nBlog 2007-08-29

A can of worms

Before the genome sequence, only one cloning vector has been reported. pCY104 is an epoch-making vector but it has hardly ever been used. In fact, only one report using this vector can be found in the PubMed database. Some reasons are thought why pCY104 was not used so far. I guess pCY104 is large in size and has only a few cloning site. Therefore, we decided to develop a new vector. pNV vectors have many good features: small in size, multi-cloning site, blue/white screening, and broad host range.

So, gene knockout is a crucial technique for analyzing the function of the gene. We have established a gene knockout system in Nocardia. We employed pK18mobsacB as a deletion-allele-delivery vector. pK18mobsacB has been developed for corynebacteria but extensively used in Gram-negative bacteria, for example Pseudomonas, Rhizobium, etc. We have successfully knocked out several nocardia genes so far, but, at present, the efficiency of gene knockout in Nocardia is very low. Because, yielding of legitimate recombinants is less than 10%. Thus, gene knockout experiment in Nocardia is a time-consuming and laborious work. It takes about one month or more per gene. So, it may be necessary to develop more efficient strategy.

Recent our works have revealed that genetic manipulation of Nocardia is possible and easier than previously thought. I am certain that our system will facilitate Nocardia genetics. But we may have opened a can of worms.