Conditionally Amplifiable BACs: Switching From Single-Copy to High-Copy Vectors and Genomic Clones

Abstract

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.

Proteorhodopsin photosystem gene expression enables photophosphorylation in a heterologous host

“…Although large-insert libraries increase the probability of capturing complete metabolic pathways in a single clone, their low copy number decreases the sensitivity of detecting heterologous gene expression. We show here that increasing fosmid copy number (13) can significantly enhance detectable levels of recombinant gene expression and therefore increases the detection rate of desired phenotypes in metagenomic libraries. The PR photosystem recombinants we characterized could be detected visually by pigment production and exhibited light-dependent proton translocation and subsequent photophosphorylation, only when the fosmid vector was induced to high copy number.…”

“…Based on these observations, we screened for PR-containing clones on retinal-containing LB agar plating medium, which we expected would display an orange to red phenotype under these conditions. To enhance assay sensitivity, we used the copycontrol system present in our fosmid vector that allowed a controlled transition from one copy per cell to multiple (up to 100) vector copies upon addition of the inducer L-arabinose (13).…”

Hydrocarbon monooxygenase in Mycobacterium: recombinant expression of a member of the ammonia monooxygenase superfamily

“…Full details of the construction and evaluation of this vector are reported elsewhere , but in brief, this vector combines the E. coli replication and stabilization functions of the pCC1FOS fosmid (Epicentre Biotechnologies) (Wild et al, 2002) with the Mycobacterium replicon and acetamidase promoter regions from the pJAM2 shuttle vector (Triccas et al, 1998)-this allows control of the copy number in E. coli via arabinose induction, and expression of cloned genes in Mycobacterium via acetamide induction.…”

New methods for tightly regulated gene expression and highly efficient chromosomal integration of cloned genes forMethanosarcinaspecies

“…Several of these plasmids are derivatives of the fosmid cloning vector pWM357 and are useful for constructing genomic DNA libraries; however, they have been modified to include additional useful features. The parental plasmid was modified to include a marker for selection of puromycin resistance in Methanosarcina species and the origin of replication from plasmid RP4 (oriV) to allow induction of high-copy replication in appropriate host strains (Wild et al 2002). The plasmids also carry the phage λ attB site, which can be used to retrofit the plasmids with additional features (see below).…”

“…Escherichia coli WM3118 (F -, mcrA, Δ(mrr-hsdRMS-mcrBC), φ80lacZΔM15, ΔlacX74,recA1,endA1,araD139,Δ(ara,leu)7697,galU,galK,rpsL,nupG, was constructed by integration of pAMG27 (Table 2) into the λattB site of DH10B (Invitrogen, Carlsbad, CA) by site-specific recombination as described by Haldimann and Wanner (2001). WM3118 was used as the host strain for all plasmids containing oriV, allowing plasmid copy number to be dramatically increased by growth in a medium containing 10 mM rhamnose before plasmid purification (Wild et al 2002). BW25141 was the host strain for Π-dependent plasmids (Haldimann and Wanner 2001).…”