Part:BBa_K5071018
pACYCDuet-BGCII-gene143
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal EcoRI site found at 112
Illegal XbaI site found at 2593
Illegal PstI site found at 131 - 12INCOMPATIBLE WITH RFC[12]Illegal EcoRI site found at 112
Illegal NheI site found at 1752
Illegal PstI site found at 131
Illegal NotI site found at 149 - 21INCOMPATIBLE WITH RFC[21]Illegal EcoRI site found at 112
Illegal BglII site found at 305
Illegal BglII site found at 4209
Illegal BamHI site found at 106
Illegal XhoI site found at 354 - 23INCOMPATIBLE WITH RFC[23]Illegal EcoRI site found at 112
Illegal XbaI site found at 2593
Illegal PstI site found at 131 - 25INCOMPATIBLE WITH RFC[25]Illegal EcoRI site found at 112
Illegal XbaI site found at 2593
Illegal PstI site found at 131
Illegal NgoMIV site found at 324
Illegal NgoMIV site found at 4478
Illegal AgeI site found at 566
Illegal AgeI site found at 1838
Illegal AgeI site found at 2161
Illegal AgeI site found at 4100 - 1000COMPATIBLE WITH RFC[1000]
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Composite Part BBa_K5071017 (pRSFDuet-BGCI-gene456)
Construction Design
First, we obtained 4 target fragments using PCR technology. In order to improve the success rate of plasmid construction, we connected the 4 target fragments pairwise (by Overlap PCR), resulting in two fragments. Subsequently, we constructed a new plasmid by ligating the fragments with a vector using enzymatic digestion and ligation.
Engineering Principle
Firstly, the target fragments were obtained using PCR technology and the vector was linearized. Then, the four target fragments were overlapped pairwise to form two fragments. Subsequently, they were connected to the backbone using enzyme digestion and ligation methods to construct an expression plasmid containing three target genes, used to validate the impact of the relevant genes on the products.
Experimental Approach
Firstly, we utilized PCR technology to obtain three target genes, BGCII-1, BGCII-4, BGCII-3 (synthesized by a biotech company), with band lengths of 180 bp, 150 bp, and 1200 bp, respectively, for connection to the plasmid. Subsequently, we performed PCR to amplify the terminator of the first reading frame and the promoter of the second reading frame along with the intervening sequence in plasmid pETD (named as pACY), resulting in a 160 bp band. Figure 2 (Red marking) demonstrates bands of the expected sizes, confirming the successful acquisition of these four fragments. Gel electrophoresis was then conducted for gel extraction, which will be used in subsequent experiments.
Subsequently, we used overlap PCR technology to connect fragment BGC-4 with BGC-5, and pACY with BGC-6, resulting in band lengths of 2000 bp and 1000 bp, respectively. Figure 3A (Red marking) displays bands of the expected sizes, confirming successful connection. Following this, we performed double enzyme digestion on the plasmid using BamH1 and Xho1 restriction enzymes to linearize the plasmid, resulting in a band length of 3766 bp. Figure 3B (Red marking) shows bands of the expected size, confirming successful linearization. We recovered the gel from both of these steps of gel electrophoresis and performed the connection, followed by transformation into E. coli DH5α.
We selected multiple colonies for PCR verification, and the bands matched the expected length (1800 bp). We sent the validated bacterial strains to a biotech company for sequencing (Figure 4), selected plasmids without mutations, and successfully obtained the constructed plasmid pRSFDuet-BGCII-gene456.
Characterization/Measurement
1: Transformation of E. coli BL21-Strain-BGCII
In our target genes, the 9 genes of BGCII represent metabolic pathway 1, which are the 9 genes contained in plasmids pACYCDuet-BGCII-gene143, pETDuet-BGCII-gene792, and pRSFDuet-BGCII-gene685. We simultaneously transformed these three plasmids into E. coli BL21 for the production of terpenoid compounds. The experimental results, as shown in Figure 5, depict the transformed E. coli BL21. We conducted single colony verification to confirm the presence of both plasmids, as illustrated in Figure 17. We obtained bacterial strains that correctly harbored both transformed plasmids, which we named as BGCII.
2: Protein expression-BGCII
The treatment method for strain BGCII was consistent with BGCI, and the experimental results, as shown in Figure 6, depicted the proteins expressing our target genes (BGCII-1 is 4.4kDa, BGCII-4 is 3.9kDA, BGCII-3 is 42.9kDa, BGCII-7 is 35.8kDa, BGCII-9 is 16.5kDA, BGCII-2 is 72.9kDa, BGCII-6 is 17.4kDa, BGCII-8 is 11.2kDA, BGCII-5 is 52.8kDa).
3: The test results for Total Antioxidant Capacity (T-AOC)
Various antioxidants and antioxidant enzymes in the fermentation broth contribute to the total antioxidant level. We used a Total Antioxidant Capacity assay kit (colorimetric method) for detection. The main principle is that DPPH is a stable free radical with maximum absorption at 515nm. Upon addition of antioxidants to the DPPH solution, a decolorization reaction occurs. Therefore, the change in absorbance can be quantified using Trolox as a control system to measure the antioxidant capacity of antioxidants. We first subjected the fermentation broth after 48 hours of fermentation to ultrasonic disruption: power 200W, ultrasound 3s, interval 10s, repeated 30 times, centrifuged at 10000rpm for 10 minutes at 4℃, followed by detection. The experimental results, as shown in Figure 7 and Table 1, revealed a significant increase in the DPPH scavenging rate for our genetically modified strains, from 4.58% to 40.80% and 49.45%, respectively. This demonstrates the success of our modification.
