 37°C cultured E.coli, and correspond to the pigment, beta-Carotene. <br>
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c. Here shows color of 42°C cultured E.coli, and correspond to the pigment, Zeaxanthin. <br><br>
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<div><img src = "https://static.igem.org/mediawiki/2011/thumb/9/9d/Nctu_cp_4.PNG/800px-Nctu_cp_4.PNG" width="700"></div><br>
 
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When the temperature is at 37℃ or higher, the hairpin is denatured so  temperature-sensitive RBS <a href ="http://partsregistry.org/Part:BBa_K115002 ">BBa_K115002</a> is activated . CrtY <a href ="http://partsregistry.org/Part:BBa_K118008 ">BBa_K118008 </a>is translated, which convert red Lycopene to orange beta-Carotene. </p>
 
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<div><img src = https://static.igem.org/mediawiki/2011/1/1b/Natu_cp_17.jpg" width="250">
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<div><img src = "https://static.igem.org/mediawiki/2011/4/44/Natu_cp_18.jpg" width="250">
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<br><b>Figure 7.</b>
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Photo of the three eppendorfs based on a white background.<br>
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Photo of the three eppendorfs based on a black background. <br>
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<div><img src = "https://static.igem.org/mediawiki/2011/thumb/6/69/Nctu_cp_5.png/800px-Nctu_cp_5.png" width="700"></div><br>
 
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<p>
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In the other circuit we cotransform into. When we increase the temperature to 42℃, the CI proteins <a href ="http://partsregistry.org/Part:BBa_K098997">BBa_K098997</a>, the repressor of cI regulated promoter <a href ="http://partsregistry.org/Part:BBa_">BBa_R0051</a>, are fully denatured. Then the cI regulated promoter is activated and CrtZ <a href ="http://partsregistry.org/Part:BBa_I742158 ">BBa_I742158</a> is produced, converting the orange beta-carotene to yellow Zeaxanthin.
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Our circuits for carotenoid pathway work as expected.
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As we can see above,Lycopene is produced at 30°C , beta-carotene is present at 37°C and Zeaxanthine 
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<a href="https://2011.igem.org/Team:NCTU_Formosa/CSP_data" ><font style="Calibri, Verdana, helvetica, sans-serif" color="white" padding-left="10">NEXT >> Data</font>
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is produced at 42°C. The ruselts of Carotenoid synthesis Pathway display different colors in different steps, that verify our concept of Pathway Commander perfectly.</p>
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�

Revision as of 20:09, 4 October 2011



 Carotenoid Pathway­

Data



Figure 3. The compositions of the Carotenoid pathway circuit. The upper strand is BBa_K539151 and the down strand is BBa_K539281.

The upper strand of the Carotenoid pathway circuit BBa_K539151 is composed of (1) crtEBI BBa_K346090 that can produce Lycopene, (2) 37℃induced RBS BBa_K115002, (3) crtY BBa_K118008 that can produce beta-Carotene, and (4) Terminator BBa_J61048. When we assembled the upper strand, we found a point mutation of BBa_K118008 (iGEM08_Edinburgh) at SpeI site and we corrected it successfully.(ACCTAGT->ACTAGT). This corrected part is numbered as BBa_K539119. The down stand BBa_K539281 is composed of (1) Heat sensitive cI promoter with repressor BBa_K098995, (2) crtZ BBa_I742158 that can produce Zeaxanthin, and (3) Terminator BBa_J61048.

After both of the strands were constructed, we transferred the assembled insert BBa_K539151 from originally backbone(psb1k3) to psb3T5. The reason why we change the backbone is that the psb3T5 with low-copy-numbers is better for E.coli to express proteins. For the same reason, we transferred the complete insert BBa_K539281 from originally backbone (psb1k3) to psb4A5. (We chose different antibiotic backbone from the upper strand in order to cotransform.) Finally, we cotransformed DH5αwith BBa_K539151 on psb3T5 and BBa_K539281 on psb4A5. To test the circuit whether it can work or not, we incubate the modified E.coli in different temperatures and extract the pigments.

  1. 30℃: It is supposed to express only CrtEBI and produce Lycopene. Show red color.
  2. 37℃: It is supposed to express CrtEBI and CrtY. Produce beta-Carotene and show orange color.
  3. 42℃: It is supposed to express CrtEBI, CrtY and CrtZ. Produce Zeaxanthin and show yellow color.
    4. �

    Experiment methods

    1. The cells were cultured in LB over night.
    2. Transferred 200ul LB to fresh 200ml LB and incubated at 37°C.
    3. After 8 hrs, when OD ( optical density) reaches 0.1, then switch the temperature to 30°C, 37°C, 42°C respectively and incubate for 24hrs.
    4. Centrifuged at 4°C, 6000rpm. The pellets were added with 1 ml ddH20 and vortexed.
    5. Moved into 1.5ml eppendorf. Centrifuged for 20minutes at 4°C, 14000rpm.
    6. Added with 500ul acetone, and vortexed for 1hr to extract the pigments.


    7. Figure 4. Here shows the result of our circuit. The colored pellets represent the achievement after incubate in 30°C(left two), 37°C(middle two), 42°C(right two) for 24 hours respectively.



      Figure 5. a. A close-up of 30°C incubated E.coli . Expressing crtE, crtB and crtI and so to produce Lycopene.
      b. In addition to express crtE,crtB and crtI,37°C cultured E.coli expresses crtY so that beta-Carotene is present.
      c. Furthermore, E.coli that cultured in 42°C expresses crtE, crtB , crtI,crtY and the high-temperature regulated gene , crtZ . Zeaxanthin is prodused.

      In order to decrease the interruption caused by the color of E.coli itself, we should extract the pigment with organic solvent since the three pigments are hydrophobic. So Acetone is added and vortexed to form a homogeneous colored solution.



      Figure 6. Here shows color of 30°C cultured E.coli , and corresponds to the pigment, Lycopene.
      b. Here shows color of 37°C cultured E.coli, and correspond to the pigment, beta-Carotene.
      c. Here shows color of 42°C cultured E.coli, and correspond to the pigment, Zeaxanthin.



      Figure 7. Photo of the three eppendorfs based on a white background.
      Photo of the three eppendorfs based on a black background.


      Our circuits for carotenoid pathway work as expected. As we can see above,Lycopene is produced at 30°C , beta-carotene is present at 37°C and Zeaxanthine is produced at 42°C. The ruselts of Carotenoid synthesis Pathway display different colors in different steps, that verify our concept of Pathway Commander perfectly.



      �

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