I'm hoping for some help from the microbiologist community. I'm creating lacZ mutants from laboratory strain e.coli. I want to remove endogenous blue/white screening capability and screen libraries (fosmid and phage) for novel B-galactosidase activity. I'll be doing this by homologous recombination by using pKD46 recombineering vectors targeting the e.coli LacZ gene and thereby removing the ability to alpha complement. I will be creating mutants from three strains given below with their genotypes:
DH5α Turbo (NEB)
F´ proA+B+ lacIq ∆ lacZ M15/ fhuA2 ∆(lac-proAB) glnV gal R(zgb-210::Tn10)TetS endA1 thi-1 ∆(hsdS-mcrB)5
SOLR (Stratagene)
e14-(McrA-) Δ(mcrCB-hsdSMR-mrr)171 sbcC recB recJ uvrC umuC::Tn5 (Kanr) lac gyrA96 relA1 thi-1 endA1 λR [F’ proAB lacIqZ ΔM15]C Su-
EPI300 (EpiBio)
F- mcrA Δ(mrr-hsdRMS-mcrBC) Φ80dlacZΔM15 ΔlacX74 recA1 endA1 araD139 Δ(ara, leu)7697 galU galK λ-rpsL (StrR) nupG trfA dhfr
The strians differ in their LacZ muations in that both DH5 and SOLR carry ∆ lacZ M15 mutations on the endogenous chromosome, but epi300 carry the mutation on a prophage insertion (Φ80dlacZΔM15) while the whole endogenous lac operon was deleted ( ΔlacX74).
So here's my questions, please bear with me seeing s I'm not a microbiologist by trade. I cant find any information on this inactivated prophage information including any sequence. Can I use the same e.coli lacZ sequence for recombination in all three strains? Furthermore does the prophage insertion carry the other lac operon genes or not and how will this affect lactose metabolism by the cells.
Since they all contain the same LacZ portion, you can insert for example a selectable marker somewhere into it disrupting the LacZ fragment, without knowing what exactly is in the prophage.
Do note that all these strains are white (not blue) already since they need the alpha-LacZ fragment generally contained on your plasmid. If you remove the alpha-LacZ fragment in your library screening backbone, you should be good to go too. Then you do not have to worry about the different mutations in the genome.
To answer your specific question about the lacZ sequence in the phi80 prophage genome, yes it is the same lacZ but whether or not you can use the same sequence to recombineer depends on the sequence you intend to introduce. There is much less flanking material around the lacZ, therefore if you are using internal lacZ homology you should be ok but if you are using upstream and downstream regions for homology then you may not be. You could easily test this by a simple PCR from the EPI300 genome with your primers.
Having said that, I suggest you may wish to consider an entirely alternative approach. I am guessing that the strain you wish to use is deleted for the lacZ gene or perhaps for the entire lac operon. There are many existing cloning strains that are deleted for either lacZ only or for the entire lac operon. It would be much easier just to obtain a strain with the genotype you want than to try to build your own. For example the first two strains you list have the lac operon on the F episome and the most likely event is going to be loss of the entire F. In fact if you want to do recombineering I would recommend you begin with a lac+ strain so that you can easily detect the final lac- recombinant without difficulty.
However many strains exist already that you can directly use without modification. Which strain you use depends on whether you are going to screen your library for blue on XGal in which case the only relevant thing is the galactosidase gene. However if you intend to screen for growth on lactose then you also need the permease or some other transport function.
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