CONTRIBUTION
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[1]. Two terpene BGCs were obtained by optimization and direct cloning[2]. The first gene cluster was named BGCI, specifically composed of 6 gene fragments, named from BGCI-1 to BGCI-6.The second gene cluster was named BGCII, specifically composed of 9 gene fragments, named from BGCII-1 to BGCII-9.Then the above gene fragments were recombined on the plasmid. After successful recombination, the next fermentation, separation, purification and functional verification were carried out[3-4].
During our experiment, we added some new parts for iGEM part and new information to an existing part (Table 1)
Table 1. Part contributions
Part number Part name Contribution type Part type
BBa_K5071000 BGCI-1 basic part Coding
BBa_K5071001 BGCI-2 basic part Coding
BBa_K5071002 BGCI-3 basic part Coding
BBa_K5071003 BGCI-4 basic part Coding
BBa_K5071004 BGCI-5 basic part Coding
BBa_K5071005 BGCI-6 basic part Coding
BBa_K5071006 BGCII-1 basic part Coding
BBa_K5071007 BGCII-2 basic part Coding
BBa_K5071008 BGCII-3 basic part Coding
BBa_K5071009 BGCII-4 basic part Coding
BBa_K5071011 BGCII-6 basic part Coding
BBa_K5071012 BGCII-7 basic part Coding
BBa_K5071013 BGCII-8 basic part Coding
BBa_K5071014 BGCII-9 basic part Coding
Part contribution
- Add new basic part (BGCI-1, BBa_K5071000)
Name: BGCI-1
Base Pairs: 150 bp
Origin: Bacteriovoracaceae
Usage and Biology
BGCI-1 is a gene identified within a predicted terpene biosynthesis gene cluster, with bioinformatic tools suggesting a potential role in terpene production. Although its precise function remains unknown, sequence analysis indicates the presence of conserved domains common to enzymes involved in secondary metabolite pathways, highlighting its potential importance in the biosynthesis of terpenoids. Further experimental validation is required to elucidate its specific role.
Cultivation
We used PCR to amplify the BGCI-1 gene, with a length of 150 bp. Fig 1. shows a band consistent with the target size, indicating that the target gene were successfully amplified. After agarose gel electrophoresis and gel recovery, homologous recombination was used to obtain the recombinant plasmid pETDuet-BGCI-gene123.
Fig 1. The purpose segment of plasmid pETDuet-BGC1-gene123
- Add new basic part (BGCI-2, BBa_K5071001)
Name: BGCI-2
Base Pairs: 1200 bp
Origin: Bacteriovoracaceae
Usage and Biology
BGCI-2 is a gene identified within a predicted terpene biosynthesis gene cluster, with bioinformatic tools suggesting a potential role in terpene production. Although its precise function remains unknown, sequence analysis indicates the presence of conserved domains common to enzymes involved in secondary metabolite pathways, highlighting its potential importance in the biosynthesis of terpenoids. Further experimental validation is required to elucidate its specific role.
Cultivation
We used PCR to amplify the BGCI-2 gene, with a length of 1200 bp. Fig 1. shows a band consistent with the target size, indicating that the target gene were successfully amplified. After agarose gel electrophoresis and gel recovery, homologous recombination was used to obtain the recombinant plasmid pETDuet-BGCI-gene123.
- Add new basic part (BGCI-3, BBa_K5071002)
Name: BGCI-3
Base Pairs: 500 bp
Origin: Bacteriovoracaceae
Usage and Biology
BGCI-3 is a gene identified within a predicted terpene biosynthesis gene cluster, with bioinformatic tools suggesting a potential role in terpene production. Although its precise function remains unknown, sequence analysis indicates the presence of conserved domains common to enzymes involved in secondary metabolite pathways, highlighting its potential importance in the biosynthesis of terpenoids. Further experimental validation is required to elucidate its specific role.
