Part:BBa_K1497007

Naringenin producing operon

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. 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.

iGEM TU Darmstadt 2014 :)

Figure 2 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 BBa_K1497019 without fluorphor. Right: On agar plate with 100 µM Naringenin under UV light. The fluorescence of GFP, CFP and mKate is visible.

Functional Parameters

The iGEM Team TU Darmstadt 2014 create the naringenin biosynthesis under the control of the T7 promotor BBa_ I712074and the strong constitutive promotor BBa_J23100, respectively . They measure the naringenin production after a 16 h incucation time with the naringenin biosensor BBa_K1497020

Figure 2 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 BBa_K1497019 without fluorphor. Right: On agar plate with 100 µM Naringenin under UV light. The fluorescence of GFP, CFP and mKate is visible.

Figure 1 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 BBa_K1497019 without fluorphor. Right: On agar plate with 100 µM Naringenin under UV light. The fluorescence of GFP, CFP and mKate is visible.

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