I am currently attempting to invert my reporter gene, which is present in an inverted orientation between PhiC31 attB and attP sites. However, when I performed the gene inversion experiment using PhiC31 integrase, no inversion was observed under fluorescence multi mode reader. The PhiC31 integrase is under the control of the PLtetO1 promoter, and the bacterial strain used is E. coli DH5αZ1.
Your inversion might not be working for a few main reasons:
The sites or their orientation might not be right PhiC31 only works on attB and attP in opposite directions, not attL/attR.
The site sequences or spacing could have small errors that stop recombination.
After flipping, the reporter gene might not actually be in a readable form (promoter or RBS ends up backwards, or the protein doesn’t mature/detect properly).
The integrase might not be expressed strongly enough in DH5αZ1, since the promoter you’re using is repressed by default. On the flip side, if expression is too high, the cells can lose it because it’s toxic.
Plasmid context matters, if copy number is very low, or if the DNA piece is too big/awkward, recombination may be inefficient.
PhiC31 is one-way once it flips, it stays locked (attL/attR), so you won’t see dynamic switching unless you add the RDF.
Sometimes PhiC31 will hit “look-alike” sites elsewhere on the plasmid, so the intended flip doesn’t happen
Mohammed Abdelouahab Tabal Thank you for your suggestion. I have tried multiple experiments by changing different bioparts of the plasmids, but currently I am facing a different kind of problem where I observed huge EGFP expression even in the absence of induction, while fluorescence levels decreased upon induction. As a control, cells containing only the attB-attP cassette (without the integrase plasmid) showed no EGFP expression, as expected. I am planning to change RBS for PhiC31 integrase gene containing plasmids. Do you have any further suggestions? I would be grateful if you could share them with me.
For reference, the switch plasmids are designed as follows:
1. J23 promoter + attB + reverse complement of EGFP + reverse complement of RBS + reverse complement of RiboJ + reverse complement of attP + terminator.
2. PltetO1_phiC31 integrase.
It looks like the main issue may be basal expression of PhiC31. A few quick checks and fixes you'd try
Run PCR across the attB–attP region (± induction) to see if the switch is already flipped
Test PltetO1 leakiness by swapping integrase for a GFP reporter
Add a constitutive TetR cassette or move integrase to a low-copy plasmid; also try weaker RBS or a degradation tag.
Simplify the switch cassette (remove reverse RiboJ/RBS) and add strong terminators to rule out cryptic transcription
Titrate aTc carefully, since too much integrase can cause plasmid loss or off-target recombination
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