Difference between revisions of "Part:BBa K1497007"
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Naringenin is the mayor flavone from grapefruits. In plants, it is synthesized from tyrosine and is one of the central metabolite in the flavone biosynthesis. It is able to reduce the oxdiative stress and inhibit some P450 enzymes. One of these cytochrome P450 enzymes are involved in the degradation of caffeine and increase the effect of caffeine after the inhibition with naringenin.  
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<b>Naringenin</b> is the main flavone from grapefruits. In plants, it is synthesized from tyrosine and is one of the central metabolite in the flavone biosynthesis. It is able to reduce the oxidative stress and inhibit some P450 enzymes. One of these cytochrome P450 enzymes are involved in the degradation of caffeine and increase the effect of caffeine after the inhibition with naringenin. The biosynthesis of naringenin is encoded by four genes and these proteins convert L-tyrosine to the bioactive enantiomer S-naringenin.    
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The biosynthesis of naringenin is encoded by four genes and these proteins convert L-tyrosine to the bioactive enantiomer S-naringenin.
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   <li>Chalchone synthase - CHS          <a href="/Part:BBa_K1497101">BBa_K1497101</a></li>
 
   <li>Chalchone synthase - CHS          <a href="/Part:BBa_K1497101">BBa_K1497101</a></li>
 
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<br>
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Together, these genes build the naringenin biosynthesis operon without a promotor.  In addition of a promotor part the device is able to build S-naringenin. <br><br>
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Together, these genes build the naringenin biosynthesis operon without a promotor.  In addition of a promotor part the device is able to build S-naringenin. These device is working in <i>E. coli</i> K and B strains.<br><br>
 
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       <p class="MsoCaption" align="text-align:justify"><span lang="EN-US"><b>Figure 2</b></span></a><span lang="EN-US">
 
       <p class="MsoCaption" align="text-align:justify"><span lang="EN-US"><b>Figure 2</b></span></a><span lang="EN-US">
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  E. coli Top10 with different Naringenin biosensors. Left: On agar plate without naringenin no colour is visible. Middle: On agar plate with 100 µM naringenin colour is visible, except of negative sample <a href="/Part:BBa_K1497019">BBa_K1497019</a> without fluorphor. Right: On agar plate with 100 µM Naringenin under UV light. The fluorescence of GFP, CFP and mKate is visible. <br></span></p>
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  Genetic map of the naringenin operon with T7 promoter (<a href="/Part:BBa_K1497017">BBa_K1497017</a>). This device build naringenin in <i>E. coli</i> BL21(DE3) in present of the inductor IPTG . <br></span></p>
 
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===Functional Parameters===
 
===Functional Parameters===
 
<partinfo>BBa_K1497007 parameters</partinfo>
 
<partinfo>BBa_K1497007 parameters</partinfo>
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   The iGEM Team TU Darmstadt 2014 create the naringenin biosynthesis under the control of the T7 promotor <a href="/Part:BBa_ I712074">BBa_ I712074</a>and the strong constitutive promotor <a href="/Part:BBa_J23100">BBa_J23100</a>, respectively . They measure the naringenin production after a 16 h incucation time with the naringenin biosensor <a href="/Part:BBa_K1497020">BBa_K1497020</a>
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   The iGEM Team TU Darmstadt 2014 create the naringenin biosynthesis under the control of the T7 promoter <a href="/Part:BBa_ I712074">BBa_I712074</a> and the strong constitutive promoter <a href="/Part:BBa_J23100">BBa_J23100</a>, respectively . They measure the naringenin production after a 16 h incubation time with the naringenin biosensor <a href="/Part:BBa_K1497020">BBa_K1497020</a>. <br><br>The cell pellets from E. coli BL21(DE3) – pSB1C3-fdeR-gfp with and without T7-naringenin operon (<a href="/Part:BBa_K1497017">BBa_K1497017</a>) are in figure 3 shown. Only in the cell pellet with <a href="/Part:BBa_K1497017">BBa_K1497017</a> is a GFP fluorescence visible. <br><br>The Darmstadt Team was also able to measure the GFP fluorescence quantitatively and to calculate with a calibrate curve for the naringenin sensor the production yield of both operons (Figure 4). For <a href="/Part:BBa_K1497017">BBa_K1497017</a> was 3 µM naringenin calculated and for the operon with the constitutive promoter <a href="/Part:BBa_J23100">BBa_J23100</a> (<a href="/Part:BBa_K1497016">BBa_K1497016</a>) was 1.9 µM naringenin calculated.</td>
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<font color="#FFFFFF">iGEM TU Darmstadt 2014 :)</font><br>
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       <p class="MsoCaption" align="text-align:justify"><span lang="EN-US"><b>Figure 2</b></span></a><span lang="EN-US">
 
       <p class="MsoCaption" align="text-align:justify"><span lang="EN-US"><b>Figure 2</b></span></a><span lang="EN-US">
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  E. coli Top10 with different Naringenin biosensors. Left: On agar plate without naringenin no colour is visible. Middle: On agar plate with 100 µM naringenin colour is visible, except of negative sample <a href="/Part:BBa_K1497019">BBa_K1497019</a> without fluorphor. Right: On agar plate with 100 µM Naringenin under UV light. The fluorescence of GFP, CFP and mKate is visible. <br></span></p>
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  Cell pellets with and without T7-Naringenin operon from <i>E. coli</i> BL21(DE3)-pSB1C3-<i>fdeR-gfp</i>. By using ultraviolet light the pellet containing the naringenin operon shows a GFP fluorescence.<br></span></p>
 
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       <p class="MsoCaption" align="text-align:justify"><span lang="EN-US"><b>Figure 1</b></span></a><span lang="EN-US">
 
