_A_SxPAlternativeAcetyltransferases-GCMS

Short Name: SxPAlternativeAcetyltransferases-GCMS Assay Class: WET Assay Type: GCMS Title: Assay of transient expression of alternative acetyltransferases Description: The purpose of this assay is to test the efficiency of conversion of Z11-16OH to Z11-16OAc, in the search of a more efficient alternative to the already used EaDact acetyltransferase gene, chosen for the generation of the SxPv10 and SxPv12. The expression is assayed in a transient expression assay on WT Nicotiana benthamiana plants, agroinfiltrated with the combination of the two previous genes required in the metabolic pathway of moth sex pheromones plus the assayed acetyltransferase gene. Samples were finally measured by GC-MS. pISA Assay creation date: 2021-11-25 pISA Assay creator: RMF Lab manager: DO Sample collection protocol: Between 5 and 6 disks were collected 5 days postinfiltration using a 1.5-2 cm corkborer and snap frozen in liquid nitrogen. 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 ‘L 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 ‘m 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 ‘m) 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 160›C for 2 min, 7›Cmin-1 ramp until 280›C, and a final hold at 280›C for 6 minutes. Mass spectrometry protocol: Electron impact ionization (EI), 70 eV ionization energy, MS source temperature 230őC, MS quadrupole temperature 150őC, single quadrupole detector, m/z range 35-300. Phenodata: ../../phenodata_20200723.txt Featuredata: Creation date: 2020-07-23 Extract ID: $_extr Extraction Method: HS-SPME Date Extraction: 2020-07-23 Derivatization or Labelling: none Date Derivatization or Labelling: 2020-07-23 Derivatized or labeled Extract ID: $_extrD Other Post Extraction Procedures: Storage: Date GC-MS Run: 2020-07-23 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.5810507