Difference between revisions of "Part:BBa K2819206"

Revision as of 19:28, 15 October 2018

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 our composite parts 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)

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.

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

Conclusion

The above characterisation results indicate that the FNS gene is being produced, however, further optimization is needed to better understand the gene.

Sequence and Features