iGEM 2024 — Team subcat-china

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NOTEBOOK
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ENGINEERING
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[Overview](#nav1)
[Reference](#nav2)

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Overview

This project will integrate metagenomic samples from deep-sea ecosystems for data analysis, construct a set of deep-sea microbial genomes and a set of biosynthetic gene clusters, systematically analyze and sort out the genetic resources of secondary metabolites, and directionally explore new terpene secondary metabolites with high value biological activity and their biosynthetic gene clusters. Two terpene BGCs were obtained by optimization and direct cloning. Using a high-throughput biosynthesis system, we achieved the synthesis and expression of two terpene secondary metabolites and attempted to analyze the product structure. Specifically, we established two metabolic pathways in E.coli BL21, and produced new terpene secondary metabolites by constructing recombinant plasmids, transforming BL21, and fermentation.

Our team conducted metagenomic sequencing of deep-sea ecosystem samples obtained from 800 sampling sites around the world, and integrated deep-sea metagenomic samples in the public database to obtain a total of 2,138 deep-sea metagenomic sequencing data. MEGAHIT software was used to assemble the metagenomic data after quality control, and metaWRAP software was used to sort the microorganisms. The average nucleotide similarity ( gANI ) of the gene composition of the strains was calculated by fastANI, and the strains were divided by 95 % similarity. Using GTDB-tk to assign an objective phylogenetic position to each genome, a genome set of deep-sea metagenomic data can be obtained. Genome set based on deep-sea metagenome.AntiSMASH was used to identify and analyze the biosynthetic gene clusters and extract the Pfam domain features. Combined with the sampling geographical location and genomic phylogenetic status, the distribution of terpene biosynthetic gene clusters derived from deep-sea microorganisms was analyzed. The terpene biosynthetic gene cluster sequence and core gene protein sequence were extracted, and the sequence alignment was performed by DIAMOND software with the non-redundant protein sequence database in NCBI. The gene cluster with a similarity of less than 50 % of the core gene protein sequence was screened. Then, the gene cluster similarity was compared with the antiSMASH database and the MIBiG standard database, and the gene cluster with a similarity of less than 30 % was further screened as a potential new terpene biosynthetic gene cluster.Finally, BGCI and BGCII were selected, and then we carried out the wet experiment.

Part 1. pETDuet-BGCI-gene123, BBa\_K5071016

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.

![](https://static.igem.wiki/teams/5071/eng122/1.png)

Figure 1. The plasmid map of pETDuet-BGCI-gene123

Build

Firstly, we utilized PCR technology to obtain three target genes, BGCI-1, BGCI-2, BGCI-3 (synthesized by a biotech company), with band lengths of 150 bp, 1200 bp, and 500 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 123-mid), resulting in a 200 bp band. Figure 2 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.

![](https://static.igem.wiki/teams/5071/eng122/2.png)

Figure 2. The purpose segment of plasmid pETDuet-BGC1-gene123

Subsequently, we used overlap PCR technology to connect fragment BGCI-1 with BGCI-2, and 123-mid with BGCI-3, resulting in band lengths of 1300bp and 700bp, respectively. Figure 3A 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 4000bp. Figure 3B 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.

![](https://static.igem.wiki/teams/5071/eng122/3.png)

Figure 3. The results of the overlap connection and plasmid linearization

We selected multiple colonies for PCR verification, and the bands matched the expected length (1200 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 pETDuet-BGC1-gene123.

![](https://static.igem.wiki/teams/5071/eng122/4.png)

Figure 4. Single clone verification of pETDuet-BGC1-gene123 transformed E. coli DH5α. A. The results of colony PCR; B: The clones

Part 2. pRSFDuet-BGCI-gene456, BBa\_K5071017

Design

The design concept is consistent with the above description.

![](https://static.igem.wiki/teams/5071/eng122/5.png)

Fig. 5. The plasmid map of pRSFDuet-BGCI-gene456

Build

Firstly, we utilized PCR technology to obtain three target genes, BGCI-4, BGCI-5, BGCI-6 (synthesized by a biotech company), with band lengths of 1300 bp, 800 bp, and 800 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 456-mid), resulting in a 150 bp band. Figure 6 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.

The design concept is consistent with the above description.

![](https://static.igem.wiki/teams/5071/eng122/6.png)

Figure 6. The purpose segment of plasmid pRSFuet-BGC1-gene456

Subsequently, we used overlap PCR technology to connect fragment BGCI-4 with BGCI-5, and 456-mid with BGCI-6, resulting in band lengths of 2000 bp and 1000 bp, respectively. Figure 7A displays bands of the expected sizes, confirming successful connection. Following this, we performed double enzyme digestion on the plasmid using EcoR1 and Xho1 restriction enzymes to linearize the plasmid, resulting in a band length of 3500bp. Figure 7B 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.

![](https://static.igem.wiki/teams/5071/eng122/7.png)

Figure 7. The results of the overlap connection and plasmid linearization

We selected multiple colonies for PCR verification, and the bands matched the expected length (800 bp). We sent the validated bacterial strains to a biotech company for sequencing (Figure 8), selected plasmids without mutations, and successfully obtained the constructed plasmid pRSFuet-BGC1-gene456.

![](https://static.igem.wiki/teams/5071/eng122/8.png)

Figure 8. Single clone verification of pRSFuet-BGC1-gene456 transformed E. coli DH5α. A. The results of colony PCR; B: The clones on the plate; C: Sequencing results

Part 3. pACYCDuet-BGCII-gene143, BBa\_K5071018

Design

The design concept is consistent with the above description.

![](https://static.igem.wiki/teams/5071/eng122/9.png)

Fig. 9. The plasmid map of pACYCDuet-BGCII-gene143

Build

Firstly, we utilized PCR technology to obtain three target genes, BGCII-1, BGCII-4, BGC11-3 (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 10(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.

![](https://static.igem.wiki/teams/5071/eng122/10.png)

Figure 10. The purpose segment of plasmid pACYCDuet-BGCII-gene143

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 11A (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 11B (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.

![](https://static.igem.wiki/teams/5071/eng122/11.png)

Figure 11. The results of the overlap connection and plasmid linearization

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 12), selected plasmids without mutations, and successfully obtained the constructed plasmid pACYCDuet-BGCII-gene143.

![](https://static.igem.wiki/teams/5071/eng122/12.png)

Figure 12. Single clone verification of pACYCDuet-BGCII-gene143 transformed E. coli DH5α. A. The results of colony PCR; B: The clones on the plate; C: Sequencing results

Part 4. pETDuet-BGCII-gene792, BBa\_K5071019

Design

The design concept is consistent with the above description.

