donny

Ok, I think I'll try adapting this method using a combination of RED and P1 transduction.
http://www.ncbi.nlm....pubmed/22127754

Still the question, where to insert? I will be generating foreign metaboites so there no way to know if non-essential genes, ie those in the KEIO collection, are actually important for the tolerance towards these metabolites. I'm thinking of inserting the genes into "barren" region in the genome. For example, as shown in the attached photo, there is a ~5kbp region that has apparently no genes. Is it safe to insert genes into this region?

 e coli genome.jpg (109.32K)
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It is common for protein expression to decrease with subculturing. As protein expression stresses the cells and reduces the cell growth rate, cells with spontaneous mutations that reduce protein expression will have a growth advantage and gradually dominate the population. For example, it has been shown that the amount of functional T7 RNA polymerases in BL21(DE3) strain decreases after multiple subculturing due to spontaneous mutation (http://www.microbial...m/content/4/1/3). This in turn reduces overexpression of the target protein from a pET vector. Even if it's not a pET system, mutations to the promoter that reduces overexpression will give the same effect. Since the antibiotic resistance gene is usually constitutively expressed and independent of the protein expression, your cells would still be resistant to puromycine but the protein expression ability is impaired.

I actually moisten the paper with plain LB and incubated for 1 h before I placed them on selective plate/medium. I guess that was not enough. In retrospect, I probably should have grown them up in plain LB and streak the overnight culture on selective plates. Btw, the plates were incubated for 2 days.

I didn't use agar slant as I wanted the most convenient and inconspicuous way to bring it with me onto the plane. I was afraid agar slant would be a bit suspicious on the X-ray. I read a paper that recovery from freeze dried cells is lower than air dried ones.

I would have tried but it was a hasty departure and I didn't have the time to test it out. Also, I was searching the web and it seems like a routine procedure. I have seen a colleague recover cells from a disc sent by CGSC and she got a lawn of colonies.

With reference to leelee's comment, the link given is for short oligos. For a vector, this link is more apt because linearised plasmids would have one longer flanking end that may be sufficient for digestion. However, since SmaI is not listed in the link, it may "require 6 base pairs on either side of their recognition site to cleave efficiently" as stated in the link. Therefore the long flanking end may or may not be enough for cleavage of the SmaI site.

When NEB suggests sequential digestion, it is usually to minimise star activity. In this case, it may be due to the difference in digestion temperature as well (25C for SmaI and 37C for EcoRI). Since they both have 100% activity in NEBuffer 4, I suggest you try double digestion in the buffer first (first at 25C then 37C) and see if it works [I've worked against the sequential digestion advice with no problem ]. But since EcoRI seems to have less problem cleaving a site with short flanking ends, it may be better to digest with SmaI at 25C, then add EcoRI and digest at 37C. This way, SmaI would be digesting the plasmid with two long ends flanking the restriction site and EcoRI will be left to do the "tough work" of digesting the linearised vector with the EcoRI site flanked by 3bp. Make sure the glycerol concentration is kept below 5%.