pKIL18 and pKIL19, contained the ccdB gene in frame with an MCS. E. coli that were transformed with the empty vectors expressed the ccdB gene and were therefore unable to propagate because CcdA wasn’t available to counteract the toxin. If, however, an investigator were to successfully clone an insert into the vector, the ccdB reading frame would be disrupted allowing cells expressing the recombinant plasmid to propagate. Any cells that contained non-recombinant vectors (re-ligated empty vectors, for instance) would still express ccdB and therefore would die. This procedure dramatically reduces the number of clones that do not contain the recombinant plasmid and therefore makes the cloning process much more efficient, as one does not have to thoroughly screen colonies for the insert.

Gateway? technology (developed by InvitrogenTM) is essentially a more modern version of this older system, with added advantages that will be discussed in detail in a separate post. Focusing on the ccdB aspect, Gateway takes advantage of the same principle that cells will not propagate while expressing the gene. Briefly, the vector “backbone” in this system contains ccdB. A successful insertion will completely replace ccdB with the investigator’s insert of interest. Hence correct clones are identified much more efficiently, as those that do not contain the desired insert should not grow.

Figure 1: Image from Bernard, P., et al. showing the pKIL 18/19 vectors and demonstrating the concept of restriction enzyme-mediated disruption of ccdB, leading to positive identification of desired clones.

生產(chǎn)廠家Supplier:

BioVector NTCC質(zhì)粒載體菌株細(xì)胞蛋白抗體基因保藏中心

E-mail:BioVector@163.com

http://www.biovector.net