To generate an alternative source of EPA through synthetic biology, we cloned the DHA-producing pfa genes, pfa A, pfa C and pfa D from the deep-sea bacteria Moritella marina. The biobricks of pfa B (Moritella marina), pfa B' (Shewanella pneumatophori) and pfa E (Moritella marina) were ordered from IDT. The pfa A and pfa D genes were cloned into pColdI vector under the control of CspA promoter, while the pfa B, pfa C and pfa E genes are cloned into pSTV28 vector under the control of lac operon.
To enhance the yield of EPA, we ordered the biobricks for AccBC, AccD1 and AccE genes (Corynebacterium glutamicum) from IDT, and cloned these genes into pET28a vector.
These three vectors will be co-transformed into E. coli and the EPA production will be examined by High Performance Liquid Chromatography (HPLC).
To clone the pfa genes into the selected vectors, we PCR-amplified the pfa A1 (the first 4.8 kb region of pfa A), pfa A2 (the last 4 kb region of pfa A), pfa C and pfa D from the genome of Moritella marina using pfu DNA polymerase.
The pfa B (Moritella marina), pfa B' (Shewanella pneumatophori) and pfa E are amplified from ordered biobricks by PCR using pfu DNA polymerase.
The amplicons of pfa genes were cloned into TA vector for sequencing confirmation. The sequencing results of TA-pfa D and TA-pfa E are shown.
The AccBC biobrick and AccD1E biobrick (containing the AccD1 and AccE genes) from IDT were amplified by PCR using pfu DNA polymerase.
The amplicons of Acc biobricks were cloned into TA vector for sequencing confirmation. The sequencing results of TA-AccBC and TA-AccD1E are shown.
We digested the pColdI, pSTV28 and pET28a vectors.
The sequence confirmed pfa D was digested from TA-pfa D and ligated into pColdI vector with selected restriction enzymes. The pColdI-pfa D was subjected to sequencing and the result is shown below.
To induce pfa D protein expression, we cultured the E. coli BL21 strain with pColdI-pfa D vectors in Terrific Broth with ampicillin and 1 mM IPTG at 16 °C. The bacteria were harvested at 9, 11 and 13 hr and subjected to PAGE analysis and Coomassie blue staining. The predicted size of pfa D protein is around 60 kDa, but we observed clear protein induction with size around 45 kDa. We have confirmed that there is no premature stop codon in pColdI-pfa D. We will perform experiments to confirm whether there is unexpected protease activity or we may optimize the codon near the suspected premature ending site.
The sequence confirmed pfa E was digested from TA-pfa E and ligated into pSTV28 vector with selected restriction enzymes. The pSTV28-pfa E was subjected to sequencing and the result is shown below.
To induce pfa E protein expression, we cultured the E. coli BL21 strain with pSTV28-pfaE vectors in Terrific Broth with chloramphenicol and 5 mM IPTG at 37 °C. The bacteria were harvested at 8, 18 and 24 hr and subjected to PAGE analysis and Coomassie blue staining. The predicted size of the pfa E protein is around 32 kDa. The staining result showed the induction of pfa E proteins.
The sequence confirmed AccD1E was digested from TA-AccD1E and ligated into pET28a vector with selected restriction enzymes. The pET28a-AccD1E was subjected into sequencing and the result is shown below.
To induce ACCD1 protein expression, we cultured the E. coli BL21 strain with pET28a-AccD1E vectors in Terrific Broth with kanamycin and 0.4 mM IPTG at 37 °C. The bacteria were harvested at 2, 3 and 4 hr and subjected to PAGE analysis and Coomassie blue staining. The predicted sizes of ACCD1 proteins are around 60 kDa. The staining results clearly showed the induction of ACCD1 protein.
To confirm that pColdI (Ampicillin resistant) and pSTV28 (Chloramphenicol resistant) plasmids can be co-expressed in bacteria to establish the pfa megasynthase expression system, we first performed a double antibiotics selection for pColdI and pSTV28 vectors.
We start by fixing the concentration of ampicillin at 50 μg/ml, which is generally applied to select plasmids with Amp-resistance. The transformed copy number of the plasmid was fixed at 109. A range of chloramphenicol concentrations from 5 to 20 μg/ml were selected according to reports using pSTV28 vector as expression vector.
The selection results showed an apposite number of colonies at 10 μg/ml of chloramphenicol. Therefore, we selected the Amp (50 μg/ml) and Cm (10 μg/ml) for the double antibiotics selection.
| Copy number of each plasmid: 109 ; Ampicillin: 50 μg/ml | |||||
| Chloramphenicol | Colony number | Mean | SD | ||
| 5 μg/ml | 232* | 1240 | 1244 | 1242 | 2.82 |
| 10 μg/ml | 255 | 360 | 542 | 385.67 | 145.21 |
| 20 μg/ml | 206 | 437 | 937* | 321.5 | 163.34 |
* outliers excluded from the average
To further confirm that both kinds of plasmids are co-expressed in the growing colonies, we randomly selected some of the colonies and purified the plasmids from them. The purified plasmids were digested with restriction enzyme SapI. The gel electrophoresis results showed the sizes of the digested fragments of pColdI (5.7 kb) and pSTV28 (4.2, 0.9 kb) as expected.
To confirm that the pfa-expressing vectors (pColdI and pSTV28) can be co-expressed with pET28a (kanamycin resistant) vector, we performed triple antibiotics selection.
Based on the result of double antibiotics selection, we started the triple antibiotics selection by fixing the concentrations of Amp (50 μg/ml) and Cm (10 μg/ml). However, these conditions were too harsh when adding kanamycin as the third antibiotic, and only a few colonies survived. We then decreased the concentration of chloramphenicol to 5 μg/ml, and performed triple antibiotics selection with 1.25 μg/ml, 2.5 μg/ml and 5 μg/ml kanamycin.
The result showed an apposite number of colonies at 5 μg/ml of kanamycin. Therefore, we selected Amp (50 μg/ml), Cm (5 μg/ml) and Kan (5 μg/ml) for the triple antibiotics selection.
| Copy number: 109 ; Ampicillin: 50 μg/ml, Chloramphenicol: 5 μg/ml | |||||
| kanamycin | Colony number | Mean | SD | ||
| 1.25 μg/ml | > 1000 | > 1000 | > 1000 | X | X |
| 2.5 μg/ml | 134 | 252 | 534 | 193 | 83.43 |
| 5 μg/ml | 79 | 138 | 179 | 132 | 50.26 |
To further confirm that all three kinds of plasmids are co-expressed in the grown colonies, we randomly selected some of the colonies and purified the plasmids from these colonies. The purified plasmids were digested with restriction enzymes XbaI and BamHI. The gel electrophoresis result showed the sizes of the digested fragments of pColdI (4.4, 1.2 kb), pSTV28 (3.2, 1.4, 0.5 kb) and pET28a (5.6 kb), as expected. The five colonies have been conducted plasmid extraction and digestion, and the results are shown in below.
Through the double and triple antibiotics selection, we set up conditions which allow pColdI and pSTV28, or pColdI, pSTV28 and pET28a vectors to co-exist in E. coli. Therefore, we can co-transform our engineered plasmids into E. coli and co-express the pfa megasynthase with ACC enzyme to produce EPA.