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Lab notebooks

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April 2023

  • Lab introduction
  • Cloning to validate HopAO1 FLS2 interaction using LexA Y2H

May 2023

  • Outgrowth of yeast strains (EGY48, OVY216) for Y2H and rY2H
  • Yeast transformations for HopAO1-FLS2 trials
  • Flow cytometry to test HopAO1-FLS2 interaction using GFP reporter
  • LexA Y2H repeated
  • Cloning for rdY2H
  • HopAO1-FLS2 interaction was not reproducible, alternatives were BAK1 and BIR2
  • Cloning for split-ubiquitin Y2H to prove interaction between BAK1 and BIR2
  • EFR alternative also tested for interaction with HopAO1

June 2023

  • Generate mutations on AvrPto and AvrPtoB
  • Golden Gate of AvrPto, AvrPtoB for split ubiquitin testing
  • Localisation test of AvrPto, AvrPtoB, Snrk2.3 interaction via GFP and mCherry
  • Golden gate to assemble AvrPto-Nub and BAK1- Cub for split ubiquitin
  • Started testing CERK1-AvrPtoB interaction

July 2023

  • Confocal microscopy for localisation check of AHA1 and AvrPto in FRP795
  • rdY2H of AvrPtoB-CERK1 and AvrPtoB-Snrk2.8
  • Introduction of E2A and GS-linker with BleoR using p53 and antigen-T positive control
  • Computational analysis of CERK1-AvrPtoB interaction to find variants

August 2023

  • Cloning of CERK1 and AvrPtoB into E2A-BleoR Y2H system
  • Split ubiquitin Y2H for BAK1-BIR2
  • Introduction of CERK1 kinase dead mutation
  • Preparation of Est-AvrPtoB for transient expression using agrobacteria
  • Infiltration of N.benthamiana and ROS burst assay
  • Cloning of truncated and artificial STOP codon on CERK1 for truncation resistance testing of E2A-BleoR system

September 2023

  • Introduction of computationally generated mutations into CERK1
  • Error prone PCR to generate random mutants
  • rY2H of STOP and truncation mutants of CERK1 using E2A-BleoR system
  • Infiltration of N.benthamiana with agrobacteria for VIGS (CERK1 silencing)
  • Test of alternative self-cleaving peptide (P2A, O2A)
  • Optimisation of ROS burst assays using transient AvrPtoB expression
  • Western blot to check E2A, P2A and O2A cleavage
  • Construction of CERK1 vector for plant infiltration
  • Cloning of 35S, estradiol and native promoters into CERK1 plant vector
  • rY2H of epPCR generated CERK1 variants
  • Sequencing of positive epPCR results
  • Test of CERK1 VIGS using ROS burst
  • Flow cytometry for ymUKG GFP reporter
  • Cell death assay using 35S promoter