Table 1: DPPH scavenging rates of the genetically modified strains
| Strain | Absorbance | STD | DPPH Free Radical Clearance (%) |
|---|---|---|---|
| Control | 0.146 | 0.0191 | 4.58 |
| BGCII | 0.076 | 0.0088 | 49.45 |
4: The test of the fermentation product antibacterial experiment
For the antibacterial activity testing of the fermentation broth, we utilized the double-layer agar plate method, with the bottom layer containing 1.5% LB solid medium and the top layer containing 0.8% LB solid medium poured after the bottom layer had cooled. Once the top layer reached an appropriate temperature, it was mixed with the cultured K-12 strain and poured into petri dishes. As shown in Figure 8, 4µL of the respective liquid was pipetted into each position. Each column represents three parallels of the same experimental group: 1. Positive control with ciprofloxacin concentration of 1g/L; 2. Positive control with ciprofloxacin concentration of 0.5g/L; 3. Concentrated 5-fold lysate supernatant after cell disruption; 4. Original lysate supernatant after cell disruption; 5. Squalene at 200mg/L. Our experimental results indicate that the concentrated 5-fold fermentation broth of strain BGCI exhibits some antibacterial effects, but we cannot determine the identity of this substance.
5: Determination of squalene in the fermentation broth by HPLC
To determine if our target terpenoid compound is squalene, we conducted testing on the fermentation broth of the bacterial strains. The detection method involved the following steps: Fermentation was carried out using a biphasic fermentation method, with 10% volume of normal heptane added on top of the LBG medium. After fermentation, 1 mL of the 24-hour whole-cell catalytic liquid was taken, centrifuged at 13,000×g for 10 minutes, and the supernatant was discarded. Then, 400 μL of saline solution was added to wash the fermentation cells, centrifuged at 13,000×g for 10 minutes, and the supernatant was discarded. Next, ddH2O was added, thoroughly mixed, and brought to a volume of 400 μL. The cells were disrupted by ultrasonication at a working power of 20%, for 2 minutes with 3-second on and 5-second off cycles. Subsequently, 600 μL of ethyl acetate was added, mixed well, and subjected to ultrasonic cleaning twice for 15 minutes each. The mixture was then centrifuged, and 400 μL of the extract phase was obtained. The extract was concentrated using a vacuum centrifuge to evaporate the solvent, then re-dissolved in 200 μL of methanol, filtered through a 0.22 μm filter membrane, and ready for analysis.
Squalene yield detection was performed using high-performance liquid chromatography (HPLC) under the following conditions: Column: Waters XBridgeTM C18 (3.5 μm 4.6 mm×150 mm); Column temperature: 35 ℃; Mobile phase: 100% pure acetonitrile; Flow rate: 1 mL/min; Detector: Photodiode array detector at 196 nm wavelength. The experimental results, as shown in Figure 9, indicated that after our modification, our target gene was extracted from a deep-sea metagenome terpenoid biosynthetic gene cluster. However, we could not determine the specific terpenoid compound produced through this pathway from the genetic information. Hence, we presumed it to be squalene. Yet, upon comparing the peak retention time of the fermentation broth with squalene standard, we found that squalene was not produced in our bacterial strains.
6: Determination of squalene in the fermentation broth by LC-MS
The extraction method for squalene involves taking 50 mg of freeze-dried bacterial cells in a grinding tube, adding 2 grinding beads and 500 µL of methanol to each tube, and grinding in a grinder for 4 minutes. After removal, 1 mL of chloroform is added to each tube, and they are extracted in a constant-temperature shaker at 30°C and 200 rpm for 12 hours. The supernatant is collected after centrifugation at 12000 rpm for 10 minutes, dried using a nitrogen evaporator, re-dissolved in 1 mL of n-hexane, vortexed for 5 minutes, centrifuged at 12000 rpm for 10 minutes, and the supernatant is collected and filtered through a 0.22 µm organic membrane, then placed in brown gas chromatography vials.
The determination method for squalene uses gas chromatography to detect squalene with the following gas phase conditions: the chromatographic column is an Rtx-5 capillary column (30 m × 0.32 mm × 0.25 µm); the injector temperature is set at 300°C; the detector temperature is set at 330°C; the carrier gas is nitrogen at a flow rate of 2 mL/min; the injection volume is 1 µL with a split ratio of 10:1; the detector used is a Flame Ionization Detector (FID); the column initial temperature is set at 200°C, maintained for 1 minute, then increased at a rate of 20°C/min to 280°C and maintained for 5 minutes.
As shown in Figure 10 and Table 2, the squalene content was measured in the bacterial strain through LC-MS, with a yield of 6.60 mg/L. Due to the higher detection accuracy of LC-MS compared to HPLC, it is more precise and suitable for testing substances at low concentrations.
Table 2: Detection results of squalene in the fermentation broth of the bacterial strains
| Strain | Squalene concentration (mg/L) | STD |
|---|---|---|
| BGCI | 6.60 | 0.5056 |
| BGCII | 0.00 | 0.0000 |
| None |