High-throughput cloning and expression in recalcitrant bacteria.
We developed a generic method for high-throughput cloning in bacteria that are less amenable to conventional DNA manipulations. The method involves ligation-independent cloning in an intermediary Escherichia coli vector, which is rapidly converted via vector-backbone exchange (VBEx) into an organism-specific plasmid ready for high-efficiency transformation. We demonstrated VBEx proof of principle for Lactococcus lactis, but the method can be adapted to all organisms for which plasmids are available.
Geertsma
Eric R
ER
Department of Biochemistry, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Nijenborgh 4, 9747 AG, Groningen, The Netherlands.
Poolman
Bert
B
D001483:Base Sequence; D003001:Cloning, Molecular / Q000379:methods*; D004269:DNA, Bacterial / Q000235:genetics*; D015252:Deoxyribonucleases, Type II Site-Specific / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D015964:Gene Expression Regulation, Bacterial; D005822:Genetic Vectors; D013294:Lactococcus lactis / Q000235:genetics; D008969:Molecular Sequence Data; D010957:Plasmids
D004269:DNA, Bacterial; C041749:endodeoxyribonuclease SfiI; D015252:Deoxyribonucleases, Type II Site-Specific