Part:BBa_K2819000

Coding Region for F3'H

This part is the coding sequence of flavonoid 3′-hydroxylase (F3′H). This gene comes from Gerbera hybrida, and the sequence was obtained after codon optimization.

Usage and Biology

- This engineered part can be used to produce Eriodictyol, a flavonoid.
- To achieve optimal expression of this coding region, our modelling results suggested to use a strong promoter as its K_cat value is low.
- In addition, 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

To characterize this part, we expressed it under an EL222 blue light repressible promoter, BBa_K2332019. To obtain the sequence of this composite part, refer to BBa_K2819200.

Real-time Polymerase Chain Reaction (qPCR)
[http://2018.igem.org/Team:NTU-Singapore NTU-Singapore] has kindly carried out qPCR on BBa_K2819200, which contains this coding region, 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 F3'H gene

Figure 1 shows that the mRNA of F3'H is transcribed, suggesting that our part is working as intended. Induction was carried out in dark, hence, P_BLrep would behave like a constitutive promoter. However, exponential fold change of the F3’H gene seemed to increase in higher aTc concentration, when it should not be affected by the inducer concentration. This suggests that further optimization is required for this part.

Biosynthesis of Luteolin
F3’H is involved in the luteolin-synthesis pathway. We use co-transformed this part with BBa_K2819206 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 F3'H and FNS 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
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 713
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]