![](https://static.igem.wiki/teams/5071/eng122/13.png)

Figure 13. The plasmid map of pETDuet-BGCII-gene792

Build

Firstly, we utilized PCR technology to obtain three target genes, BGCII-7, BGCII-9, BGC11-2 (synthesized by a biotech company), with band lengths of 1000 bp, 750 bp, and 2000 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 pETD), resulting in a 200 bp band. Figure 14(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.

![](https://static.igem.wiki/teams/5071/eng122/14.png)

Figure 14. The purpose segment of plasmid pETDuet-BGCII-gene792

Subsequently, we used overlap PCR technology to connect fragment BGC-4 with BGC-5, and pETD with BGC-6, resulting in band lengths of 1700 bp and 2200 bp, respectively. Figure 15A (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 5178 bp. Figure 15B (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.

![](https://static.igem.wiki/teams/5071/eng122/15.png)

Figure 15. The purpose segment of plasmid pETDuet-BGCII-gene792

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 16), selected plasmids without mutations, and successfully obtained the constructed plasmid pETDuet-BGCII-gene143.

![](https://static.igem.wiki/teams/5071/eng122/16.png)

Figure 16. Single clone verification of pETDuet-BGCII-gene143 transformed E. coli DH5α. A. The results of colony PCR; B: The clones on the plate; C: Sequencing results

Part 5. pRSFDuet-BGCII-gene685, BBa\_K5071020

Design

The design concept is consistent with the above description.

![](https://static.igem.wiki/teams/5071/eng122/17.png)

Figure 17. The plasmid map of pRSFDuet-BGCII-gene685

Bbuild

Firstly, we utilized PCR technology to obtain three target genes, BGCII-6, BGCII-8, BGCII-5 (synthesized by a biotech company), with band lengths of 500 bp, 350 bp, and 1500 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 pRSF), resulting in a 200 bp band. Figure 18(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.

![](https://static.igem.wiki/teams/5071/eng122/18.png)

Figure 18. The purpose segment of plasmid pRSFuet-BGCII-gene685

Subsequently, we used overlap PCR technology to connect fragment BGCII-6 with BGCII-8, and pRSF with BGCII-5, resulting in band lengths of 850 bp and 1700 bp, respectively. Figure 19A (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 3587 bp. Figure 19B (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.

![](https://static.igem.wiki/teams/5071/eng122/19.png)

Figure 19. The purpose segment of plasmid pRSFuet-BGCII-gene685

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 20), selected plasmids without mutations, and successfully obtained the constructed plasmid pRSFDuet-BGCII-gene143.

![](https://static.igem.wiki/teams/5071/eng122/20.png)

Figure 20. Single clone verification of pRSFDuet-BGCII-gene143 transformed E. coli DH5α. A. The results of colony PCR; B: The clones on the plate; C: Sequencing results

Test：

1: Transformation of E. coli BL21

1.1 Strain-BGCI

In our target genes, the 6 genes of BGCI represent metabolic pathway 1, which are the 6 genes contained in plasmids pETDuet-BGCI-gene123 and pRSFDuet-BGCI-gene456. We simultaneously transformed these two plasmids into E. coli BL21 for the production of terpenoid compounds. The experimental results, as shown in Figure 21, depict the transformed E. coli BL21. We conducted single colony verification to confirm the presence of both plasmids, as illustrated in Figure 16. We obtained bacterial strains that correctly harbored both transformed plasmids, which we named as BGCI.

![](https://static.igem.wiki/teams/5071/eng122/21.png)

Figure 21. Colony PCR results of strain BGCI

1.2 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 22, 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.

![](https://static.igem.wiki/teams/5071/eng122/22.png)

Figure 22. Colony PCR results of strain BGCII

2: Protein expression

2.1 Strain-BGCI

To assess the gene expression in the bacterial strains, we lysed the cells. To run a protein gel, start by preparing protein samples with a loading buffer and loading them into the gel wells. Run the gel at a constant voltage to separate proteins by size, then stain the gel to visualize the separated proteins. Finally, analyze the protein bands to interpret the results. We performed protein electrophoresis at different time points after IPTG induction, as shown in Figure 23, to detect the proteins expressing our target genes (BGCI-2 is 43.3kDa, BGCI-1 is 3.1kDA, BGCI-2 is 18.4kDa, BGCI-5 is 26.1kDa, BGCI-4 is 48.4kDA, BGCI-6 is 29.2kDa).

![](https://static.igem.wiki/teams/5071/eng122/23.png)

Figure 23. Protein gel results of strain BGCI

2.2 Strain-BGCII

The treatment method for strain BGCII was consistent with BGCI, and the experimental results, as shown in Figure 24, 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).

![](https://static.igem.wiki/teams/5071/eng122/24.png)

Figure 24. Protein gel results of strain BGCII

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 25 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  Absorbancy  STD  DPPH free radical clearance (%)

Control  0.146  0.0191  4.58

BGCI  0.089  0.0039  40.80

BGCII  0.076  0.0088  49.45

![](https://static.igem.wiki/teams/5071/eng122/25.png)

Figure 25. DPPH scavenging rates of the genetically modified strains

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 26, 4uL 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.

![](https://static.igem.wiki/teams/5071/eng122/26.png)

Figure 26. Results of the antibacterial experiment on the bacterial strains

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 27, 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.

![](https://static.igem.wiki/teams/5071/eng122/27.png)

Figure 27. Detection results of squalene in the fermentation broth of the bacterial strains

5: Determination of squalene in the fermentation broth by GC-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

![](https://static.igem.wiki/teams/5071/eng122/image-2.png)

Figure 28. Detection results of squalene in the fermentation broth of the bacterial strains

Learn

For the production of terpenoid compounds, we should consider knocking out some metabolic pathways within the bacterial strains to redirect more carbon flux towards the production of the desired compounds, thereby reducing the production of by-products.The exploration of the metagenome should be more in-depth to increase the chances of obtaining effective production components, which can better guide subsequent production processes.

![](https://static.igem.wiki/teams/5071/home/fvv.png)
Reference

[1] Alma'abadi AD, Gojobori T, Mineta K. Marine Metagenome as A Resource for Novel Enzymes. Genomics Proteomics Bioinformatics. 2015 Oct;13(5):290-5. doi: 10.1016/j.gpb.2015.10.001. Epub 2015 Nov 10. PMID: 26563467; PMCID: PMC4678775.

