Search results for "Shuttle vector"
showing 5 items of 5 documents
Artificial chromosome libraries of Streptomyces coelicolor A3(2) and Planobispora rosea
2003
Using an Escherichia coli-Streptomyces shuttle vector derived from a bacterial artificial chromosome (BAC), we developed methodologies for the construction of BAC libraries of filamentous actinomycetes. Libraries of Streptomyces coelicolor, the model actinomycete, and Planobispora rosea, a genetically intractable strain, were constructed. Both libraries have an average insert size of 60 kb, with maximal insert larger than 150 kb. The S. coelicolor library was evaluated by selected hybridisations to DraI fragments and by end sequencing of a few clones. Hybridisation of the P. rosea library to selected probes indicates a good representation of the P. rosea genome and that the library can be u…
Genetics for Pseudoalteromonas provides tools to manipulate marine bacterial virus PM2
2008
ABSTRACT The genetic manipulation of marine double-stranded DNA (dsDNA) bacteriophage PM2 ( Corticoviridae ) has been limited so far. The isolation of an autonomously replicating DNA element of Pseudoalteromonas haloplanktis TAC125 and construction of a shuttle vector replicating in both Escherichia coli and Pseudoalteromonas enabled us to design a set of conjugative shuttle plasmids encoding tRNA suppressors for amber mutations. Using a host strain carrying a suppressor plasmid allows the introduction and analysis of nonsense mutations in PM2. Here, we describe the isolation and characterization of a suppressor-sensitive PM2 sus2 mutant deficient in the structural protein P10. To infect an…
Advanced Strategies for Food-Grade Protein Production: A New E. coli/Lactic Acid Bacteria Shuttle Vector for Improved Cloning and Food-Grade Expressi…
2019
Food-grade production of recombinant proteins in Gram-positive bacteria, especially in LAB (i.e., Lactococcus, Lactobacillus, and Streptococcus), is of great interest in the areas of recombinant enzyme production, industrial food fermentation, gene and metabolic engineering, as well as antigen delivery for oral vaccination. Food-grade expression relies on hosts generally considered as safe organisms and on clone selection not dependent on antibiotic markers, which limit the overall DNA manipulation workflow, as it can be carried out only in the expression host and not in E. coli. Moreover, many commercial expression vectors lack useful elements for protein purification. We constructed a &ld…
Molecular Co-operation between Protein PAM and Streptokinase for Plasmin Acquisition by Streptococcus pyogenes
1998
Bacterial surface-associated plasmin formation is believed to contribute to invasion, although the underlying molecular mechanisms are poorly understood. To define the components necessary for plasmin generation on group A streptococci we used strain AP53 which exposes an M-like protein ("PAM") that contains a plasminogen-binding sequence with two 13-amino acid residues long tandem repeats (a1 and a2). Utilizing an Escherichia coli-streptococcal shuttle vector, we replaced a 29-residue long sequence segment of Arp4, an M-like protein that does not bind plasminogen, with a single (a1) or the combined a1a2 repeats of PAM. When expressed in E. coli, the purified chimeric Arp/PAM proteins both …
Plasmid conjugation from Proteobacteria as evidence for the origin of xenologous genes in Cyanobacteria
2014
Comparative genomics have shown that 5% of Synechococcus elongatus PCC 7942 genes are of probable proteobacterial origin. To investigate the role of interphylum conjugation in cyanobacterial gene acquisition, we tested the ability of a set of prototype proteobacterial conjugative plasmids (RP4, pKM101, R388, R64, and F) to transfer DNA from Escherichia coli to S. elongatus. A series of BioBrick-compatible, mobilizable shuttle vectors was developed. These vectors were based on the putative origin of replication of the Synechococcus resident plasmid pANL. Not only broad-host-range plasmids, such as RP4 and R388, but also narrower-host-range plasmids, such as pKM101, all encoding MPFT-type IV …