I made a plant viral vector expressing GFP. I got this GFP from a previously made construct (a viral vector of the same type) which I PCR amplified the GFP from, and used restriction enzyme cloning to clone the GFP into the new viral vector I made. This was confirmed via sequencing. However, after infiltrating my new vector into benthamiana, the GFP expression is very pale and weak. As in, the viral vector is definitely replicating and moving systemically, however, the GFP fluorescence is a pale, light green. This is in comparison to what I was expecting, the characteristic bright green fluorescence usually seen when using a GFP-expressing viral vector. The original vector that I got GFP from before I cloned it into my new vector produces the characteristic bright green fluorescence, as well. Does anybody have any ideas as to why the vector I made expresses GFP that is paler and weaker instead of the normal bright green GFP fluorescence? I attached two photos for reference. Photo 1 is a plant infiltrated with my new vector. Photo 2 is the characteristic GFP expression I was expecting, from the original GFP vector.
Hi April, nice images! The expression intensity might depend on the type of promoter you use in different systems. Have you re-cloned the whole cassette including the original promoter?
Hi April, nice images.
You can increase your protein expression by adding codon Kozak consensus sequence before your start codon.
Another problem could be that your target organism has more GC-containing DNA and your GFP version is more AT-based. This would result in less tRAN being available for protein synthesis.
Hi April DeMell , it looks like the one which was newly created has less within-host multiplication. Assuming it is a viral RNA vector, if you have the WT clone without any modification, you may want to check the viral RNA accumulation and compare it that to the old vector. This is kind of expected from viruses, though they are the same strain still, there is huge variations in replication capacity. You may want to check this paper of mine that shows variations in virus isolates replication belonging to the same genus: https://doi .org/10.1128/JVI.01268-18. The reasons can be variation in 5'/3' UTR, RNA secondary structures, or polymerase.
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