I am working with ATCC 43816 Klebsiella pneumoniae and am having extreme difficulty in creating knockouts using a lambda red recombination system. This system has been successful in other strains of K. pneumoniae but I have had extreme difficulty in getting successful transformants thus far. If anyone has advice on improving transformation efficiency in a high capsule producing strain, or has heard of issues with using this particular strain in molecular cloning please let me know!
Thanks in advance!
This protocol worked pretty well for me to transform a plasmid of ~5kb. However, it didn't work for 10kb. Good luck. plasmid. http://www.sciencedirect.com/science/article/pii/0167701295000534
Have you tried electroporation? Harvest 50 ml of bacterial culture at an OD of 0.6 and wash them 4 or more times with cold destiled water, resuspend them in 200 ul of cold water and electroporate them with the PCR.
Hope this works
Claudia
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