[2] Sunagawa S, Coelho LP, Chaffron S, Kultima JR, Labadie K, Salazar G, Djahanschiri B, Zeller G, Mende DR, Alberti A, Cornejo-Castillo FM, Costea PI, Cruaud C, d'Ovidio F, Engelen S, Ferrera I, Gasol JM, Guidi L, Hildebrand F, Kokoszka F, Lepoivre C, Lima-Mendez G, Poulain J, Poulos BT, Royo-Llonch M, Sarmento H, Vieira-Silva S, Dimier C, Picheral M, Searson S, Kandels-Lewis S; Tara Oceans coordinators; Bowler C, de Vargas C, Gorsky G, Grimsley N, Hingamp P, Iudicone D, Jaillon O, Not F, Ogata H, Pesant S, Speich S, Stemmann L, Sullivan MB, Weissenbach J, Wincker P, Karsenti E, Raes J, Acinas SG, Bork P. Ocean plankton. Structure and function of the global ocean microbiome. Science. 2015 May 22;348(6237):1261359. doi: 10.1126/science.1261359. PMID: 25999513.

[3] Sunagawa S, Coelho LP, Chaffron S, Kultima JR, Labadie K, Salazar G, Djahanschiri B, Zeller G, Mende DR, Alberti A, Cornejo-Castillo FM, Costea PI, Cruaud C, d'Ovidio F, Engelen S, Ferrera I, Gasol JM, Guidi L, Hildebrand F, Kokoszka F, Lepoivre C, Lima-Mendez G, Poulain J, Poulos BT, Royo-Llonch M, Sarmento H, Vieira-Silva S, Dimier C, Picheral M, Searson S, Kandels-Lewis S; Tara Oceans coordinators; Bowler C, de Vargas C, Gorsky G, Grimsley N, Hingamp P, Iudicone D, Jaillon O, Not F, Ogata H, Pesant S, Speich S, Stemmann L, Sullivan MB, Weissenbach J, Wincker P, Karsenti E, Raes J, Acinas SG, Bork P. Ocean plankton. Structure and function of the global ocean microbiome. Science. 2015 May 22;348(6237):1261359. doi: 10.1126/science.1261359. PMID: 25999513.

[4] Schempp FM, Drummond L, Buchhaupt M, Schrader J. Microbial Cell Factories for the Production of Terpenoid Flavor and Fragrance Compounds. J Agric Food Chem. 2018 Mar 14;66(10):2247-2258. doi: 10.1021/acs.jafc.7b00473. Epub 2017 Apr 18. PMID: 28418659.

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EXPERIMENTS
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[Outline](#nav1)
 [Preparation of LB (Luria-Bertani) culture medium](#nav2)
 [Construction of recombination plasmids](#nav3)
[Co-transformation of E. coli BL21](#nav4)
[Protein expression](#nav5)
[Functional test](#nav6)

![](https://static.igem.wiki/teams/5071/home/fvv.png)
Outline

###### 1. Preparation of LB (Luria-Bertani) culture medium

###### 2. The construction of plasmid

2.1 pETDuet-BGCI-gene123

2.2 pRSFDuet-BGCI-gene456

2.3 pACYCDuet-BGCII-gene143

2.4 pETDuet-BGCII-gene792

2.5 pRSFDuet-BGCII-gene685

###### 3. Co-transformation of E. coli BL21

3.1 Strain-BGCI

3.2 Strain-BGCII

###### 4. Protein expression

4.1 Strain-BGCI

4.2 Strain-BGCII

###### 5. Functional test

5.1 The test results for Total Antioxidant Capacity (T-AOC)

5.2 The test of the fermentation product antibacterial experiment

5.3 Determination of squalene in the fermentation broth by HPLC

![](https://static.igem.wiki/teams/5071/home/fvv.png)
Preparation of LB (Luria-Bertani) culture medium

Goal: To create a functional and nutritional liquid or solid medium for bacteria growing. Activation, cultivation, and fermentation for

microorganisms.

Materials:

1) Conical flask

2) Autoclave

3) Tryptone

4) Yeast Extract

5) Sodium Chloride

6) Double distilled H2O

7) Agar

Procedures:

1) Add 10 g tryptone, 5 g yeast extract, and 10 g of sodium chloride per liter of the culture medium. (Note: 1.5% agar powder was added to the solid culture medium);

2) Add into 1L conical flask;

3) Maintain at 121 ℃ for 20 minutes in an autoclave for sterilization.

![](https://static.igem.wiki/teams/5071/home/fvv.png)
Construction of recombination plasmids

###### The construction strategy in this chapter takes pETDuet-BGCI-gene123 as an example, and the construction methods of other recombination plasmids are the same as pETDuet-BGCI-gene123.

###### Ⅰ. Obtain target gene fragments by 1st PCR

Goal: To amplify BGCI-gene1, BGCI-gene2, BGCI-gene3, and backbone middle fragment for 2nd overlap PCR.

Materials:

1) 2x PrimeStarMix

2) Gene template

3) Double distilled water (ddH2O)

4) Primer-F

5) Primer-R

6) PCR tube

7) PCR thermal cycler

\* PrimeStarMix includes buffer and dNTPs for PCR. The extention rate for PrimeStarMix (2x) is 1000bp/10 s.

Procedures:

1) Add 2x PrimerStarMix 25µl, gene template 1µl, primer-F 1µl, and primer-R 1µl to centrifuge tube. Add ddH2O to the centrifuge tube up to 50µl. If bubbles are observed in the mixture, centrifuge the mixture for a few seconds to remove the bubbles;

2) Insert PCR tubes into PCR thermal cycler. Pre-denature the fragments at 95˚C for 3 minutes;

3) Perform 35 cycles of 30 seconds 95˚C denaturing, 30 seconds 55˚C annealing, and 2 minutes extension 72˚C extension;

4) Extend the fragments for 1 minutes at 72˚C;

5) If not use immediately, preserve the fragments at 4˚C;

6) Recover PCR fragments by agarose gel electrophoresis.

###### Ⅱ. Agarose gel construction, deployment and recycle

Goal: To produce a functional and well-shaped gel for electrophoresis.

Materials:

1) Casting Tray

2) Well comb

3) Microwave

4) 1x TAE buffer

5) Agarose

6) 10000x Nucleic acid gel stain

7) Conical flask

Procedures:

1) Dissolve 1.5g Agarose in 200mL Conical flask;

2) Add 100mL TAE buffer;

3) Heat the material with microwave;

4) Melt the mixture at 30 sec interval;

5) See whether bubbles are forming, if did succesfully mixed;

6) Add 3μl nucleic acid gel stain (10000x);

7) Let it cool for 5~10 minutes;

8) Pour the agarose mixture into the casting tray;

9) Place the appropriate well comb to create the wells;

10) Wait to solidify ~30 minutes;

11) Remove the comb and place the gel in the gel box.

