Bacterial artificial chromosome (BAC) vectors enable stable cloning of large DNA fragments from single genomes or microbial assemblages. A novel shuttle BAC vector was constructed that permits replication of BAC clones in diverse Gram-negative species. The "Gram-negative shuttle BAC" vector (pGNS-BAC) uses the F replicon for stable single-copy replication in E. coli and the broad-host-range RK2...

Shuttle vectors (pMS3 and pMS4) which replicated in Escherichia coli and in gram-positive Acetobacterium woodii were constructed by ligating the replication origin of plasmid pAMbeta1 with the E. coli cloning vector pUC19 and the tetM gene of streptococcal transposon Tn916. Electrotransformation of A. woodii was achieved at frequencies of 4.5 x 10 transformants per mug of plasmid DNA. For conju...

INTRODUCTION One of our projects involved the development of a transformation and transposon mutagenesis systems for the bacterium that causes Pierce's disease (PD), Xylella fastidiosa (Xf). We now have a random transposon mutagenesis system working for Xf (Guilhabert, et al. 2001) and recently we have developed an E. coli/Xf plasmid shuttle vector. We are currently assessing the stability of t...

A stable shuttle vector which replicates in Escherichia coli and Clostridium perfringens was constructed by ligating a 3.6-kilobase (kb) fragment of plasmid pBR322 with C. perfringens plasmid pHB101 (3.1 kb). The marker for this shuttle plasmid originated from the 1.3-kb chloramphenicol resistance gene of plasmid pHR106. The resulting shuttle vector, designated pAK201, is 8 kb in size and codes...

The construction of an integrative shuttle expression vector and potential utility was reported in Escherichia coli and Anabaena (Nostoc) sp. strain PCC 7120. The vector comprised of the following elements: (a) an intergenic non-coding region from Anabaena to facilitate its genomic integration (b) a strong functional PpsbAI promoter from Anabaena for desired gene expression and (c) neomycin pho...

Trinucleotide repeats (TNRs) undergo high frequency mutagenesis to cause at least 15 neurodegenerative diseases. To understand better the molecular mechanisms of TNR instability in cultured cells, a new genetic assay was created using a shuttle vector. The shuttle vector contains a promoter-TNR-reporter gene construct whose expression is dependent on TNR length. The vector harbors the SV40 ori ...