Field of Science

AAACCCKKK!!! - I screwed up the selection

I did Day 1 of the big experiment yesterday.  But because the NovR DNA fragment the postdoc gave m hadn't transformed very well in the test, and I didn't have enough of it for the large volumes I was transforming, I used MAP7 DNA for the transformations that select for hypercompetent mutants.  At the time I thought the only disadvantages were a slightly lower overall transformation frequency and the 1-2% risk (for each new mutant) that transformation would also replace the mutation itself.

I forgot that, because the MAP7 DNA carries resistance to all the antibiotics we commonly select for, the pool of NovR cells I'd get would contain 1-2% cells resistant to teach of the other antibiotics.  This means that I can't just reselect the cells in the NovR pool for hypercompetence using DNA with another mutation, because this will just select those cells in the pool that got that mutation in the first round of selection for hypercompetence.

I see several options:

Option 1:  Yesterday I froze lots of mutagenized cells that hadn't yet been incubated with any DNA.  I can thaw these out, grow them for an hour at low density, and transform them to NovR with either the NovR DNA fragment or chromosomal NovR DNA that doesn't carry any other antibiotic resistances.  (I may have an old stock of this DNA.)   Then I can grow the pooled cells in medium with novobiocin for 6 hr, dilute them, and do the second-round transformation (NalR or KanR DNA)) and plate.  If I'm using the NovR fragment I may need to do this transformation on cells that have been concentrated - this will increase background, but the next round of hypercompetence-selection should take care of this.

Option 2 (BAD): I can plate the NovR transformants and manually check single colonies for resistance to the antibiotic I want to use next, by toothpicking them onto other antibiotic plates.  Then I could either test them individually for hypercompetence or pool the ones that are sensitive to whatever antibiotic I plan to use next.  Testing them individually would make the planned pool-sequencing unnecessary, and pooling them would be a lot of work.  But I might as well freeze my pools of NovR cells for later manual screening, just in case we  want to do this.

Option 3 (BAD):  I can make DNA from the pooled NovR colonies from the strain carrying the StrR mutation linked to sxy and the strain carrying the CmR mutation lnked to murE, and use this DNA to transform wildtype cells to StrR and CmR. Then I can do another round of hypercompetence selection, transforming the pooled cells with a marker that's not linked to the one I selected for.  BUT, these new pools will probably (maybe) include cells with the unselected antibiotic resistances from the MAP7 DNA.

Expanded plans for Option 1: 
  1. Thaw 1 vial of each of the 7 cultures (wildtype treated with 0, 0.05 and 0.08 mM EMS, StrR and CmR treated with 0.05 and 0.08 mM EMS).  
  2. Dilute way down in sBHI, to a density equivalaent to that used yesterday.  Grow 1 hr or more at low density.  
  3. Look for NovR chromosomal DNA.  If using the PCR fragment, concentrate the cells by filtration just before adding the DNA.
  4. Incubate cells with DNA for 15 or 30 min, then DNase-I-treat, filter, wash, and resuspend in sBHI + Novobiocin.  
  5. Grow 6 hr or more, keeping OD600 below 0.1.  
  6. Transform a fraction of this culture (no benefit from using it all).  Add KanR chromosomal DNA or NalR PCR fragment.  Incubate 15 min, DNase-treat and plate (if KanR) or grow for 90 min before plating (if NalR).

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