###### Ⅲ. Agarose gel electrophoresis

Goal: To verify the DNA bands' lengths and thus preparing for the next step of gel extraction to recover the bands that are of the correct length.

Procedures:

1) Extract 50µl of every sample; add 5µl of loading buffer (10x) to each sample;

2) Place the gel in the horizontal electrophoresis system;

3) Extract 50µl of the mixed solution containing the sample, mix them till their colors are almost uniform;

4) Add the marker to the first well and load 50µl of each sample into the wells;

5) The procedure runs at 120V for 20min;

6) Take out the gel from the device and proceed to following steps.

###### Ⅳ. DNA gel extraction

Goal: To recover DNA fragments in agarose gel.

Materials:

1) Agarose gel with PCR products obtaining from 1st PCR;

2) 1.5 mL Eppendorf (EP) tube

3) Agarose Gel Electrophoresis Recovery Kit

Procedures:

1) Cut the slice of gel containing PCR products of BGCI-gene1, BGCI-gene2, BGCI-gene3, and backbone middle fragment obtaining from 1st PCR, cutting off as much unneeded gel as possible, and place it in an 1.5 mL EP tube;

2) Add 500 µl buffer B2 and put the tube into a 50°C hot water bath until gel has completely melted;

3) Transfer the solution containing melted gel into a purification column that's in a collection tube and perform centrifugation at 12000 rpm for 30 seconds;

4) Empty the collection tube, put the purification column back in, and add 500 µl wash solution containing pure ethanol. Perform centrifugation at 12000 rpm for 30 seconds, and empty collection tube again;

5) Repeat steps 4;

6) Perform centrifugation one more time at 12000 rpm for 2 minutes, then open the cap of the tube and let it sit for one minute to allow the ethanol to evaporate;

7) Transfer purification column to a new 1.5 mL EP tube. Add 50 µl double distilled water (ddH2O) at the center of the purification column;

8) Cap the lid and let the tube sit for 1 minute. Perform centrifugation at 12000 rpm for 1 minute;

9) Discard the purification column. Store DNA fragments in the EP tube at 4°C.

###### Ⅴ. Obtain recombination fragments by 2nd overlap PCR

Goal: To amplify recombination fragments of BGCI-gene1 with BGCI-gene2 and backbone middle fragment with BGCI-gene3 by 2nd overlap PCR for further Infusion assay.

Materials:

1) 2x PrimeStarMix

2) Gene template from 1st PCR products

3) Double distilled water (ddH2O)

4) Primer-F

5) Primer-R

6) PCR tube

7) PCR thermal cycler

Procedures:

1) Take recombination process of BGCI-gene1 and BGCI-gene2 as an example, the same as backbone middle fragment and BGCI-gene3;

2) Add 2x PrimerStarMix 25µl, BGCI-gene1 1st PCR product 1µl, and BGCI-gene2 1st PCR product 1µl to PCR tube. Add ddH2O to the centrifuge tube up to 46µl (Except primer-F and primer-R). If bubbles are observed in the mixture, centrifuge the mixture for a few seconds to remove the bubbles;

3) Insert PCR tubes into PCR thermal cycler. Pre-denature the fragments at 95˚C for 3 minutes;

4) Perform 35 cycles of 30 seconds 95˚C denaturing, 30 seconds 55˚C annealing, and 2 minutes extension 72˚C extension;

5) After performing 5 cycles for self-annealing, then pause the PCR thermal cycler;

6) Add primer-F of BGCI-gene1 and primer-R of BGCI-gene2 each 2µl to PCR reaction solutions;

7) Restart PCR thermal cycler for the rest steps;

8) Extend the fragments for 1 minutes at 72˚C;

9) If not use immediately, preserve the fragments at 4˚C;

10) Recover recombination PCR fragments by agarose gel electrophoresis.

###### Ⅵ. Linearize pETDuet/pRSFDuet/pACYCDuet backbone by restriction endonucleases

Goal: To linearize pETDuet/pRSFDuet/pACYCDuet backbone for Infusion recombination.

Materials:

1) pETDuet backbone plasmid

2) pRSFDuet backbone plasmid

3) pACYCDuet backbone plasmid

4) 10x Cutting buffer

5) EcoRI endonuclease

6) XhoI endonuclease

7) BamHI endonuclease

8) Double distilled water (ddH2O)

Procedures:

1) Use BamHI and XhoI endonucleases to digest pETDuet backbone, use EcoRI and XhoI endonucleases to digest pRSFDuet backbone, and use BamHI and XhoI endonucleases to digest pACYCDuet backbone;

2) Add 5µg backbone plasmid, 10x Cutting buffer 5µl, endonuclease I 5µl, and endonuclease II 5µl in PCR tube. Add ddH2O to PCR tube up to 50µl;

3) Mix by pipette and remove the bubbles by low-speed centrifuge;

4) Insert PCR tubes into PCR thermal cycler. Digest the plasmid at 37˚C for 30 minutes;

5) Recover linearized plasmid backbone fragments by agarose gel electrophoresis.

###### Ⅶ. Homologous recombination of linearized plasmid backbone with 2nd overlap PCR products

Goal: Perform homologous recombination between the linearized plasmid backbone and 2nd overlap PCR products to generate the complete plasmid. Take pETDuet-BGCI-gene123 as an example.

Materials:

1) CloneExpress Mix (2x)

2) 2nd overlap PCR products of BGCI-gene1 with BGCI-gene2 and backbone middle fragment with BGCI-gene3

3) Linearized pETDuet backbone

Procedure:

1) Measure the concentration of the gel-recovered fragments;

2) Prepare the system for the construction of pETDuet-BGCI-gene123 in PCR tubes: 5μl CloneExpress Mix, 2μl Linearized pETDuet backbone, 1.5μl 2nd overlap PCR products of BGCI-gene1 with BGCI-gene2, and 1.5μl 2nd overlap PCR products of backbone middle fragment with BGCI-gene3;

3) Insert PCR tubes into PCR thermal cycler. Ligase the fragments at 50˚C for 30 minutes;

###### Ⅷ. Transformation

Materials:

1) Ligated recombination plasmid

2) Liquid LB solution without antibiotics

3) LB Agar plates with corresponding antibiotics

Procedures:

1) Add recombination plasmid into E.Coli DH5α strain;

2) Placed on ice for 20 minutes;

3) Heat shock at 42˚C for 45 seconds, then immediately put on ice for 2-3 minutes;

4) Add 400μL liquid LB solution without antibiotics, incubate at 37 ˚C for 30 minutes;

5) Centrifuge at 5000 rpm for 3 minutes, extract 300μL of supernatant then discard;

6) Apply bacterial liquid to LB Agar plates with corresponding antibiotics, incubate for 12-16 hours at 37˚C.

