Difference between revisions of "Part:BBa K3279001"

Revision as of 05:19, 19 October 2019

Phytoene Synthase (CrtB) from Rhodobacter sphaeroides

This part is a phytoene synthase which transforms geranylgeranyl diphosphate into 15-cis-phytoene. It is a crucial enzyme in lycopene synthesis pathway and always works together with geranylgeranyl diphosphate synthase (CrtE) and phytoene desaturase (CrtI). This sequence is optimized for use in E.coli.

Usage and Biology

Firstly, the CrtB gene was amplified from Rhodobacter sphaeroides's genomic sequence by PCR (see Figure 1a) and was cloned into pET-30a(+) plasmid. Then, colony PCR was run to check the success of ligation (see Figure 1b).

Fig. 1 pET-CrtB plasmid construction. (a) Amplified CrtB from Rhodobacter sphaeroides's genomic sequence; (b) Colony PCR verified that CrtB was successfully assembled into the pET-30a (+) vector.

In order to determine the optimal induction condition of CrtB, we transferred the constructed plasmid pET-30a-CrtB into E. coli BL21 (DE3) and induced it under gradient IPTG concentration and different temperature conditions (see Figure 2). We found that CrtB could be induced at both 25℃ and 30℃, and the minimum IPTG concentration to induced CrtB was 0.02 mM.

Fig. 2 SDS-PAGE assay for CrtB protein. (a) Induce CrtB at 25℃; (b) Induce CrtB at 30℃.

Since the product and substrate of CrtB are colorless and have no special color reaction, measuring the enzymatic activity of CrtB requires isotopic labeling. Our team members do not yet have an isotope operation license, so we constructed the CrtB, CrtE (original squence, not the part BBa_K3279000) and CrtI (BBa_K3279002) genes into the same plasmid (pACYC184-M) to see if they can produce lycopene. There are no other genes in E. coli that can replace the functions of these three genes to produce lycopene. So if the engineered E.coli could produce lycopene, it means all of the three genes worked. Figure 3 shows our construction results: the colonies (2, 3, 4, 8) with the successfully constructed plasmid turned red significantly, while those colonies (1, 5, 6, 7) with misconnected plasmid remained white. Then we extracted the plasmid of strain 8 and sequenced it. The sequencing results confirmed that the plasmid we constructed is correct.

Sequence and Features