High-throughput screening of high protein producer budding yeast using gel microdrop technology

The need for protein production has been growing over the years in various industries. We here present a high-throughput screening strategy to isolate high producer budding yeast clones from a mutagenized cell population using gel microdrop (GMD) technology. We use a microfluidic water-in-oil (W/O) emulsion method to produce monodisperse GMDs and a microfluidic cell sorter for damage-free sorting of GMDs by fluorescently quantifying secreted proteins. As a result, this high-throughput GMD screening method effectively selects high producer clones and improves protein production up to five-fold. We speculate that this screening strategy can be applied, in principle, to select any types of high producer cells (bacterial, fungal, mammalian, etc.) which produce arbitrary target protein as it does not depend on enzymes to be produced.

Evidence and characterization of a gene cluster required for the production of viscosin, a lipopeptide biosurfactant, by a strain ofPseudomonas fluorescens

The genetic control of viscosin production was examined in a strain of Pseudomonas fluorescens (PfA7B) that causes broccoli head rot. Viscosin is a potent lipopeptide biosurfactant that enables the bacteria to come into intimate contact with the difficult-to-wet waxy heads of broccoli. Tn5 mutagenesis completely disrupted viscosin production as shown by HPLC analysis of the mutagenized cell lysates. The Vis–mutants retained their pectolytic capability and were able to decay potato slices. On broccoli, however, the Vis–mutants caused decay of wounded florets, but the decay failed to spread to adjacent nonwounded florets as had occurred with the wild-type PfA7B. Triparental matings of the Vis–mutants with their corresponding wild-type clones and the helper Escherichia coli HB101 carrying the mobilization plasmid pPK2013 resulted in three stable viscosin-producing transconjugants that caused typical decay of broccoli tissue. Linkage maps of clones and protein profiles showed that a 25-kb chromosomal DNA region of PfA7B affected the production of three high molecular mass proteins required for viscosin synthesis. These proteins, approximately 218, 215, and 137 kDa in size, likely compose a synthetase complex that assembles the nine amino acid peptide of viscosin and subsequently attaches this to the hydrophobic fatty acid component of the molecule. A probe made from this DNA region hybridized with DNA fragments of other phytopathogenic pseudomonads to varying degrees.Key words: virulence factor, head rot, broccoli.

CYTOPLASMIC INHERITANCE OF CHLORAMPHENICOL RESISTANCE IN TETRAHYMENA

ABSTRACT Chloramphenicol-resistant mutants of Tetrahymena pyriform's were obtained after mutagenesis with nitrosoguanidine at an estimated frequency of 10-3 mutants per mutagenized cell. The mutants are still partially sensitive to chloramphenicol and have a lowered growth rate, compared to the wild type strain, in rich medium without chloramphenicol. The genetic analysis described here indicates that chloramphenicol resistance is inherited as a cytoplasmic determinant that is not exchanged during conjugation. This represents the first simple cytoplasmic genetic determinant described in this species. A number of arguments favor a mitochondrial localization for this genetic determinant. In addition to the possible utility of such mutants for studies of mitochondrial structure and function in Tetrahymena, analogous mutations might serve as cytoplasmic "tags" in other ciliate species where the results of selfing need to be distinguished from those of outcrossing.