Experiments

Introduction

Our 2022 project had a wide variety of demanding experimental procedures which were successfully carried out. These procedures can be divided in three main categories: Preparation, Construct’s production/expression and Final experiment.

Preparation

During the preparation stage, we had to do all the work for the initiation of our main experiments.

  • Preparation of the necessary solid and liquid growth media.

  • Preparations of chemically competent E.coli DH10b and Agrobacterium Tumefaciens C58C1 cells.

  • Preparation and aliquoting of primers for our main parts: OmpA, Nsm115-135, LRR, NB-LRR and OmpA:LRR/OmpA:NB-LRR.

  • Inspection of the 2021 iGEM Distribution Kit for DNA quality and failed attempts to transform competent E.coli cells (probably due to inappropriate storing conditions)

  • PCR to amplify our preferred part (OmpA) right from iGEM’s 2021 Distribution Kit
  • Gel Electrophoresis to verify that we multiplied OmpA and DNA purification
  • PCR to amplify LRR, NB-LRR and Nsm115-135 genes.
  • Gel Electrophoresis for LRR, NB-LRR and Nsm115-135 genes.

Constructs's production-expression

In this experimental stage, our main goal was to gather all of the constructs that were needed in the final experiment

  • Golden Gate Cloning to assemble OmpA:LRR and OmpA:NB-LRR constructs to pLCH86988

  • Transformation of pLCH86988:OmpA:LRR and pLCH86988:OmpA:NB-LRR into E.coli DH10bcells

  • Plasmid DNA extraction and purification

  • Diagnostic restriction enzyme digestion and gel electrophoresis

  • Golden Gate Cloning to assemble Nsm115-135:YFP to pLCH86988

  • Transformation of pLCH86988:Nsm115-135:YFP into Agrobacterium Tumefaciens C58C1 cells

  • Agroinfiltration of Agrobacterium Tumefaciens C58C1 cells with pLCH86988:Nsm115-135:YFP to N. Sylvestris

  • Protein extraction from N. Sylvestris leaves

  • Colony PCR for all constructs

  • PCR to amplify OmpA:LRR and OmpA:NB-LRR

  • Cloning with restriction enzymes SacI and NdeI to assemble pET-2B:OmpA:LRR and pET-2B:OmpA:NB-LRR

  • Cloning product clean-up

  • Transformation of pET-2B:OmpA:LRR and pET-2B:OmpA:NB-LRR into E.coli DH10b cells

  • Plasmid DNA extraction and purification

  • Restriction enzyme digestion and gel electrophoresis

Final experiment

This is our last experiment and essentially the one that will determine the success of our project.

  • Incubation of E.coli cells surface displaying OmpA:NB-LRR and OmpA:LRR with protein extract from Benthamiana leaves expressing Nsm115-135:YFP for 10 min, 20

  • Cell washes with BS (2x)
  • Cell testing for fluorescence in biomolecular imager, indicating binder efficacy.

See more of our Protocols

See more of our Results