Part:BBa_K2819206

Arabinose Inducible Production of Flavone Synthase (FNS)

This part contains the coding sequence of Flavone Synthase (FNS), inducible by arabinose.
This gene comes from Petroselinum crispum, and the sequence was obtained after codon optimization.

Usage and Biology

- This engineered part can be directly used to produce Apigenin, a yellow flavonoid.
- We recommend BL21* (DE3) as a chassis for this composite part. This is because BL21* (DE3) is an RNase knockout strain, therefore the half-life of mRNA is prolonged in this strain, which can help in the biomanufacturing of the E. coli.

Characterization

Real-time Polymerase Chain Reaction (qPCR)
[http://2018.igem.org/Team:NTU-Singapore NTU-Singapore] has kindly carried out qPCR on this composite part for us as part of our collaboration. BL21 (DE3) carrying this plasmid was induced according to our protocol.

Exponential Fold Change was calculated according to the following formula:
ΔCt1= Ct(wild type) - Ct (16S gene)
ΔCt1 = Ct(cell with genes) - Ct (16S gene)
ΔΔCt = ΔCt2 - ΔCt1
Exponential fold of gene = 2^-(ΔΔCt)

Figure 1: Exponential Fold Change of FNS gene

As seen from figure 1, the mRNA of FNS is transcribed, this suggests that our part is working as intended. However, mRNA seems to be transcribed even without arabinose, indicating that PBAD is leaky. However, the expression level of FNS without arabinose seems to be higher or equal to than when 10mM and 20mM of arabinose are present. This suggests that further optimization is required for this part.

Biosynthesis of Luteolin
FNS is involved in the luteolin-synthesis pathway. We use co-transformed this part with BBa_K2819200 into BL21* (DE3) for the biosynthesis of luteolin. We not only carried out the biosynthesis in flasks, but also in a bioreactor.

Figure 2: Luteolin in the supernatant of the cell cultures from biosynthesis using a bioreactor

High Performance Liquid Chromatography (HPLC)
To confirm that we have produced luteolin, we carried out HPLC, and the following shows the results.

Figure 3: Comparison of Luteolin concentration in BL21* (DE3) wild type and BL21* (DE3) with our parts

Conclusion

As both FNS and F3'H are needed in the biosynthesis of luteolin, these characterisation results indicate that the F3'H gene is being produced, however, further optimization is needed to better understand the gene.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 1205
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 1144
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 979
  Illegal AgeI site found at 1983
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal SapI site found at 961