Cultivation
We used PCR to amplify the BGCI-3 gene, with a length of 500 bp. Fig 1. shows a band consistent with the target size, indicating that the target gene were successfully amplified. After agarose gel electrophoresis and gel recovery, homologous recombination was used to obtain the recombinant plasmid pETDuet-BGCI-gene123.
- Add new basic part (BGCI-4, BBa_K5071003)
Name: BGCI-4
Base Pairs: 1300 bp
Origin: Bacteriovoracaceae
Usage and Biology
BGCI-4 is a gene identified within a predicted terpene biosynthesis gene cluster, with bioinformatic tools suggesting a potential role in terpene production. Although its precise function remains unknown, sequence analysis indicates the presence of conserved domains common to enzymes involved in secondary metabolite pathways, highlighting its potential importance in the biosynthesis of terpenoids. Further experimental validation is required to elucidate its specific role.
Cultivation
We used PCR to amplify the BGCI-4 gene, with a length of 1300 bp. Fig 2. shows a band consistent with the target size, indicating that the target gene were successfully amplified. After agarose gel electrophoresis and gel recovery, homologous recombination was used to obtain the recombinant plasmid pETDuet-BGCI-gene456.
Fig 2. The purpose segment of plasmid pRSFuet-BGC1-gene456
- Add new basic part (BGCI-5, BBa_K5071004)
Name: BGCI-5
Base Pairs: 800 bp
Origin: Bacteriovoracaceae
Usage and Biology
BGCI-5 is a gene identified within a predicted terpene biosynthesis gene cluster, with bioinformatic tools suggesting a potential role in terpene production. Although its precise function remains unknown, sequence analysis indicates the presence of conserved domains common to enzymes involved in secondary metabolite pathways, highlighting its potential importance in the biosynthesis of terpenoids. Further experimental validation is required to elucidate its specific role.
Cultivation
We used PCR to amplify the BGCI-5 gene, with a length of 800 bp. Fig 2. shows a band consistent with the target size, indicating that the target gene were successfully amplified. After agarose gel electrophoresis and gel recovery, homologous recombination was used to obtain the recombinant plasmid pETDuet-BGCI-gene456.
- Add new basic part (BGCI-6, BBa_K5071005)
Name: BGCI-6
Base Pairs: 800 bp
Origin: Bacteriovoracaceae
Usage and Biology
BGCI-6 is a gene identified within a predicted terpene biosynthesis gene cluster, with bioinformatic tools suggesting a potential role in terpene production. Although its precise function remains unknown, sequence analysis indicates the presence of conserved domains common to enzymes involved in secondary metabolite pathways, highlighting its potential importance in the biosynthesis of terpenoids. Further experimental validation is required to elucidate its specific role.
Cultivation
We used PCR to amplify the BGCI-6 gene, with a length of 800 bp. Fig 2. shows a band consistent with the target size, indicating that the target gene were successfully amplified. After agarose gel electrophoresis and gel recovery, homologous recombination was used to obtain the recombinant plasmid pETDuet-BGCI-gene456.
- Add new basic part (BGCII-1, BBa_K5071006)
Name: BGCII-1
Base Pairs: 180 bp
Origin: Paceibacterales
Usage and Biology
BGCII-1 is a gene identified within a predicted terpene biosynthesis gene cluster, with bioinformatic tools suggesting a potential role in terpene production. Although its precise function remains unknown, sequence analysis indicates the presence of conserved domains common to enzymes involved in secondary metabolite pathways, highlighting its potential importance in the biosynthesis of terpenoids. Further experimental validation is required to elucidate its specific role.
Cultivation
We used PCR to amplify the BGCII-1 gene, with a length of 180 bp. Fig 3. shows a band consistent with the target size, indicating that the target gene were successfully amplified. After agarose gel electrophoresis and gel recovery, homologous recombination was used to obtain the recombinant plasmid pACYCDuet-BGCII-gene143.
