We've been using DNA uptake assays with fragments having specific differences from the USS consensus to measure how differences at each USS position affect uptake. But these are a lot of work, and I'm considering an alternative approach that would gather all the information in one big experiment.
We'd start with the plasmid that has a perfect USS insert, and subject it to high-efficiency random mutagenesis in the USS segment (say about 40-50bp). This would be done using a mutagenesis kit and a batch of degenerate oligos for this segment. Each degenerate oligo would have a small probability (say 9%) of having a 'wrong' base at each position (3% of each 'wrong' base), so that on average each oligo in the batch would have about 3-4 differences from the consensus. But the distribution would be broad, so a small fraction of the oligos would have one or no changes, and some would have 5 or more.
We'd then use competent cells to select, from the pool of mutagenized plasmid inserts, ones that can be bound or taken up (depending on whether or not we add DNase I to destroy DNA on the outside of the cells).
Then we'd use PCR to amplify the USS inserts of the taken-up sequences, and analyze their genetic diversity. I'm not up on the technology that would be most appropriate - I'll need to ask and search for genome analysis tools. In principle this could be done in two ways - we'd probably want to do both. The first way would be to use some sort of chip or array (?) to determine the proportion of each base at each position in the 40-50bp we've mutagenized. Because this wouldn't tell us anything about the correlations between differences at different positions, we'd also want to sequence some (say 1000?) of the mutagenized segments.
In principle this is just a high-tech version of analysis Sol Goodgal did about 15 years ago.
End of summer &
7 hours ago in The Phytophactor