Most of the old P1 lysates I had left on my bench were dead, though one was OK and the ones in the fridge were still pretty good. So I made fresh lysates yesterday on the wildtype strain W3110, but the titers are lower than I expected, and maybe not even high enough to give acceptable transduction.
So I'm going to do one more try. I found what looks to be an excellent protocol on the Open Wetware pages. I'll follow their instructions exactly (no more "I'm so smart I can safely cut corners"). I'll also try the lysate I generated yesterday, and make fresh lactose plates for the selection. Two days ago I streaked the donor and recipient strains on lactose plates to check how the donor and recipient grow. The donor grows well, giving small colonies after 24 hrs and big ones after 48 hrs. The recipient does grow a bit on lactose, giving tiny colonies after 48 hours, presumably because it lacks the lactose uptake protein but can grow very slowly on lactose that leaks in to the cell. These tiny colonies are what I saw in my last transduction.
Remember why I'm doing these transductions? I want to combine the ppdD::lacZ fusion (indicator of CRP-S promoter activity and thus of Sxy and CRP activity) with a sxy knockout and with a crp knockout to find out whether the high constitutive expression of the fusion depends on its CRP-S promoter. And I'm using the ppdD::lacZ fusion because I want to find out how to induce Sxy activity and CRP-S promoter activity in E. coli. If I find that the constitutive activity is dependent on Sxy and CRP, I will suspect that such promoters are normally moderately active. If the activity is not Sxy and CRP dependent, I will conclude that the fusion is not a good indicator and not work with it any more.
In Tuesday's post I described checking some plasmids, to see that they had the expected inserts. We asked for these plasmids (gift from other researchers) because they too contain fusions of CRP-S promoters to lacZ, and can be used in the same way as the ppdD chromosomal fusion. The two promoters are from the ppdA gene and the hofM gene, which are both homologs of CRP-S genes that H. influenzae requires for DNA uptake.
So yesterday I compared the amount of beta-galactosidase (product of lacZ) produced by cells carrying these plasmids to that produced by the ppdD fusion. Even though cells have many more copies of the plasmids than of a chromosomal fusion, they produced quite a bit less beta-galactosidase. I don't need to use P1 transduction to check whether this expression is dependent on Sxy and CRP. Because I already have preps of the plasmids, and I have strains carrying the sxy and crp knockouts, I can just transform the plasmids into the knockout strains, selecting for the ampicillin resistance of the plasmids and the kanamycin resistance of the knockouts. Even better, one of the post-docs has offered me some already-competent cells of the crp knockout.
1 day ago in Variety of Life