A Novel Vector for Positive Selection of Inserts Harboring an Open Reading Frame by Translational Coupling

Ohashi-Kunihiro, Sumiko; Yohda, Masafumi; Hara, Masaki; Machida, Masayuki

doi:10.2144/000112629

Cited by 5 publications

(3 citation statements)

References 13 publications

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“…The promoters for these operons (marked P1 and P3 in ) are shown upstream of the CC_0969–CC_0968 and CC_1754–CC_1757 gene clusters with LacI-family transcription regulators (CC_0969 and CC_1753, respectively). Widely spaced genes in operons are known to be highly expressed and are usually expressed under complex regulatory conditions (Ohashi-Kunihiro et al , 2007; Pradhan et al , 2009; Price et al , 2006). Identification of the regulatory sequences and characterization of corresponding inducers/repressors of these genes would further reveal the genetic regulation associated with these BGLs and TBDRs in terms of broader gluco-oligosaccharide metabolism in C. crescentus .…”

Section: Discussionmentioning

confidence: 99%

Extracellular gluco-oligosaccharide degradation by Caulobacter crescentus

et al. 2014

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The oligotrophic bacterium Caulobacter crescentus has the ability to metabolize various organic molecules, including plant structural carbohydrates, as a carbon source. The nature of b-glucosidase (BGL)-mediated gluco-oligosaccharide degradation and nutrient transport across the outer membrane in C. crescentus was investigated. All gluco-oligosaccharides tested (up to celloheptose) supported growth in M2 minimal media but not cellulose or CM-cellulose. The periplasmic and outer membrane fractions showed highest BGL activity, but no significant BGL activity was observed in the cytosol or extracellular medium. Cells grown in cellobiose showed expression of specific BGLs and TonB-dependent receptors (TBDRs). Carbonyl cyanide 3-chlorophenylhydrazone lowered the rate of cell growth in cellobiose but not in glucose, indicating potential cellobiose transport into the cell by a proton motive force-dependent process, such as TBDR-dependent transport, and facilitated diffusion of glucose across the outer membrane via specific porins. These results suggest that C. crescentus acquires carbon from cellulose-derived gluco-oligosaccharides found in the environment by extracellular and periplasmic BGL activity and TBDR-mediated transport. This report on extracellular degradation of gluco-oligosaccharides and methods of nutrient acquisition by C. crescentus supports a broader suite of carbohydrate metabolic capabilities suggested by the C. crescentus genome sequence that until now have not been reported.

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Section: Discussionmentioning

confidence: 99%

Extracellular gluco-oligosaccharide degradation by Caulobacter crescentus

et al. 2014

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“…Likewise, the fundamental aspects of other published gain-of-function positive selection vectors also limit their scope of usability. These include reliance on a specific host strain for selection of clones (Banerjee et al 2010), on noncanonical type II or other specific restriction enzymes for DNA insertion (Ohashi-Kunihiro et al 2007;Miura et al 2011), or on complete inhibition of expression of the inserted gene of interest in a manner that precludes use in expression library formation and enzyme activity screening (Malo and Husain 2003).…”

Section: Introductionmentioning

confidence: 99%

A gain-of-function positive-selection expression plasmid that enables high-efficiency cloning

et al. 2014

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Directed enzyme evolution is now a routine approach to improve desirable biocatalytic properties. When only a low-throughput screen is available to detect improved variants from a mutant gene library, it is imperative that cloning efficiency be maximized during library synthesis to avoid wasting effort screening empty plasmids. To achieve this we developed pUCXKT, a gain-of-function positive selection expression vector. Insertion of genes amplified using a specialized downstream PCR primer restores key regulatory and genetic elements necessary for co-expression of a kanamycin resistance marker adjacent to the pUCXKT cloning region. We show that pUCXKT enables 100 % cloning efficiency as well as high-level expression of inserted genes. Unlike previous positive selection expression plasmids, the strategy we used to design pUCXKT is readily adaptable to different vector backbones, antibiotic marker genes, and multiple cloning regions.

