pPSX: a novel vector for the cloning and heterologous expression of antitumor antibiotic gene clusters.
A cosmid cloning vector has been constructed that demonstrates high levels of segregational stability in Escherichia coli K12. pPSX is a 14-kilobase vector derived from the IncW plasmid pR388. pPSX is highly stable in E. coli in the absence of antibiotic selection, even while expressing the toxic indolocarbazole antitumor antibiotic violacein. The incorporation of the lambdacos sequence enables construction of cosmid libraries with inserts ranging from 24 to 36kb. The inclusion of a lacZalpha multiple cloning site (MCS) allows blue/white screening. pPSX cosmids can be extracted from the host cell with commercial plasmid extraction kits facilitating downstream analysis, sequencing and sub-cloning. pPSX can be transferred to a variety of heterologous hosts by either electroporation or mobilization from E. coli S17-1. While it is unstable in non-E. coli hosts without antibiotic selection, heterologous host strains such as Rhodobacter sphaeroides and Pseudomonas stutzeri will maintain the plasmid under antibiotic selection to allow screening of expressed inserts. pPSX provides the benefits of large insert sizes with high stability to allow cloning of chemotherapeutic gene clusters in E. coli and a range of other heterologous hosts.
Sarovich
Derek S
DS
Department of Microbiology and Parasitology, University of Queensland, Brisbane, Australia.
Pemberton
John M
JM
D000903:Antibiotics, Antineoplastic / Q000096:biosynthesis*; D000970:Antineoplastic Agents / Q000138:chemical synthesis*; D003001:Cloning, Molecular; D003227:Conjugation, Genetic; D003360:Cosmids / Q000235:genetics*; D047169:Cyclotides; D048168:Escherichia coli K12 / Q000187:drug effects / Q000235:genetics* / Q000254:growth & development; D005822:Genetic Vectors; D005810:Multigene Family; D010957:Plasmids; D044223:Pseudomonas stutzeri / Q000235:genetics; D012242:Rhodobacter sphaeroides / Q000235:genetics
D000903:Antibiotics, Antineoplastic; D000970:Antineoplastic Agents; D047169:Cyclotides; C510720:violacin A