Difference between revisions of "Part:BBa K1497017"

 
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<partinfo>BBa_K1497017 short</partinfo>
 
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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 metabolites 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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      <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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Reaction Scheme of the naringenin producing operon. The substrate for the reaction is L-tyrosine. The substrate is metabolized to S-naringenin in serval steps.  <br></span></p>
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===Usage and Biology===
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<td style="padding: 0cm 5.4pt; width: 336.7pt; height: 214.9pt;"> This part is a composite of four genes each with the strong RBS (<a href="/Part:BBa_B0034">BBa_B0034</a>).
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  <li>4-coumaryl ligase - 4-CL         <a href="/Part:BBa_K1033001">BBa_K1033001</a></li>
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  <li>Tyrosine ammonia lyase - TAL  <a href="/Part:BBa_K1033000">BBa_K1033000</a></li>
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  <li>Chalchone isomerase - CHI            <a href="/Part:BBa_K1497100">BBa_K1497100</a></li>
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  <li>Chalchone synthase - CHS          <a href="/Part:BBa_K1497101">BBa_K1497101</a></li>
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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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<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">
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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===
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<partinfo>BBa_K1497007 parameters</partinfo>
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  The iGEM Team TU Darmstadt 2014 created the naringenin biosynthesis operons 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 measured 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 shown in figure 3. Only in the cell pellet with <a href="/Part:BBa_K1497017">BBa_K1497017</a> exhibited GFP fluorescence. <br><br>The Darmstadt team was also able to measure the GFP fluorescence quantitatively and to calculate with the help of a calibration 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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      <img style="width: 350px; height: 38,4px;" alt=""
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src="https://static.igem.org/mediawiki/parts/e/e6/Naringeninoperont7coli.png"></p>
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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">
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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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Transformed into BL21(DE3) and induced with IPTG this composite is able to produce naringenin from tyrosine.
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It consists of 4 genes:
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4-CL (BBa_K801093)
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TAL (BBa_K1033000)
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CHI (BBa_K1497000)
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CHS (BBa_K1497001)
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For a strong and stable translation in front of each gene the RBS Bba_B0034 is implemented.  
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src="https://static.igem.org/mediawiki/parts/b/b5/NaringenBALK.png"></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">
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Fluorescence of cells with and without the 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. <i>E. coli</i> BL21(DE3)-pSB1C3-<i>fdeR-gfp</i> without T7-naringenin operon showed no detectable fluorescence. Only in the cells with the functional operon is the GFP fluorescence measurable. The estimated yields are 3 µM this part <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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For characterization the naringenin biosensor K1497020 was used. After 24 h incubation GFP fluorescence was visible to the unaided eye. We measured the relative GFP fluorescence by use of OptimaFluoroStar.
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Revision as of 21:19, 14 October 2014

Naringenin producing operon under the T7 promoter BBa_I712074

Naringenin is the main flavone from grapefruits. In plants, it is synthesized from tyrosine and is one of the central metabolites 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. The substrate is metabolized to S-naringenin in serval steps.

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 created the naringenin biosynthesis operons under the control of the T7 promoter BBa_I712074 and the strong constitutive promoter BBa_J23100, respectively . They measured 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 shown in figure 3. Only in the cell pellet with BBa_K1497017 exhibited GFP fluorescence.

    The Darmstadt team was also able to measure the GFP fluorescence quantitatively and to calculate with the help of a calibration 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 the 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. E. coli BL21(DE3)-pSB1C3-fdeR-gfp without T7-naringenin operon showed no detectable fluorescence. Only in the cells with the functional operon is the GFP fluorescence measurable. The estimated yields are 3 µM this part (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 1158
      Illegal NheI site found at 4685
    • 21
      INCOMPATIBLE WITH RFC[21]
      Illegal BglII site found at 1725
      Illegal BglII site found at 4694
      Illegal XhoI site found at 3692
    • 23
      COMPATIBLE WITH RFC[23]
    • 25
      INCOMPATIBLE WITH RFC[25]
      Illegal NgoMIV site found at 1831
      Illegal NgoMIV site found at 2663
      Illegal NgoMIV site found at 4665
      Illegal NgoMIV site found at 5241
      Illegal AgeI site found at 1926
      Illegal AgeI site found at 2092
    • 1000
      INCOMPATIBLE WITH RFC[1000]
      Illegal BsaI site found at 3121
      Illegal BsaI site found at 4614
      Illegal BsaI.rc site found at 1357
      Illegal BsaI.rc site found at 4096