###### Ⅸ. Colony PCR verification

Goal: E.Coli DH5α strain containing constructed plasmids has multiplied. We aimed to identify mono-colony E.coli DH5α containing correct recombination plasmids by colony PCR.

Materials:

1) Taq Master Mix (2x)

2) Double distilled water (ddH2O)

3) Culture plates post-transformation

Procedures:

1) Add 10μl Taq Master Mix, 1μl primer-F, 1μl primer-R, and 8μl ddH2O into PCR tubes;

2) Pick a single colony with a 10μl sterile pipette tip and perform aspiration and dispensing in PCR tubes;

3) Insert mixture into a PCR thermal cycler, design the PCR program according to the manufacturer's instructions.

4) Perform agarose gel electrophoresis, and the presence of the correct bands indicates the successful construction of recombination plasmids.

###### Ⅹ. Plasmid extraction and sequencing

Goal: To prepare the recombination plasmids and then to verify the correctness of sequences.

Materials:

1) Bacterial fluid of pETDuet-BGCI-gene123

2) Centrifuge

3) 1.5mL centrifuge tubes and absorption columns

4) Sangon small-scale plasmid extraction kit

Procedures:

1) Take all bacterial fluid into centrifuge for 2 minutes at 12000 rpm and discard all supernatant;

2) Add 250μl Buffer P1 to the bacteria and let it resuspend;

3) Add 250μl Buffer P2 and gently turn the centrifuge tube upside down for 5-10 times to let it mix. Leave the centrifuge tube at room temperature for 2-4min;

4) Add 350μl Buffer P3, immediately and gently invert the centrifuge tube for 5-10 times to fully mix;

5) Centrifuge the tube at 12000 rpm for 15min, and carefully move all the supernatant into the adsorption column and centrifuge at 12000 rpm for 30 sec. Drain the liquid in the collection tube;

6) Add 700μl Wash Solution into the adsorption column, centrifuge at 12000 rpm for 30 sec. Drain the liquid in the collection tube.;

7) Repeat step 6 once;

8) Put empty adsorption column and the collection tube together and centrifuge them at 12000 rpm for 1 min;

9) Add 50μl double distilled water to the center of the adsorption membrane. Leave the tube at room temperature for 1-2min.

10) Transfer the absorption membrane to a new EP tube and then centrifuge it at 12000 rpm for 1min;

11) Store plasmid in the EP tube at 4°C;

12) Sequence the plasmid by specific primers in Sangon Biotech Co., Ltd.

![](https://static.igem.wiki/teams/5071/home/fvv.png)
Co-transformation of E. coli BL21

###### The transformation strategy in this chapter takes Strain-BGCI as an example, and the transformation methods of Strain-BGGII are the same as Strain-BGCI. The only exception difference between Strain-BGCI and Strain-BGCII is the types of antibiotics using for selecting positive colony.

Goal: To co-transform correct combination plasmid into E. coli BL21 for further protein expression. For strain-BGCI, verified pETDuet-BGCI-gene123 and pRSFDuet-BGCI-gene456 plasmids are co-transformed into E. coli BL21. For strain-BGCII, verified pACYCDuet-BGCII-gene143, pETDuet-BGCII-gene792, AND pRSFDuet-BGCII-gene685 are co-transformed into E. coli BL21.

###### Ⅰ. Co-transformation of E.coli BL21

Materials:

1) Verified pETDuet-BGCI-gene123 and pRSFDuet-BGCI-gene456 plasmids

2) Liquid LB solution without antibiotics

3) LB Agar plates with corresponding double antibiotics

Procedures:

1) Add each 1μl verified pETDuet-BGCI-gene123 and pRSFDuet-BGCI-gene456 plasmids into E.Coli BL21 strain;

2) Placed on ice for 20 minutes;

3) Heat shock at 42˚C for 45 seconds, then immediately put on ice for 2-3 minutes;

4) Add 400μL liquid LB solution without antibiotics, incubate at 37 ˚C for 30 minutes;

5) Centrifuge at 5000 rpm for 3 minutes, extract 300μL of supernatant then discard;

6) Apply bacterial liquid to LB Agar plates with corresponding antibiotics, incubate for 12-16 hours at 37˚C.

7) For strain-BGCI, use double antibiotics of kanamycin and ampicillin to select positive colonies. For strain-BGCII, use triple antibiotics of kanamycin, ampicillin, and chloramphenicol to select positive colonies.

###### Ⅱ. Colony PCR verification for multi plasmids

Materials:

1) Taq Master Mix (2x)

2) Double distilled water (ddH2O)

3) Culture plates co-transformation

Procedures:

1) For verification of multi plasmids in E.coli BL21 colonies, primers for each plasmids are used to amplify target bands, respectively;

2) Add 10μl Taq Master Mix, 1μl primer-F, 1μl primer-R, and 8μl ddH2O into PCR tubes;

3) Pick a single colony with a 10μl sterile pipette tip and perform aspiration and dispensing in PCR tubes;

4) Insert mixture into a PCR thermal cycler, design the PCR program according to the manufacturer's instructions.

5) Perform agarose gel electrophoresis, and the presence of the correct bands indicates the successful construction of plasmids.

![](https://static.igem.wiki/teams/5071/home/fvv.png)
Protein expression

###### Induction of protein expression for strain-BGCI and strain-BGCII

###### Ⅰ. Amplification culture for correct colony

Goal: To acquire large amounts of target proteins for SDS-PAGE

Materials:

1) Liquid LB solution

2) E.coli BL21 of strain-BGCI and strain-BGCII bacterial fluid

3) Antibiotics: kanamycin, ampicillin, and chloramphenicol

4) Inducer: IPTG

5) Iso-thermic shaker

Procedures:

1) Add 100μl of strain-BGCI and strain-BGCII bacterial fluid and 100μl of corresponding multi antibiotics into 100mL of liquid LB solutions, respectively;

2) Culture bacterial liquid in iso-thermic shaker for 3 hours until absorbance OD600nm to 0.4-0.6;

3) Add IPTG into bacterial liquid with 1mM final concentration;

4) Sample for each bacterial liquid cultured at 25°C and 37°C for 0.5h, 1h, 3h, and 6h, respectively.

