screwcap flasks  (Corning Life Sciences)

Journal: bioRxiv

Article Title: Engineering genetically-encoded synthetic biomarkers for breath-based cancer detection

doi: 10.1101/2021.09.01.456741

Figure Lengend Snippet: ( A) Schematic of experimental methodology. (Top) Cultured HeLa cells were transfected with a vector containing LS and eGFP genes under the control of a CAG promoter. Antibiotic and FACS selection for stably transfected clones (sorting on eGFP-expressing cells) resulted in a HeLa cell line containing both LS and eGFP (HeLa-LS-eGFP cells, subsequently referred to as HeLa-LS cells). ( Bottom ) HeLa-LS cells were subsequently transfected with a vector containing the tHMGR and tRFP genes under the control of an EF1α promoter. Antibiotic and FACS selection (based on dual expression of eGFP and tRFP) resulted in a HeLa cell line containing LS, tHMGR, eGFP, and tRFP (HeLa-LS-tHMGR-eGFP-tRFP, subsequently referred to as HeLa-LS-tHMGR). Solid phase microextraction (SPME) fibers were used to sample the culture headspace of confluent stably transfected HeLa-LS and HeLa-LS-tHMGR cells for 30 minutes, and were then analyzed for limonene by GC-MS. (B) (i) Piggybac transposon DNA vector containing truncated limonene synthase (LS) and enhanced green fluorescent protein (eGFP) driven by a CAG promoter, and puromycin resistance gene driven by a CMV promoter. (ii) Piggybac transposon DNA vector containing truncated HMG CoA reductase (tHMGR) and turbo red fluorescent protein (tRFP) driven by an EF1α promoter, and hygromycin resistance gene driven by a CMV promoter. (C) Bright-field and fluorescence images showing HeLa-LS and HeLa-LS-tHMGR cells after antibiotic selection and FACS sorting, compared with untransfected control HeLa cells. Scale bar = 200 um for HeLa control and 400 μm for HeLa-LS and HeLa-LS-tHMGR. (D) Mass spectrum from an SPME fiber exposed to the headspace of confluent HeLa-LS cells (top, red) compared with the reference spectrum of limonene from a mass spectrum library (Mnova database) (bottom, black). Note the characteristic peaks at m/z = 68, 93, and 136. (E) Selected ion monitoring (SIM) mode chromatogram of an SPME headspace sample from HeLa-LS cells (left) and from a pure limonene standard (right), showing matching ion ratios and retention times. (F) Calibration curve relating headspace limonene concentration as measured by SIFT-MS to the quantity of limonene spiked into culture media in a T75 flask ( y = 0.62 x 0.86 , R 2 =0.99). Over the range of limonene production by cultured cells (1 to 1000 ng, red bracket), the relationship is well-modeled by y = 0.28 x (R 2 =0.99). (G) Headspace concentration of limonene as a function of cell number for HeLa-LS ( y = [1.56×10 −6 ] x + 1.06, R 2 = 0.99) and HeLa-LS-tHMGR cells ( y = [3.21×10 −6 ] x + 2.70, R 2 = 0.98) after incubation at 37°C for 24 hours. Limonene measured from HeLa-LS-tHMGR cells was approximately double that from HeLa-LS cells over the cell density range examined.

Article Snippet: Serial dilutions of pure limonene (Sigma Aldrich, St. Louis, MO) in ethanol were prepared in Eppendorf tubes and spiked into 10 mL of media (DMEM with 10% FBS) to final concentrations ranging from 0.01 ng to 100 μg in T75 flasks with screwcap septa (MIDSCI, St. Louis, MO).

Techniques: Cell Culture, Transfection, Plasmid Preparation, Selection, Stable Transfection, Clone Assay, Expressing, Solid-phase Microextraction, Gas Chromatography-Mass Spectrometry, Fluorescence, Concentration Assay, Incubation