_A_AgroinfGuidedPathwaywithConstitutivedCas9EV-GCMS

Short Name: AgroinfGuidedPathwaywithConstitutivedCas9EV-GCMS Assay Class: WET Assay Type: GCMS Title: Agroinfiltration of guided pathway for moth pheromones production with constitutive dCas9EV system in Nicotiana benthamiana leaves Description: Two constructs were made that comprised the three genes required for moth pheromone production (AtrD11, HarFar and EaDAct/ATF1) under the regulation of the synthetic promoters created in T11. In the constructs it was also included the guideRNA1 and nptII genes for their use in stable transformation. The constructs were first tested transiently in Nicotiana benthamiana leaves via agroinfiltration, together with a plasmid containing the dCas9EV system expressed constitutively or a plasmid containing just P19 as a negative control. Leaf samples were collected after 5 days and the pheromone production was assayed via GC-MS pISA Assay creation date: 2021-01-07 pISA Assay creator: ElenaMG Lab manager: DO Sample collection protocol: Leaf samples were collected at 5 days post-infiltration, frozen in liquid nitrogen immediately, 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 μL 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 μm 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 80°C 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 250°C 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 250°C for 5 min under a helium flow. Chromatography protocol: Chromatography was performed on a DB5ms (60 m, 0.25 mm, 1 μm) capillary column (JandW) with helium as the carrier gas at a constant flow of 1.2mLxmin-1. The oven programming conditions were an initial temperature of 160°C for 2 min, 7°Cmin-1 ramp until 280°C, and a final hold at 280°C for 6 minutes to reduce the overall running time without losing resolution of the desired compounds. Mass spectrometry protocol: Electron impact ionization (EI), 70 eV ionization energy, MS source temperature 230ºC, MS quadrupole temperature 150ºC, single quadrupole detector, m/z range 35-300. Phenodata: ../../phenodata_20210107 Featuredata: Creation date: 2021-01-07 Extract ID: $_extr Extraction Method: HS-SPME Date Extraction: 2021-01-07 Derivatization or Labelling: none Date Derivatization or Labelling: 2021-01-07 Derivatized or labeled Extract ID: $_extrD Other Post Extraction Procedures: Storage: Date GC-MS Run: 2021-01-07 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 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: ElenaMG Notes: Data: https://doi.org/10.5281/zenodo.5810257