Knocking out non-essential gene - (Jan/15/2012 )
We have been trying to knock out PagP from the E. Coli genome by using Datensko and wanner process. Basically we use to amplify the kanamycin cassette in pkD13 plasmid by designing the primer that contains kanamycin terminal primer and some homologous region (50 bp) upstream and downstream of PagP. Later through the homologous recombination we try to knock out PagP (we also use pKD46, a lambda recombinase plasmid to make our strain MG1655 able to take up linear DNA) and replace it with kanamycin cassette. These transformed cells can grow on kanamycin containing LB plate but when we try to verify the transformed cell with PCR by using dcuC forward (which is 700 bp upstream of starting codon of PagP) and cspE reverse (which is 250 bp downstream of stop codon of PagP), we use to get two bands: one correspond to the PagP present on it and another correspond to the kanamycin cassette present on it. We also use PagP terminal primers which shows the PagP band and also got confirmation by sequencing. This things happens not only to the strain we prepared in our lab but to the other strains from keio library, strains from Princeton University which they stated as delta PagP. We tried with MG1655, BW25113, Mc4100 and all behaving the same.
I would be grateful if you comment on my technique stated above and if possible, provide me the solution of my problem. Please suggest me how can I verify where kanamycin cassette is inserted in the genome?
My thought is that you are getting single crossovers. I am guessing the pkD13 backbone has an ampicillin resistance cassette? In which case you can verify if your colonies are single crossovers by checking for AmpR - if they are resistant, then clearly they still have the pKD13 backbone inserted.
So now, after selecting the Km resistant colonies, you need to passage in non-antibiotic containing media and then check for any isolates which are Km resistant and Amp sensitive. These would be true double crossovers, and would have the pagP gene deleted
Hey man. I used the same datsenko/waner process for my library of KOs. Although, I use pKD4 to amplify the kan cassette and pKM208 to induce the recombinase for homologous recombination. After I transform the kan product into my cell, I first select it on Kan20 plates (lower concentration) and then passage a whole bunch of isolated colonies from the Kan20 onto Kan50 plates. When you look at your Kan 50, you will see that some colonies, have not grown at all, some grow weakly and some show heavy growth. The heavily growing colonies are almost always true KOs. This extra step eliminates selection of any random mutation that might get selecting on Kan50 plates directly after your transformation. After this you can do your regular PCR and restriction validation.
Now another thing you also have to remember is that, you don't overinduce the recombinase for too long. This can sometime result in recombination into wrong positions within the chromosome.