First, we added some new documentation to an existing Part BBa_25F2FZCH on that Part's Registry page.
1. Introduction
Figure 1 shows the restriction enzyme sites on the gene of the BBa_25F2FZCH part.
Fig.1 All the restriction enzyme sites on the part BBa_25F2FZCH.
Geranylgeranyl Pyrophosphate Synthase (GPPS) is a key enzyme for Geranylgeranyl Pyrophosphate (GPP) synthesis and also a gatekeeping enzyme linking central metabolism (MVA/MEP pathways) to the biosynthesis of the large monoterpenoid family[1]. The reaction catalyzed by GPPS is showed in below (Fig.2).
Fig.2 Synthesis of GPP catalyzed by GPPS using IPP and DMAPP.
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2. Construction of pYES2-GPPS-GES recombinant plasmid
GPP is converted to geraniol catalyzed by geraniol synthase (GES). To produce more geraniol in Saccharomyces cerevisiae (S. cerevisiae), we constructed the two genes (GPPS and GES) under the control of a single promoter of pYES2 plasmid.
Fig.3 GPPS and GES genes were constructed into the pYES2 plasmid, followed by transformation into S. Cerevisiae.
To reduce the metabolic burden on yeast cells and enable co-expression of GPPS and GES in cells, we linked these two genes by a ribosomal binding site (RBS) and constructed them together under the control of the galactose-inducible promoter PGAL1. This design allows the two genes to be transcribed simultaneously while being translated into two independent proteins.
Fig.4 Construction and identification of pYES2-GPPS-GES recombinant vector using BamH I and EcoR I restriction enzymes.
M: Marker; 1: pYES2 plasmid; 2: pYES2-GPPS-GES recombinant plasmid; 3: Digestion of pYES2-GPPS-GES using BamH I and EcoR I; 4: PCR product of GPPS using pYES2-GPPS-GES as template; 5: PCR product of GES using pYES2-GPPS-GES as template.
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3. Detection geraniol production using gas chromatography
To investigate whether yeast cells can synthesize geraniol after transformation of GPPS and GES, gas chromatography (GC) was employed for analysis. When the yeast cells grew to a certain concentration (OD₆₀₀ = 0.8), 2% galactose was used to induce the expression of GPPS and GES, followed by continued cultivation for 24 h. A two-phase extraction method was adopted: n-dodecane was used to extract geraniol from the culture, and geraniol standard was used as a control for analysis via gas chromatography. Two samples were included in the experiment: Sample 1 was yeast (without ERG20 gene knockdown) overexpressing GPPS and GES; Sample 2 was yeast (with ERG20 gene knockdown) overexpressing GPPS and GES.
Fig.5 Gas chromatography analysis for geraniol production by S. cerevisiae.
The retention time of geraniol standard was 5.65 min; the retention time of geraniol produced by S. cerevisiae was 5.63 min.
The experimental results showed that the retention time of the geraniol standard was 5.65 min. Sample 1 contained little to no geraniol. Sample 2 exhibited a peak with a retention time (5.63 min) essentially consistent with that of the geraniol standard, indicating geraniol synthesis. The concentration of geraniol in Sample 2 was determined to be 8.58 mg/L. However, this concentration is relatively low, necessitating further optimization of the synthesis conditions.
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Second, we created 3D-printed aroma diffuser to demonstrate the utility of our product.
To accelerate the transformation of our project into a market-ready product, we have designed practical product application examples, developed a mini aroma diffuser, and 3D-printed 2 diffuser prototypes.
Users can customize essential oil products with specific ratios of citronellol and linalool based on their personal preferences and needs - such as "pre-sleep relaxation", "morning awakening", or "forest pulse" - and they can even create their own unique types.
Simply place our product directly into the aroma diffuser, and it can be used after dilution. Users can set the aromatherapy mode independently, including the mist output level, aromatherapy duration, and whether to enable the neon light companion function. For more details on product development, please refer to our "Entrepreneurship".
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Fig.6 The Process of Making an Aroma Diffuser via 3D Printing.
Fig.7 Demonstrating the utility of 3D-Printed Aroma Diffuser.
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References:
[1]. Song S, Jin R, Chen Y, et al. The functional evolution of architecturally different plant geranyl diphosphate synthases from geranylgeranyl diphosphate synthase. Plant Cell. 2023;35(6):2293-2315. doi:10.1093/plcell/koad083