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“…The feasibility of this type of cloning has been demonstrated in several publications (339)(340)(341)(342), however many of the positive selection vectors described in these examples are limited in their scope of use due to either the requirement of unique restriction enzymes (340; 341), dependence on a specific selectable marker (340), or generation of translational fusions between the cloned gene product and selectable marker (342). The first aim of this chapter was therefore to develop and test a positive selection vector that was compatible with the cloning methods used in the lab and that also permitted inducible expression of inserted genes, enabling its use in SOS assays and other activity screens.…”

Section: ) As It Has Been Wellmentioning

confidence: 99%

Discovery and Optimisation of Bacterial Nitroreductases for Use in Anti-Cancer Gene Therapy

Prosser¹

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<p>Nitroaromatic prodrugs are biologically inert compounds that are attractive candidates for anti-cancer therapy by virtue of their ability to be converted to potent DNA alkylating agents by nitroreductase (NTR) enzymes. In gene-directed enzyme-prodrug therapy (GDEPT), NTR-encoding therapeutic transgenes are delivered specifically to tumour cells, whereupon their expression confers host cell sensitivity to subsequent systemic administration of a nitroaromatic prodrug. The most well studied NTR-GDEPT system involves reduction of the aziridinyl dinitrobenzamide prodrug CB1954 by the Escherichia coli NTR NfsB. However, low affinity of this enzyme for CB1954 has so far limited the clinical efficacy of this GDEPT combination. The research described in this thesis has primarily sought to address this limitation through identification and optimisation of novel NTR enzymes with improved nitroaromatic prodrug reductase activity. Efficient assessment of NTR activity from large libraries of candidate enzymes requires a rapid and reliable screening system. An E. coli-based assay was developed to permit indirect assessment of relative rates of prodrug reduction by over-expressed NTRs via measurement of SOS response induction resulting from reduced prodrug-induced DNA damage. Using this assay in concert with other in vitro and in vivo tests, more than 50 native bacterial NTRs of diverse sequence and origin were assessed for their ability to reduce a panel of clinically attractive nitroaromatic prodrugs. Significantly, a number of NTRs were identified, particularly in the family of enzymes homologous to the native E. coli NTR NfsA, which displayed substantially improved activity over NfsB with CB1954 and other nitroaromatic prodrugs as substrates. This work also examined the roles of E. coli DNA damage repair pathways in processing of adducts induced by various nitroaromatic prodrugs. Of particular interest, nucleotide excision repair was found to be important in the processing of DNA lesions caused by 4-, but not 2-nitro group reduction products of CB1954, which suggests that there are some parallels in the mechanisms of CB1954 adduct repair in E. coli and mammalian cells. Finally, a lead NTR candidate, YcnD from Bacillus subtilis, was selected for further activity improvement through site-directed mutagenesis of active site residues. Using SOS screening, a double-site mutant was identified with 2.5-fold improved activity over the wildtype enzyme in metabolism of the novel dinitrobenzamide mustard prodrug PR-104A. In conclusion, novel NTRs with substantially improved nitroaromatic prodrug reducing activity over previously documented enzymes were identified and characterised. These results hold significance not only for the field of NTR-GDEPT, but also for other biotechnological applications in which NTRs are becoming increasingly significant, including developmental studies, antibiotic discovery and bioremediation. Furthermore, the in vitro assays developed in this study have potential utility in the discovery and evolution of other GDEPT-relevant enzymes whose prodrug metabolism is associated with genotoxicity.</p>

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A Novel Vector for Positive Selection of Inserts Harboring an Open Reading Frame by Translational Coupling

Cited by 5 publications

References 13 publications

Extracellular gluco-oligosaccharide degradation by Caulobacter crescentus

Extracellular gluco-oligosaccharide degradation by Caulobacter crescentus

A gain-of-function positive-selection expression plasmid that enables high-efficiency cloning

Discovery and Optimisation of Bacterial Nitroreductases for Use in Anti-Cancer Gene Therapy

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