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Waclaw Szybalski

Publications and source records attributed to Waclaw Szybalski.

8 recordsLinked to original sources
PubMed2004

Copy-control pBAC/oriV vectors for genomic cloning.

The use of the improved BAC system for cloning genomic DNA and library constructions is described. This system retains all the advantages of the original BACs but, in addition, permits, on command, amplification of the BAC plasmids and cloned DNA. This system consists of (1) plasmid pBAC/oriV containing an additional replication origin, oriV, and (2) a host carrying the up-mutants of the trfA replicator gene expressed from the l-arabinose-inducible Para promoter. The pBAC/oriV clones are always maintained in the single-copy state, but if more DNA is required, they could be amplified up to 100-fold, depending on the size of the cloned insert.

Chromosomes, Artificial, Bacterial↗
PubMed2004

Copy-control tightly regulated expression vectors based on pBAC/oriV.

A novel type of expression vectors with a dual regulation of both the plasmid copy number and gene expression, is described. The most important and beneficial feature of these vectors is that when they are not induced, they are maintained as a single-copy plasmid, and therefore, any residual expression is much more tightly regulated than for the conventional multicopy expression vectors. The simplest version of these copy-control expression vectors is based on the pBAC/oriV plasmid that carries the trfA up-mutant gene under control of the l-arabinose-inducible Para promoter (araC-PBAD). The same promoter controls expression of a gene cloned into MCS. Thus, addition of the inducer (l-arabinose) simultaneously turns on amplification of the plasmid and expression of the cloned gene. Net result is about a 50,000-fold increase in the cloned gene expression. However, when not induced, background expression level is very low, which is important for the maintenance of any "toxic" genes. This vector could be used in most E. coli hosts. Similar versions of the described vector employ the rhamnose-inducible Prha promoter (rhaS-Prha). Other expression systems allow independent regulation of the plasmid amplification and of the cloned gene expression, and some also use the PLtetO-1 promoter. Copy-control expression vector pETcoco, based on the pT7lacO promoter, is commercially available.

Chromosomes, Artificial, Bacterial↗
PubMed2002

Conditionally amplifiable BACs: switching from single-copy to high-copy vectors and genomic clones.

The widely used, very-low-copy BAC (bacterial artificial chromosome) vectors are the mainstay of present genomic research. The principal advantage of BACs is the high stability of inserted clones, but an important disadvantage is the low yield of DNA, both for vectors alone and when carrying genomic inserts. We describe here a novel class of single-copy/high-copy (SC/HC) pBAC/oriV vectors that retain all the advantages of low-copy BAC vectors, but are endowed with a conditional and tightly controlled oriV/TrfA amplification system that allows: (1) a yield of ~100 copies of the vector per host cell when conditionally induced with L-arabinose, and (2) analogous DNA amplification (only upon induction and with copy number depending on the insert size) of pBAC/oriV clones carrying >100-kb inserts. Amplifiable clones and libraries facilitate high-throughput DNA sequencing and other applications requiring HC plasmid DNA. To turn on DNA amplification, which is driven by the oriV origin of replication, we used copy-up mutations in the gene trfA whose expression was very tightly controlled by the araC-P(araBAD) promoter/regulator system. This system is inducible by L-arabinose, and could be further regulated by glucose and fucose. Amplification of DNA upon induction with L-arabinose and its modulation by glucose are robust and reliable. Furthermore, we discovered that addition of 0.2% D-glucose to the growth medium helped toward the objective of obtaining a real SC state for all BAC systems, thus enhancing the stability of their maintenance, which became equivalent to cloning into the host chromosome

Arabinose↗