Fig 3. The purpose segment of plasmid pACYCDuet-BGCII-gene143
- Add new basic part (BGCII-3, BBa_K5071008)
Name: BGCII-3
Base Pairs: 1200 bp
Origin: Paceibacterales
Usage and Biology
BGCII-3 is a gene identified within a predicted terpene biosynthesis gene cluster, with bioinformatic tools suggesting a potential role in terpene production. Although its precise function remains unknown, sequence analysis indicates the presence of conserved domains common to enzymes involved in secondary metabolite pathways, highlighting its potential importance in the biosynthesis of terpenoids. Further experimental validation is required to elucidate its specific role.
Cultivation
We used PCR to amplify the BGCII-3 gene, with a length of 1200 bp. Fig 3. shows a band consistent with the target size, indicating that the target gene were successfully amplified. After agarose gel electrophoresis and gel recovery, homologous recombination was used to obtain the recombinant plasmid pACYCDuet-BGCII-gene143.
- Add new basic part (BGCII-4, BBa_K5071009)
Name: BGCII-4
Base Pairs: 150 bp
Origin: Paceibacterales
Usage and Biology
BGCII-4 is a gene identified within a predicted terpene biosynthesis gene cluster, with bioinformatic tools suggesting a potential role in terpene production. Although its precise function remains unknown, sequence analysis indicates the presence of conserved domains common to enzymes involved in secondary metabolite pathways, highlighting its potential importance in the biosynthesis of terpenoids. Further experimental validation is required to elucidate its specific role.
Cultivation
We used PCR to amplify the BGCII-4 gene, with a length of 150 bp. Fig 3. shows a band consistent with the target size, indicating that the target gene were successfully amplified. After agarose gel electrophoresis and gel recovery, homologous recombination was used to obtain the recombinant plasmid pACYCDuet-BGCII-gene143.
- Add new basic part (BGCII-7, BBa_K5071012)
Name: BGCII-7
Base Pairs: 1000 bp
Origin: Paceibacterales
Usage and Biology
BGCII-7 is a gene identified within a predicted terpene biosynthesis gene cluster, with bioinformatic tools suggesting a potential role in terpene production. Although its precise function remains unknown, sequence analysis indicates the presence of conserved domains common to enzymes involved in secondary metabolite pathways, highlighting its potential importance in the biosynthesis of terpenoids. Further experimental validation is required to elucidate its specific role.
Cultivation
We used PCR to amplify the BGCII-7 gene, with a length of 1000 bp. Fig 4. shows a band consistent with the target size, indicating that the target gene were successfully amplified. After agarose gel electrophoresis and gel recovery, homologous recombination was used to obtain the recombinant plasmid pETDuet-BGCII-gene792.
Fig 4. The purpose segment of plasmid pETDuet-BGCII-gene792
- Add new basic part (BGCII-9, BBa_K5071014)
Name: BGCII-9
Base Pairs: 750 bp
Origin: Paceibacterales
Usage and Biology
BGCII-9 is a gene identified within a predicted terpene biosynthesis gene cluster, with bioinformatic tools suggesting a potential role in terpene production. Although its precise function remains unknown, sequence analysis indicates the presence of conserved domains common to enzymes involved in secondary metabolite pathways, highlighting its potential importance in the biosynthesis of terpenoids. Further experimental validation is required to elucidate its specific role.
Cultivation
We used PCR to amplify the BGCII-9 gene, with a length of 750 bp. Fig 4. shows a band consistent with the target size, indicating that the target gene were successfully amplified. After agarose gel electrophoresis and gel recovery, homologous recombination was used to obtain the recombinant plasmid pETDuet-BGCII-gene792.
- Add new basic part (BGCII-2, BBa_K5071007)
Name: BGCII-2
Base Pairs: 2000 bp
Origin: Paceibacterales
Usage and Biology
BGCII-2 is a gene identified within a predicted terpene biosynthesis gene cluster, with bioinformatic tools suggesting a potential role in terpene production. Although its precise function remains unknown, sequence analysis indicates the presence of conserved domains common to enzymes involved in secondary metabolite pathways, highlighting its potential importance in the biosynthesis of terpenoids. Further experimental validation is required to elucidate its specific role.