###### Ⅱ: Protein crude extraction and SDS-PAGE

Goal: To verify the expression of desired proteins.

Materials for protein crude extraction:

1) 500μl bacterial liquid for different induced conditions

2) Centrifuge

3) Double distilled water (ddH2O)

4) 6x Protein loading buffer

Procedures for protein crude extraction:

1) Centrifuge samples at 12000 rpm for 1 minute;

2) Discard supernatant and resuspend precipitation with 50μl 1x Protein loading buffer diluted by ddH2O;

3) Boiled samples at 95°C for 15 minutes in PCR thermal cycler.

Materials for SDS-PAGE:

1) Sangon 12.5% SDS-PAGE Color Preparation kit

2) Electrophoresis buffer (Tris-Glycine)

3) Protein ladder

4) 6x Protein loading buffer

5) Vertical electrophoresis system

6) Coomassie blue staining solution

Procedures for SDS-PAGE：

1) Add and mix 2.2mL 2x separating gel solution, 2.2mL 2X separating gel buffer, and 44μL catalyst in centrifugation tubes for separating gel;

2) Slowly pipetting mixture into casting stand and frame to avoid bubbles;

3) Add 1 mL ddH2O at the top of separating gel;

4) Wait the separating gel to solidify for 8 minutes;

5) Discard the water that was added previously;

6) Add and mix 825μL 2x stacking gel solution, 825μL 2X stacking gel buffer, and 11μL catalyst in centrifugation tubes for stacking gel;

7) Add stacking gel mixture until the cast is completely filled, then slowly insert comb without producing any air bubbles;

8) Wait the stacking gel to solidify for 12 minutes, then carefully remove the comb, and wash the wells with electrophoresis buffer;

9) Add 10μL protein ladder into the first well, then load samples in each successive well;

10) Transfer gel into vertical electrophoresis system;

11) Run at 120V for 90 minutes;

12) Stain the gel with Coomassie blue staining solution for 10 minutes, then destain the gel using destaining solution repeatedly until gel background become transparent.

![](https://static.igem.wiki/teams/5071/home/fvv.png)
Functional test

###### 1 The test results for Total Antioxidant Capacity (T-AOC)

Goal: Total antioxidant capacity represents the levels of antioxidant substances and antioxidant enzymes in sample. We used bacterial fluids expressing strain-BGCI and strain-BGCII to test the total antioxidant capacity via DPPH method. DPPH is a stable substance that is soluble in ethanol and has a maximum absorption at 515 nm. When antioxidants are added to DPPH solutions, the antioxidant capacity of antioxidant substances can be quantified by changes in absorbance value.

Materials:

1) Bacterial fluids expressing strain-BGCI or strain-BGCII

2) Extraction buffer

3) DPPH solution

4) Centrifuge

5) 96-wells plate

6) Microplate reader

7) Double distilled water(ddH2O)

Procedures:

1) Add 2mL bacterial fluids to centrifuge tubes, then centrifuge at 12000 rpm for 2 minutes;

2) Resuspend precipitation with 1mL extraction buffer;

3) Put on ice for 30 minutes;

4) Use ultrasonic apparatus to pre-treat samples: power set at 200W, ultrasound 3s, interval 10s, repeated 30 times;

5) Centrifuge at 10000 rpm for 10 minutes;

6) Transfer supernatant to a new tube for measurement;

7) Prepare tubes for blank and test. Add 20μL extraction buffer and 380μL DPPH solution to blank tube. At the same time, add 20μL bacterial crude extraction and 380μL DPPH solution to test tube;

8) React in dark at room temperature for 20 minutes;

9) Add 200μL reaction solutions to 96-well plate;

10) Use microplate reader to measure the absorption value of samples at 515nm;

11) Calculate the total antioxidant capacity: DPPH free radical clearance rate (%) = (Ablank-Atest)÷Ablank×100%

###### 2 The test of the fermentation product antibacterial experiment

Goal: Use the double-layer agar plate to test the antibacterial activity of fermentation products expressing strain-BGCI or strain-BGCII via observe antibacterial plaque.

Materials:

1) Bacterial fluids expressing strain-BGCI or strain-BGCII

2) Test strain K-12

3) LB solid medium plate without antibiotics

4) Positive control: 1g/L Ciprofloxacin

5) Standard sample: 200mg/L Squalene

Procedures:

) Prepare the double-layer agar plate, 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.

2) Mix and culture strain K-12 bacterial fluids in double-layer agar plate. Streak the swab evenly across the surface of the nutrient agar plate to create a bacterial lawn.

3) Put the double-layer agar plate in the incubator at 37℃ for 24 hours;

4) Add 4μL drops of each antibacterial agent onto the surface of the inoculated agar plates;

5) At the same time, add 4μL drops of Ciprofloxacin and Squalene onto the surface of the inoculated agar plates;

6) Cover the plates with lids and incubate them upside down at 37°C for 24 hours.

7) After incubation, observe the plates for zones of inhibition (clear areas where bacteria did not grow).

###### 3 Determination of squalene in the fermentation broth by HPLC

Goal: To determine if our target terpenoid compound is squalene, we conducted testing on the fermentation broth of the bacterial strains.

Materials:

1) Bacterial fluids expressing strain-BGCI or strain-BGCII

2) LBG medium

3) Sonicator

4) Centrifuge

5) Double distilled water(ddH2O)

6) 0.22μm filter membrane

7) Methanol

8) Ethyl acetate

9) Pure acetonitrile

10) Waters HPLC system

11) Waters XBridgeTM C18 HPLC column

Procedures:

1) Fermentation was carried out using a biphasic fermentation method, with 10% volume of normal heptane added on top of the LBG medium;

2) 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;

3) 400μL of saline solution was added to wash the fermentation cells, centrifuged at 13,000×g for 10 minutes, and the supernatant was discarded;

4) ddH2O was added, thoroughly mixed, and brought to a volume of 400μL;

5) The cells were disrupted by ultrasonication at a working power of 20%, for 2 minutes with 3-second on and 5-second off cycles;

6) Subsequently, 600μL of ethyl acetate was added, mixed well, and subjected to ultrasonic cleaning twice for 15 minutes each;

7) The mixture was then centrifuged, and 400μL of the extract phase was obtained;

8) The extract was concentrated using a vacuum centrifuge to evaporate the solvent, then re-dissolved in 200μL of methanol, filtered through a 0.22μm filter membrane, and ready for analysis;

9) Squalene yield detection was performed using high-performance liquid chromatography (HPLC) under the following conditions: Column: Waters XBridgeTM C18 (3.5μm 4.6mm×150mm); Column temperature: 35℃; Mobile phase: 100% pure acetonitrile; Flow rate: 1 mL/min; Detector: Photodiode array detector at 196 nm wavelength.

