_A_SxPGuidedPathway-GCMS

Short Name: SxPGuidedPathway-GCMS Assay Class: WET Assay Type: GCMS Title: SxP Guided Pathway (GP) Description: Generation and analysis of the stable Nicotiana benthamiana transgenic plants with the moth sex pheromone biosynthetic pathway genes regulated by synthetic promotors and provided with the specific guide in stable. The purpose of this assay is to measure the volatile moth sex pheromone production by HSPME GCMS of the generated stable Nicotiana benthamiana plants. The activation of the pathway was performed by agroinfiltration of the dCasEV in trans, so the system is complete and the production of the pheromones is triggered. pISA Assay creation date: 2021-12-17 pISA Assay creator: RMF Lab manager: DO Sample collection protocol: Leaf samples were collected 5 days after agroinfiltration with the dCasEV, at 4 weeks after transplant for young stage and at 7 weeks for adult stage (early flowering). Samples were collected between 4 and 6 pm, frozen in liquid nitrogen immediately after collection, and ground afterwards. Extraction protocol: 50mg of frozen, ground leaf samples were weighed in a 10mL headspace screw-cap vial and stabilized by adding 1mL of 5M CaCl2 and 150 ćL of 500mM EDTA (pH = 7.5), after which they were sonicated for 5 minutes. Volatile compounds were captured by means of headspace solid phase microextraction (HS-SPME) with a 65 ćm polydimethylsiloxane/divinylbenzene (PDMS/DVB) SPME fiber (Supelco, Bellefonte, PA, USA). Volatile extraction was performed automatically by means of a CombiPAL autosampler (CTC Analytics). Vials were first incubated at 80řC for 3 minutes with 500 rpm agitation. The fiber was then exposed to the headspace of the vial for 20 min under the same conditions of temperature and agitation. Desorption was performed at 250řC for 1 minute (splitless mode) in the injection port of a 6890N gas chromatograph coupled to a 5975B mass spectrometer (Agilent Technologies). After desorption, the fiber was cleaned in a SPME fiber conditioning station (CTC Analytics) at 250řC for 5 min under a helium flow. Chromatography protocol: Chromatography was performed on a DB5ms (60 m, 0.25 mm, 1 ćm) capillary column (JandW) with helium as the carrier gas at a constant flow of 1.2mLxmin-1. The oven conditions started with an initial temperature of 160řC for 2 min, 7řCmin-1 ramp until 280řC, and a final hold at 280řC for 6 minutes. Mass spectrometry protocol: Electron impact ionization (EI), 70 eV ionization energy, MS source temperature 230řC, MS quadrupole temperature 150řC, single quadrupole detector, m/z range 35-300. Phenodata: ../../phenodata_20210920.txt Featuredata: Creation date: 2021-12-17 Extract ID: $_extr Extraction Method: HS-SPME Date Extraction: 2021-09-20 Derivatization or Labelling: none Date Derivatization or Labelling: 2021-09-20 Derivatized or labeled Extract ID: $_extrD Other Post Extraction Procedures: Storage: Date GC-MS Run: 2021-09-20 GC Instrument: 6890N gas chromatograph (Agilent Technologies) GC Autosampler Model: CombiPAL autosampler (CTC Analytics) GC Column model: DB5ms (60m, 0.25 mm, 1um) capillary column (JandW) GC Column type: capillary column Guard Column: MS Scan polarity: positive MS Scan mz range: 35-300 MS Instrument: 5975B mass spectrometer (Agilent Technologies) MS Ion source: electron ionization (EI) Mass analyzer: quadrupole mass filter Operator: RMF Notes: Data: https://doi.org/10.5281/zenodo.5811855