I want to insert a reporter gene (eg, GFP) right after a promoter on a plasmid by overlap PCR. However, this method will add some non-coding spacer sequences (~20bp) in between the promoter and GFP. Do you think it will affect the expression of the GFP?
Usually the presence of spacer does not drastically affect the expression level, however, make sure there is no ATG in the spacer region otherwise the ORF will change.
If it changes the secondary structure of the mRNA around the translational initiation site (and it almost certainly will) it may affect expression, negatively or possitively. For a relatively well-informed guess, compare the score of your proposed construct to one without the spacer using something like the RBS Calculator of the Salis group (http://salis.psu.edu/software/).
Are you adding the spacer to create the overlapping region? If yes, could you instead append the first 20 bp of your reporter gene to the plasmid and use those as overlapping region?
It should not be a problem. We added three different 72bp ER - signal sequences in front of GFP respectively and GFP expression level in 2 out of 3 constructs were higher than that from the control and one construct with the ER- signal sequence got lower level GFP expression.
Usually the presence of spacer does not drastically affect the expression level, however, make sure there is no ATG in the spacer region otherwise the ORF will change.
As far as ribosomal binding site/SD sequence upstream to start codon taken care than there should not be an issue. Also see non-coding spacer seq should not have these seq.
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