The methylotrophic yeast Pichia pastoris has gained widespread acceptance as a system of choice for heterologous protein expression in part because of the simplicity of techniques required for its molecular genetic manipulation (1). Several different procedures are available for introducing DNA into P. pastoris-spheroplast generation (2), electroporation (3), alkali cation (3,4), or polyethylene glycol (PEG) treatment (5). Here we describe a condensed protocol for cell preparation and transformation that works reliably with either auxotrophic markers or antibiotic selection.The introduction of exogenous DNA into an organism requires two steps: (i) the preparation of competent cells for DNA uptake and (ii) the transformation of the cells with the DNA. Transformation of P. pastoris by electroporation is a quick procedure. However, preparation of conventional electroporation-competent cells requires hours of work involving several washes, incubations, and centrifugations. In contrast, competent cell preparation for the heatshock method is short, but transformation requires approximately 2 h (4). The heat-shock procedure gives approximately 100-fold lower transformation efficiency than electroporation with plasmids containing auxotrophic marker genes such as HIS4. Additionally, the selection of zeocin-resistant transformants using the heat-shock transformation protocol does not work reliably.We have modified the preparation of competent cells from the heat-shock procedure (5) and combined it with transformation by electroporation (3) to yield a condensed protocol that works consistently with auxotrophic markers or antibiotic selection. The main modification of the heat-shock procedure is the addition of a step in which P. pastoris cells are incubated in an optimized concentration of dithiothreitol (DTT). The cells prepared by this "hybrid" method are then electroporated using the same parameters as conventional electroporation.Transformation efficiencies using the condensed protocol are comparable to the conventional electroporation procedure using auxotrophic markers but are approximately 20-fold lower using the zeocin resistance marker. However, the condensed protocol provides sufficient transformants, including multicopy integrants, for protein expression studies and has several advantages over the conventional electroporation and heat-shock methods. Table 1 compares the steps in cell preparation and transformation for conventional electroporation, heat shock, and our condensed protocol. Compared to the heat-shock method, the condensed protocol requires less time for the transformation step and provides much higher transformation efficiencies. Compared to the electroporation procedure, the new procedure saves both reagents

The methylotrophic yeast, Pichia pastoris, is widely used as a host organism for the expression of heterologous proteins. Currently, the Zeocin and blasticidin resistance genes are the only dominant selectable markers that can be used for primary selection of transformants. In this report we describe new expression vectors that can be used to select directly for P. pastoris transformants using G418 resistance conferred by a modified Tn903kan r gene. Compared to other dominant markers, this system is more economical and offers a higher transformation efficiency, due to the small sizes of the cloning vectors, pKAN B and pKANα B (GenBank Accession Nos EU285585 and EU285586, respectively). Additionally, multicopy transformants can be generated using these new vectors.

We describe the isolation and characterization of a new biosynthetic gene, MET2, from the methylotrophic yeast Pichia pastoris. The predicted product of PpMET2 is significantly similar to its Saccharomyces cerevisiae counterpart, ScMET2, which encodes homoserine-O-transacetylase. The ScMET2 was able to complement the P. pastoris met2 strain; however, the converse was not true. Expression vectors based on PpMET2 for the intracellular and secreted production of foreign proteins and corresponding auxotrophic strains were constructed and tested for use in heterologous expression. The expression vectors and corresponding strains provide greater flexibility when using P. pastoris for recombinant protein expression.

In spring 2016, the President’s Office at California State University-Fresno (part of the California State University system) offered grant opportunities for academic departments to create a community engagement program for students interested in supporting the Fresno community at large. Known as the Touch the Community project, the program solicited proposals that focused on a community concern and on how to address this issue. Several proposals were selected and funded ($2,000) by the President’s Office for the duration of two academic semesters. Some of these grant projects involved service-learning components: building computer labs, creating ESL programs, and engaging with K–12 students.