{% endblock %}

{% extends "layout.html" %}
{% block page\_content %}

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RESULTS
![](https://static.igem.wiki/teams/5071/home/a1.png)

[Overview](#nav1)
 [Results of our work](#nav2)
 [Future plans](#nav3)
[References](#nav4)

![](https://static.igem.wiki/teams/5071/home/fvv.png)
Overview

The various extreme environments found in the deep sea (≥1000 meters) harbor high levels of biodiversity, rich genetic resources, and biosynthetic potential[2,3]. This project aims to explore high-value skincare-active terpenoid natural products based on metagenomic data from deep-sea microorganisms. Through metagenomic analysis of samples from deep-sea ecosystems, a collection of deep-sea microbial genomes and biosynthetic gene clusters will be constructed[3]. Targeted exploration of biosynthetic gene clusters for 1-2 terpenoid natural products from deep-sea microbial communities will be conducted to elucidate the product structures. Finally, the biological activities of terpenoid natural products such as antibacterial, anti-inflammatory, and neuroprotective effects will be evaluated to provide a research foundation for their application in the skincare industry[4].

![](https://static.igem.wiki/teams/5071/home/fvv.png)
Results of our work

Outline

###### 1. The construction of plasmid

1.1 pETDuet-BGCI-gene123

1.2 pRSFDuet-BGCI-gene456

1.3 pACYCDuet-BGCII-gene143

1.4 pETDuet-BGCII-gene792

1.5 pRSFDuet-BGCII-gene685

###### 2. Transformation of E. coli BL21

2.1 Strain-BGCI

2.2 Strain-BGCII

###### 3. Protein expression

3.1 Strain-BGCI

3.2 Strain-BGCII

###### 4. Functional test

4.1 The test results for Total Antioxidant Capacity (T-AOC)

4.2 The test of the fermentation product antibacterial experiment

4.3 Determination of squalene in the fermentation broth by HPLC

###### 1. The construction of plasmid

###### 1.1 pETDuet-BGCI-gene123

Firstly, we utilized PCR technology to obtain three target genes, BGCI-1, BGCI-2, BGCI-3 (synthesized by a biotech company), with band lengths of 150 bp, 1200 bp, and 500 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 123-mid), resulting in a 200 bp band. Figure 1 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.

![](https://static.igem.wiki/teams/5071/resa11/1.png)

Figure 1. The purpose segment of plasmid pETDuet-BGC1-gene123

Subsequently, we used overlap PCR technology to connect fragment BGCI-1 with BGCI-2, and 123-mid with BGCI-3, resulting in band lengths of 1300bp and 700bp, respectively. Figure 2A 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 4000bp. Figure 2B 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.

![](https://static.igem.wiki/teams/5071/resa11/2.png)

Figure 2. The results of the overlap connection and plasmid linearization

We selected multiple colonies for PCR verification, and the bands matched the expected length (1200 bp). We sent the validated bacterial strains to a biotech company for sequencing (Figure 3), selected plasmids without mutations, and successfully obtained the constructed plasmid pETDuet-BGC1-gene123.

![](https://static.igem.wiki/teams/5071/resa11/3.png)

Figure 3. Single clone verification of pETDuet-BGC1-gene123 transformed E. coli DH5α. A. The results of colony PCR; B: The clones on the plate; C: Sequencing results

###### 1.2 pRSFuet-BGCI-gene456

Firstly, we utilized PCR technology to obtain three target genes, BGCI-4, BGCI-5, BGCI-6 (synthesized by a biotech company), with band lengths of 1300 bp, 800 bp, and 800 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 456-mid), resulting in a 150 bp band. Figure 4 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.

![](https://static.igem.wiki/teams/5071/resa11/4.png)

Figure 4. The purpose segment of plasmid pRSFuet-BGC1-gene456

Subsequently, we used overlap PCR technology to connect fragment BGCI-4 with BGCI-5, and 456-mid with BGCI-6, resulting in band lengths of 2000 bp and 1000 bp, respectively. Figure 5A displays bands of the expected sizes, confirming successful connection. Following this, we performed double enzyme digestion on the plasmid using EcoR1 and Xho1 restriction enzymes to linearize the plasmid, resulting in a band length of 3500bp. Figure 5B 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.

![](https://static.igem.wiki/teams/5071/resa11/5.png)

Figure 5. The results of the overlap connection and plasmid linearization

We selected multiple colonies for PCR verification, and the bands matched the expected length (800 bp). We sent the validated bacterial strains to a biotech company for sequencing (Figure 3), selected plasmids without mutations, and successfully obtained the constructed plasmid pRSFuet-BGC1-gene456.

![](https://static.igem.wiki/teams/5071/resa11/6.png)

Figure 6. Figure 3. Single clone verification of pRSFuet-BGC1-gene456 transformed E. coli DH5α. A. The results of colony PCR; B: The clones on the plate; C: Sequencing results

###### 1.3 pACYCDuet-BGCII-gene143

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 7(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.

![](https://static.igem.wiki/teams/5071/resa11/7.png)

Figure 7. The purpose segment of plasmid pACYCDuet-BGCII-gene143

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 8A (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 8B (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.

![](https://static.igem.wiki/teams/5071/resa11/8.png)

Figure 8. The results of the overlap connection and plasmid linearization

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 3), selected plasmids without mutations, and successfully obtained the constructed plasmid pACYCDuet-BGCII-gene143.

![](https://static.igem.wiki/teams/5071/resa11/9.png)

Figure 9. Single clone verification of pACYCDuet-BGCII-gene143 transformed E. coli DH5α. A. The results of colony PCR; B: The clones on the plate; C: Sequencing results

###### 1.4 pETDuet-BGCII-gene792

Firstly, we utilized PCR technology to obtain three target genes, BGCII-7, BGCII-9, BGC11-2 (synthesized by a biotech company), with band lengths of 1000 bp, 750 bp, and 2000 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 pETD), resulting in a 200 bp band. Figure 10(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.

![](https://static.igem.wiki/teams/5071/resa11/10.png)

Figure 10. The purpose segment of plasmid pETDuet-BGCII-gene792

Subsequently, we used overlap PCR technology to connect fragment BGC-4 with BGC-5, and pETD with BGC-6, resulting in band lengths of 1700 bp and 2200 bp, respectively. Figure 11A (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 5178 bp. Figure 11B (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.

