Results were divided into three categories, similar to the experiments. More specifically, these categories are Preparation results, Construct’s production/expression results, and Final experiment results.

Preparation’s results

  • We managed to amplify the preferred part (OmpA) from the 2021 iGEM Distribution Kit. Due to the fact that we didn’t know the amount of intact DNA in the kit, we tried multiple hypothetical concentrations of OmpA. These were 2ng, 1ng and 0.5ng.

  • After also performing a PCR for LRR and NB-LRR we verified that we indeed had the right constructs which were expected at 1089bp and 2190bp correspondingly.

Construct production results

  • After performing Golden Gate Cloning for pLCH86988:OmpA:LRR, pLCH86988:OmpA:NB-LRR, and pLCH86988:Nsm115-135:YFP assembly, we transformed E.coli cells, extracted plasmid DNA and performed gel electrophoresis to see if we actually have a plasmid.

  • Colony PCR was a success in amplifying our constructs.

  • After the last cloning in which we assembled the constructs into the expression vector, we did some restriction enzyme digestions to verify that our constructs were right.

Final experiments results

In our final experiment, we used 8 different samples.

Samples 1I, 1II: E.coli surface displaying OmpA:LRR (pET-2B:OmpA:LRR) from the first colony selected

Samples 2I, 2II: E.coli surface displaying OmpA:LRR (pET-2B:OmpA:LRR) from the second colony selected

Samples NBI, NBII: E.coli surface displaying OmpA:NB-LRR (pET-2B:OmpA:NB-LRR)

Sample Ctrl: Our negative control which is E.coli DH10b cells, not expressing anything

Sample PE: Total protein extract of N. Sylvestris leaves expressing the viral Nsm115-135 protein.

Samples 1I, 1II, 2I, 2II, NBI, NBII, and Ctrl were incubated for 2 hours with the total protein extract of N. Sylvestris

After incubation time ended, we pelleted the cells, discarded the supernatant, and washed the cells with PBS (2x). At the end of the final wash, we resuspended the cells in dH2O.

The last step was to place a drop of each sample (~4μL) between two cover glasses and checked for fluorescence in a biomolecular imager.

In this picture, we can see the image of the way the samples were placed in the biomolecular imager.

This picture clearly shows that Fluorescence is clearly visible in all of the test samples (smaller fluorescence in sample 2I). This means that the cells bind the viral Nsm115-135 protein fused with YFP and so fluorescence is visible. A minimal fluorescent signal is visible at the negative control probably due to the low-affinity binding of some E.coli membrane protein with either YFP or Nsm115-135.

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