pkD46 Recobining with itself - (Jun/24/2008 )

Dear All,

WE have been having a problem in our lab due to which none of our deletions are working. We are using Lambda Red Recombinase method to knock out a gene. HOwever, while transforming pkD46(Length 6.3KB) into BL21 strain, it is recombining with itself. Therefore, on undergoing mini prep of overnight culture, I am getting the band at around 13KB. Can anyone tell me what could be the problem. I analyzed the situation at different temperature (i. e 23C, 30C and 37C) however, I am still having the same issues.

Regards,


Metz

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Am not sure of your exact strategy, but..
1. You can go below 23'c. I have grown bugs at 18'C for four days, when unwarranted recombination was a problem.
2. If your vector has some pronounced repeats, you may want to try to remove that region first from your vector/strategy, if possible.

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Why on earth are you doing minipreps in BL21? It is a terrible strain for this, with poor transformation efficiency and very bad quality DNA from most miniprep methods.

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Because he is trying to perform a gene knockout in BL21Star. BL21Star contains the T7 DNA polymerase required for expression of genes under the T7 promoter, which in his case, is required. He's performing the mini-prep for trouble-shooting purposes. My feeling is that the arabinose promoter is too leaky which is allowing expression of the Lambda recombinase protein. The pKD46 plasmid is also undergoing the same recombination event in K12 but to a lesser extent.
pKD46 has been frequently used in the literature and was a gift.

What he is trying to say is, has anyone else seen the pKD46 plasmid under-go self-recombination? And what steps are necessary to prevent or at least lessen this from happening? So far, growing the cells at 23C or lower might be the best solution. I've also tossed around the idea of re-engineering the pKD46 plasmid to remove the arabinose promoter and replace it with the IPTG-induced Trc promoter.

As a side note, BL21Star does have poor efficiency but only about 10 fold less than that of TOP10. You learn to work around it. Besides, if they are made into electrocompetent cells, the transformation efficiency is more than sufficient. You can easily do a triple plasmid transformation with decent electrocompetent cells. As for plasmid recovery, we've had great success using kits from either Qiagen or Zymo Research regardless of the E. coli strain. The Zymo Research kits are extremely cheap (400 preps for only $160) and only take about 10 mins to perform the miniprep. It gives high yields and very clean if done correctly.

BL21Star is a great strain for expressing recombinant protein too. Plus is grows well on minimal medium while TOP10 cannot. We've experimented with other commercial strains including Rosetta, TOP10, TOP10F', XL1Blue, JM109, DH5alpha and K12. For our purposes, we require a strain that can grow on minimal salt medium, express genes under the T7 promoter and give high protein expression. All said and done, BL21Star has proven to be the best for our work.

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