Cultivation
We used PCR to amplify the BGCII-2 gene, with a length of 2000 bp. Fig 4. shows a band consistent with the target size, indicating that the target gene were successfully amplified. After agarose gel electrophoresis and gel recovery, homologous recombination was used to obtain the recombinant plasmid pETDuet-BGCII-gene792.
- Add new basic part (BGCII-6, BBa_K5071011)
Name: BGCII-6
Base Pairs: 500 bp
Origin: Paceibacterales
Usage and Biology
BGCII-6 is a gene identified within a predicted terpene biosynthesis gene cluster, with bioinformatic tools suggesting a potential role in terpene production. Although its precise function remains unknown, sequence analysis indicates the presence of conserved domains common to enzymes involved in secondary metabolite pathways, highlighting its potential importance in the biosynthesis of terpenoids. Further experimental validation is required to elucidate its specific role.
Cultivation
We used PCR to amplify the BGCII-6 gene, with a length of 500 bp. Fig 5. shows a band consistent with the target size, indicating that the target gene were successfully amplified. After agarose gel electrophoresis and gel recovery, homologous recombination was used to obtain the recombinant plasmid pRSFuet-BGCII-gene685.
Fig 5. The purpose segment of plasmid pRSFuet-BGCII-gene685
- Add new basic part (BGCII-8, BBa_K5071013)
Name: BGCII-8
Base Pairs: 350 bp
Origin: Paceibacterales
Usage and Biology
BGCII-8 is a gene identified within a predicted terpene biosynthesis gene cluster, with bioinformatic tools suggesting a potential role in terpene production. Although its precise function remains unknown, sequence analysis indicates the presence of conserved domains common to enzymes involved in secondary metabolite pathways, highlighting its potential importance in the biosynthesis of terpenoids. Further experimental validation is required to elucidate its specific role.
Cultivation
We used PCR to amplify the BGCII-8 gene, with a length of 350 bp. Fig 5. shows a band consistent with the target size, indicating that the target gene were successfully amplified. After agarose gel electrophoresis and gel recovery, homologous recombination was used to obtain the recombinant plasmid pRSFuet-BGCII-gene685.
- Add new basic part (BGCII-5, BBa_K5071010)
Name: BGCII-5
Base Pairs: 1500 bp
Origin: Paceibacterales
Usage and Biology
BGCII-5 is a gene identified within a predicted terpene biosynthesis gene cluster, with bioinformatic tools suggesting a potential role in terpene production. Although its precise function remains unknown, sequence analysis indicates the presence of conserved domains common to enzymes involved in secondary metabolite pathways, highlighting its potential importance in the biosynthesis of terpenoids. Further experimental validation is required to elucidate its specific role.
Cultivation
We used PCR to amplify the BGCII-5 gene, with a length of 1500 bp. Fig 5. shows a band consistent with the target size, indicating that the target gene were successfully amplified. After agarose gel electrophoresis and gel recovery, homologous recombination was used to obtain the recombinant plasmid pRSFuet-BGCII-gene685.
Other Contributions
Our contributions are mainly two folds :
( 1 ) By analyzing the composition and distribution of microorganisms in deep-sea ecosystems, the potential high-value terpene secondary metabolite gene resources are systematically explored, which helps to improve the understanding of deep-sea microbial functions and product diversity, and provides a research basis for the sustainability and ecological protection of marine ecosystems.
( 2 ) Exploring the biosynthesis mechanism of terpenoid secondary metabolites in deep-sea microorganisms and building a microbial synthesis expression platform can expand the research content of terpenoid secondary metabolites biosynthesis, provide important scientific basis for efficient biosynthesis and synthesis process optimization, and also provide a good example for the future iGEM team.
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.