Dr Jack writes:
It does indicate an increase in fitness, but it does not indicate that the random DNA sequence is coding for anything of consequence. They want to argue that the sequences they've introduced are doing something of consequence. I think the more obvious interpretation is that they are not doing anything in, and of, themselves but rather preventing a harmful effect.
That is a fair criticism. It would be much more helpful if they moved these genes into the bacterial chromosome so they could compare fitness to the wild type strain, and especially so for the FLAG-only version of the gene. It certainly wouldn't be feasible for the thousands of random sequences in their library, but they could have at least done this with their control plasmid.
Quite. But they measure (organism) fitness using the proxy of the number of copies of the plasmid in their population; if the plasmid varies systematically in copy number that will bias their results.
A very good point. In my own experience, plasmid stability can vary quite a bit due to different inserts in the same plasmid.
RNA can certainly have activity; however the majority of active sequences in the body are protein coding. If you're claiming that you have hundreds of beneficial sequences but only one is influenced by the STOP codon that implies a ratio of active protein sequences to RNA sequences I find implausible.
From my reading, they only tested 3 clones individually, 2 of which were not affected by the insertion of a stop codon. From the paper:
"Although two of our three individually tested clones suggest that the RNA function could be more important than the protein function, this constitutes at present only a small sample and may not be indicative of the true ratio between RNA and peptide functions."