Difference between revisions of "Part:BBa K3279001"

Latest revision as of 09:04, 21 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.

Sequence and Features

Sequence and Features

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.

Fig. 3 CrtB, together with the other two genes (CrtE and CrtI), was cloned into plasmid pACYC184-M. (a) The constructed plasmid map. (b) 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.

In order to further confirm that our engineering bacteria produced lycopene, we transferred the constructed plasmid (184M-EBI) into E. coli BL21 and induced it with IPTG, using E. coli BL21 with pACYC184-M empty plasmid as control. After induced by 0.1 mM IPTG for 10 hours, we could see that E. coli cells with the constructed plasmid (184M-EBI) turned red significantly, while those with empty vectors remained white (see Figure 4a). Then we extracted lycopene with acetone and measured its absorbance at its maximum absorption peak at 473 nm and the yield of lycopene was calibrated using a standard curve (see Figure 4b and Figure 4c).

Fig. 4 Induce the engineered E.coli cells to produce lycopene. (a) E. coli cells with the constructed plasmid (184M-EBI) turned red significantly; (b) Lycopene production of engineered E.coli cells after being induced by 0.1 mM IPTG for 10 hours; (c) Standard curve of lycopene concentration.

We also detected the yield of lycopene in engineered bacteria under different IPTG concentration conditions, and the change of lycopene production with time after IPTG induction (see Figure 5a and Figure 5b). We found that lycopene production peaked at an IPTG concentration of 0.3 mM. But to our surprise, leaky expression of the three lycopene synthesis genes was observed when there is no inducer. This suggested that the switch that controled the synthesis of lycopene may not completely shut down the expression of all the three genes. Figure 5b was measured under 0.1 mM IPTG concentration and 30℃, lycopene production increased in the first 700 minutes and remained stable in the following period.

Fig. 5 The yield of lycopene in engineered bacteria under different IPTG concentration conditions. (a) The changes of lycopene production with IPTG concentration; (b) The changes of lycopene production with induction time.