       <p class="MsoCaption" align="text-align:justify"><span lang="EN-US"><b>Figure 1</b></span></a><span lang="EN-US">
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  E. coli Top10 with different Naringenin biosensors. Left: On agar plate without naringenin no colour is visible. Middle: On agar plate with 100 µM naringenin colour is visible, except of negative sample <a href="/Part:BBa_K1497019">BBa_K1497019</a> without fluorphor. Right: On agar plate with 100 µM Naringenin under UV light. The fluorescence of GFP, CFP and mKate is visible. <br></span></p>
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  Fluorescence of Cells with and without T7-naringenin operon <a href="/Part:BBa_K1497017">BBa_K1497017</a>  from <i>E. coli</i> BL21(DE3)-pSB1C3-<i>fdeR-gfp</i> and J23100-naringenin operon (<a href="/Part:BBa_K1497016">BBa_K1497016</a>) from <i>E. coli</i> Top10-pSB1C3-<i>fdeR-gfp</i>, respectively. In the control <i>E. coli</i> BL21(DE3)-pSB1C3-<i>fdeR-gfp</i> without T7-naringenin operon is no fluorescence detectable only in the cells with the functional operon is the GFP fluorescence measurable. The estimated yields are 3 µM for <a href="/Part:BBa_K1497017">BBa_K1497017</a> and 1,9 µM for <a href="/Part:BBa_K1497016">BBa_K1497016</a>. <br></span></p>
 
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Revision as of 01:35, 12 October 2014

Naringenin producing operon

Naringenin is the main flavone from grapefruits. In plants, it is synthesized from tyrosine and is one of the central metabolite in the flavone biosynthesis. It is able to reduce the oxidative stress and inhibit some P450 enzymes. One of these cytochrome P450 enzymes are involved in the degradation of caffeine and increase the effect of caffeine after the inhibition with naringenin. The biosynthesis of naringenin is encoded by four genes and these proteins convert L-tyrosine to the bioactive enantiomer S-naringenin.

Figure 1 Reaction Scheme of the naringenin producing operon. The Substrate for the reaction is L-tyrosine. In serval steps the substrate is catalyzed to S-naringenin.

Usage and Biology

This part is a composite of four genes each with the strong RBS (BBa_B0034).

  • 4-coumaryl ligase - 4-CL BBa_K1033001
  • Tyrosine ammonia lyase - TAL BBa_K1033000
  • Chalchone isomerase - CHI BBa_K1497100
  • Chalchone synthase - CHS BBa_K1497101

    • Together, these genes build the naringenin biosynthesis operon without a promotor. In addition of a promotor part the device is able to build S-naringenin. These device is working in E. coli K and B strains.

    		 iGEM TU Darmstadt 2014 :)

    Figure 2 Genetic map of the naringenin operon with T7 promoter (BBa_K1497017). This device build naringenin in E. coli BL21(DE3) in present of the inductor IPTG .

    Functional Parameters


    The iGEM Team TU Darmstadt 2014 create the naringenin biosynthesis under the control of the T7 promoter BBa_I712074 and the strong constitutive promoter BBa_J23100, respectively . They measure the naringenin production after a 16 h incubation time with the naringenin biosensor BBa_K1497020.

    The cell pellets from E. coli BL21(DE3) – pSB1C3-fdeR-gfp with and without T7-naringenin operon (BBa_K1497017) are in figure 3 shown. Only in the cell pellet with BBa_K1497017 is a GFP fluorescence visible.

    The Darmstadt Team was also able to measure the GFP fluorescence quantitatively and to calculate with a calibrate curve for the naringenin sensor the production yield of both operons (Figure 4). For BBa_K1497017 was 3 µM naringenin calculated and for the operon with the constitutive promoter BBa_J23100 (BBa_K1497016) was 1.9 µM naringenin calculated.    		 iGEM TU Darmstadt 2014 :)

    Figure 2 Cell pellets with and without T7-Naringenin operon from E. coli BL21(DE3)-pSB1C3-fdeR-gfp. By using ultraviolet light the pellet containing the naringenin operon shows a GFP fluorescence.

    Figure 1 Fluorescence of Cells with and without T7-naringenin operon BBa_K1497017 from E. coli BL21(DE3)-pSB1C3-fdeR-gfp and J23100-naringenin operon (BBa_K1497016) from E. coli Top10-pSB1C3-fdeR-gfp, respectively. In the control E. coli BL21(DE3)-pSB1C3-fdeR-gfp without T7-naringenin operon is no fluorescence detectable only in the cells with the functional operon is the GFP fluorescence measurable. The estimated yields are 3 µM for BBa_K1497017 and 1,9 µM for BBa_K1497016.

    Sequence and Features


    Assembly Compatibility:
    • 10
      COMPATIBLE WITH RFC[10]
    • 12
      INCOMPATIBLE WITH RFC[12]
      Illegal NheI site found at 1104
      Illegal NheI site found at 4631
    • 21
      INCOMPATIBLE WITH RFC[21]
      Illegal BglII site found at 1671
      Illegal BglII site found at 4640
      Illegal XhoI site found at 3638
    • 23
      COMPATIBLE WITH RFC[23]
    • 25
      INCOMPATIBLE WITH RFC[25]
      Illegal NgoMIV site found at 1777
      Illegal NgoMIV site found at 2609
      Illegal NgoMIV site found at 4611
      Illegal NgoMIV site found at 5187
      Illegal AgeI site found at 1872
      Illegal AgeI site found at 2038
    • 1000
      INCOMPATIBLE WITH RFC[1000]
      Illegal BsaI site found at 3067
      Illegal BsaI site found at 4560
      Illegal BsaI.rc site found at 1303
      Illegal BsaI.rc site found at 4042