Part:BBa_K3279002
Phytoene desaturase (CrtI) from Rhodospirillum rubrum
This part is a phytoene desaturase which transforms phytoene into lycopene. It is a crucial enzyme in lycopene synthesis pathway and always works together with geranylgeranyl diphosphate synthase (CrtE) and phytoene synthase (CrtB). This sequence is optimized for use in E.coli.
Sequence and Features
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 779
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 766
Illegal NgoMIV site found at 1450 - 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI.rc site found at 325
Illegal BsaI.rc site found at 820
Illegal SapI.rc site found at 232
Usage and Biology
The CrtI gene was amplified from Rhodospirillum rubrum'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).
Then, the constructed plasmid was transformed into E.coli BL21 (DE3) and induced under gradient IPTG concentration. A SDS-PAGE assay was carried out to detect the expression of the CrtI protein (see Figure 2). The result showed that CrtI could be induced at a minimum IPTG concentration of 0.02 mM, but the amount of protein expressed was small at this inducer concentration. When the IPTG concentration increased to 0.04 mM, the expression amount of CrtI increased significantly. And the expression amount remained stable when the IPTG concentration get higher.
Since the substrate of CrtI were too expensive to afford, 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.
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).
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.
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