![](https://static.igem.wiki/teams/5071/resa11/11.png)

Figure 11. The purpose segment of plasmid pETDuet-BGCII-gene792

![](https://static.igem.wiki/teams/5071/resa11/12.png)

Figure 12. Single clone verification of pETDuet-BGCII-gene143 transformed E. coli DH5α. A. The results of colony PCR; B: The clones on the plate; C: Sequencing results

###### 1.5 pRSFDuet-BGCII-gene685

Firstly, we utilized PCR technology to obtain three target genes, BGCII-6, BGCII-8, BGCII-5 (synthesized by a biotech company), with band lengths of 500 bp, 350 bp, and 1500 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 pRSF), resulting in a 200 bp band. Figure 13(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.

![](https://static.igem.wiki/teams/5071/resa11/13.png)

Figure 13. The purpose segment of plasmid pRSFuet-BGCII-gene685

![](https://static.igem.wiki/teams/5071/resa11/14.png)

Figure 14. The purpose segment of plasmid pRSFuet-BGCII-gene685

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 15), selected plasmids without mutations, and successfully obtained the constructed plasmid pRSFDuet-BGCII-gene143

![](https://static.igem.wiki/teams/5071/resa11/15.png)

Figure 15. Single clone verification of pRSFDuet-BGCII-gene143 transformed E. coli DH5α. A. The results of colony PCR; B: The clones on the plate; C: Sequencing results

###### 2. Transformation of E. coli BL21

###### 2.1 Strain-BGCI

In our target genes, the 6 genes of BGCI represent metabolic pathway 1, which are the 6 genes contained in plasmids pETDuet-BGCI-gene123 and pRSFDuet-BGCI-gene456. We simultaneously transformed these two plasmids into E. coli BL21 for the production of terpenoid compounds. The experimental results, as shown in Figure 16, depict the transformed E. coli BL21. We conducted single colony verification to confirm the presence of both plasmids, as illustrated in Figure 16. We obtained bacterial strains that correctly harbored both transformed plasmids, which we named as BGCI.

![](https://static.igem.wiki/teams/5071/resa11/16.png)

Figure 16. Colony PCR results of strain BGCI

###### 2.2 Strain-BGCII

In our target genes, the 9 genes of BGCII represent metabolic pathway 2, 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 17, 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.

![](https://static.igem.wiki/teams/5071/resa11/17.png)

Figure 17. Colony PCR results of strain BGCII

###### 3. Protein expression

3.1 Strain-BGCI

To assess the gene expression in the bacterial strains, we lysed the cells. To run a protein gel, start by preparing protein samples with a loading buffer and loading them into the gel wells. Run the gel at a constant voltage to separate proteins by size, then stain the gel to visualize the separated proteins. Finally, analyze the protein bands to interpret the results. We performed protein electrophoresis at different time points after IPTG induction, as shown in Figure 18, to detect the proteins expressing our target genes (BGCI-2 is 43.3kDa, BGCI-1 is 3.1kDA, BGCI-2 is 18.4kDa, BGCI-5 is 26.1kDa, BGCI-4 is 48.4kDA, BGCI-6 is 29.2kDa).

![](https://static.igem.wiki/teams/5071/resa11/18.png)

Figure 18. Protein gel results of strain BGCI

3.2 Strain-BGCII

The treatment method for strain BGCII was consistent with BGCI, and the experimental results, as shown in Figure 19, 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).

![](https://static.igem.wiki/teams/5071/resa11/19.png)

Figure 19. Protein gel results of strain BGCII

###### 4. Functional test

###### 4.1 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 20 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.

![](https://static.igem.wiki/teams/5071/resa11/20.png)

Figure 20. DPPH scavenging rates of the genetically modified strains

Strain
Absorbancy
STD
DPPH free radical clearance (%)

Control
0.146
0.0191
4.58

BGCI
0.089
0.0039
40.80

BGCII
0.076
0.0088
49.45

###### 4.2 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 21, 4uL 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.

![](https://static.igem.wiki/teams/5071/resa11/21.png)

Figure 21. Results of the antibacterial experiment on the bacterial strains

###### 4.3 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 22, 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.

![](https://static.igem.wiki/teams/5071/resa11/22.png)

Figure 22. Detection results of squalene in the fermentation broth of the bacterial strains

###### 4.4 Determination of squalene in the fermentation broth by GC-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
0.00

BGCII
0.5056
0.0000

![](https://static.igem.wiki/teams/5071/result/image-1.png)

Figure 23. Detection results of squalene in the fermentation broth of the bacterial strains

![](https://static.igem.wiki/teams/5071/home/fvv.png)
Future plans
- For the production of terpenoid compounds, we should consider knocking out some metabolic pathways within the bacterial strains to redirect more carbon flux towards the production of the desired compounds, thereby reducing the production of by-products.
- In our experiments, we did not focus on substance detection but rather on some ancillary detection methods. In the upcoming experiments, we should prioritize the production of a single substance and work to increase its concentration.
- The exploration of the metagenome should be more in-depth to increase the chances of obtaining effective production components, which can better guide subsequent production processes.

![](https://static.igem.wiki/teams/5071/home/fvv.png)
References

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[3] Sunagawa S, Coelho LP, Chaffron S, Kultima JR, Labadie K, Salazar G, Djahanschiri B, Zeller G, Mende DR, Alberti A, Cornejo-Castillo FM, Costea PI, Cruaud C, d'Ovidio F, Engelen S, Ferrera I, Gasol JM, Guidi L, Hildebrand F, Kokoszka F, Lepoivre C, Lima-Mendez G, Poulain J, Poulos BT, Royo-Llonch M, Sarmento H, Vieira-Silva S, Dimier C, Picheral M, Searson S, Kandels-Lewis S; Tara Oceans coordinators; Bowler C, de Vargas C, Gorsky G, Grimsley N, Hingamp P, Iudicone D, Jaillon O, Not F, Ogata H, Pesant S, Speich S, Stemmann L, Sullivan MB, Weissenbach J, Wincker P, Karsenti E, Raes J, Acinas SG, Bork P. Ocean plankton. Structure and function of the global ocean microbiome. Science. 2015 May 22;348(6237):1261359. doi: 10.1126/science.1261359. PMID: 25999513.

[4] Schempp FM, Drummond L, Buchhaupt M, Schrader J. Microbial Cell Factories for the Production of Terpenoid Flavor and Fragrance Compounds. J Agric Food Chem. 2018 Mar 14;66(10):2247-2258. doi: 10.1021/acs.jafc.7b00473. Epub 2017 Apr 18. PMID: 28418659.

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