This time I'll use two different preps of the same shuttle vector pSU20 - one prep of plasmid grown in E. coli and one of plasmid grown in H. influenzae. (I would have done this the first time but couldn't find a H. influenzae-grown prep. Today I streaked out the right cells from our freezer stocks, and tomorrow I'll make a plasmid prep for this experiment.)
I expect the E. coli-grown plasmid to transform quite poorly, because the DNA will lack methylation at the HindII and HincII restriction sites. In my first experiment the difference was dramatic: I got 10,000 transformants into E. coli but none into H. influenzae. The Rd strain of H. influenzae carries both of these restriction systems (Ham Smith got a Nobel Prize for discovery of these), and its cytoplasm is chock full of the HindIII and HincII restriction enzymes. Although its own DNA is appropriately methylated and thus immune to cleavage, incoming DNA from other species is cut. The restriction enzymes only cut double-stranded DNA so they don't affect chromosomal transformation (watching the DNA uptake movie might help this make sense), but plasmids introduced by electroporation or plasmid transformation remain double-stranded, so they're vulnerable.
I'll also use E. coli-grown and H. influenzae-grown preps of the noviobiocin-resistance plasmid pRRnov1. In my quick-and-dirty experiment I used a very old tube of this plasmid, grown in H. influenzae. This gave about 100 transformants into H. influenzae but none into E. coli.
Thinking about the impact of endogenous restriction enzymes prompted the postdoc to wonder if this might be a limiting factor in the RAs' recombineering work - it might help explain why her attempts to make 'unmarked' deletion mutants sometimes succeeds and sometimes fails.
First, my understanding of the process she uses:
She starts with mutant cells she's created - in these cells a specific competence gene has been deleted and replaced with a cassette giving resistance to spectinomycin. Because this cassette can disrupt expression of downstream genes she wants to excise it from the chromosome, leaving an 'unmarked' mutation.
To excise the SpcR insert, she first electroporates a plasmid with the flp recombinase (pRSM2947?) into the H. influenzae cells carrying the SpcR-marked mutation. The SpcR cassette is flanked by DNA sequences that that are cut and rejoined by the Flp enzyme, excising the cassette. The plasmid is also temperature-sensitive (Ts) so cells must be plated at 30°C. If this electroporation works (sometimes it doesn't, perhaps due to restriction)) she gets H. influenzae cells that carry the excision plasmid.
She then induces the excision genes (by adding tetracycline), plates the cells, and screens for colonies that are now SpcS. Plating at 37°C ensures that the cells also lose the Ts plasmid. Sometimes the induction works well (more than 1% of the colonies are SpcS) and sometimes it doesn't work at all.
So the other day the postdoc pointed out that restriction and subsequent repair of an E. coli-grown plasmid transformed into H. influenzae might sometimes produce defective versions of the plasmid, versions that are able to replicate and that confer the expected antibiotic resistance but that have defects in other plasmid-borne functions.
When the RA electroporates pRSM2947 into the marked mutants, I think she uses a plasmid stock that was grown in E. coli. Cleavage of this plasmid by the endogenous restriction enzymes might sometimes give plasmids with defects in the flp recombinase gene. These cells would then fail to produce any unmarked derivatives on induction with tetracycline. Even if she routinely checks the transformants for presence of the pRSM2947 plasmid, she might not detect that the plasmid now has a defect. So maybe one way to improve the efficiency of the excision process is to start with a stock of pRSM2947 that has been grown in H. influenzae.
How is this work going to be useful for our upcoming grant proposal, you might ask? I'm asking myself that too. More on this later.
Update: The RA tells me that she switched to using H. influenzae-grown plasmid a while back, but that this didn't solve the excision problems.