High-throughput transformation of <i>Saccharomyces cerevisiae</i> using liquid handling robots

<div><p><i>Saccharomyces cerevisiae</i> (budding yeast) is a powerful eukaryotic model organism ideally suited to high-throughput genetic analyses, which time and again has yielded insights that further our understanding of cell biology processes conserved in humans. Lithium Acetate (LiAc) transformation of yeast with DNA for the purposes of exogenous protein expression (e.g., plasmids) or genome mutation (e.g., gene mutation, deletion, epitope tagging) is a useful and long established method. However, a reliable and optimized high throughput transformation protocol that runs almost no risk of human error has not been described in the literature. Here, we describe such a method that is broadly transferable to most liquid handling high-throughput robotic platforms, which are now commonplace in academic and industry settings. Using our optimized method, we are able to comfortably transform approximately 1200 individual strains per day, allowing complete transformation of typical genomic yeast libraries within 6 days. In addition, use of our protocol for gene knockout purposes also provides a potentially quicker, easier and more cost-effective approach to generating collections of double mutants than the popular and elegant synthetic genetic array methodology. In summary, our methodology will be of significant use to anyone interested in high throughput molecular and/or genetic analysis of yeast.</p></div

Plasmid and cell concentration affect transformation efficiency.

<p>(A) BY4741 cells at OD₆₀₀ 0.5 were transformed with the indicated amount of pRB1 plasmid using our automated methodology with 4-hour heat shock. After transformation, cells were spotted on a –Uracil agar plate. (B) Different OD₆₀₀ cells were transformed with 100ng pRB1 plasmid and heat shocked at 42°C for 4 hours. (C) Microscopy of BY4741 cells originally transformed either at 0.4 or 1.5 OD₆₀₀, and examined at an OD of 3.5. Numbers indicate average stress granule (Pab1-GFP, green text) or P-body (Edc3-mCh, red text) foci per cell, and the percentage of Edc3-mCh foci co-localized with Pab1-GFP (white text) (D). Quantification of stress granule (SG) and P-Body (PB) size in (C). Data is presented as mean ± standard deviation of 3 independent experiments; n.s., not significant.</p

VPS38 deletion and check primers.

<p>VPS38 deletion and check primers.</p

Successful application of high-throughput transformation method to gene deletion.

<p>(A) Generation of <i>VPS38</i> knockout strain by insertion via homologous recombination of a <i>LEU2</i> selective marker at the <i>VPS38</i> locus. (B) Different volumes of 150ng/ul of the <i>LEU2</i> cassette were transformed into yeast cells. Transformants were plated on—Leucine media. (C) <i>VPS38</i> deletion was verified using check primers (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0174128#pone.0174128.t002" target="_blank">Table 2</a>) from four single colonies (wild type, 1#: 30 μl spot, 2#: 60 μ spotl, 3#: 120 μl spot). Expected PCR product size of wild type: 1701 bp, <i>VPS38</i> knockout: 2810 bp. (D) Plate 1 from the non-essential yeast knockout library was cultured and transformed with 4500ng VPS38 knockout <i>LEU2</i> cassette using our methodology. Transformed cells were cultured in –Leucine SD media for 3 days and spotted on –Leucine media using prongs.</p

Comparison of SGA and high-throughput gene deletion methods.

<p>Comparison of SGA and high-throughput gene deletion methods.</p

Schematic model of yeast transformation using liquid handling robot.

<p>Step 1, Inoculate overnight culture to the deep well plate. Grow another 2 ½ -4 hours to recover cells to the mid-log phase. Step 2, Prepare plasmid and transformation mix to transformation. Normally, an OD₆₀₀ range of 0.4–1.5, ≥100ng plasmid or 4500 ng PCR product is optimal for our automated transformation method. Step 3, Heat shock of the transformants for 3–6 hours. Step 4, Transfer the transformed cells to a liquid selective media plate to grow another 2–4 days. Pin the transformed strains onto appropriate selective media to generate the new library.</p

Strains and plasmids used in this study.

<p>Strains and plasmids used in this study.</p

Transformation efficiency is increased with extended 42°C heat shock periods.

<p>(A) BY4741 cells at OD₆₀₀ 0.5 were transformed with the indicated amount of pRB1 plasmid and incubated at 42°C for 30 minutes (min), 1, 2, 4, and 6 hours (h). Transformants were selected on –Uracil plates. (B) Transformed cells generated from transformation reactions with different 42°C incubation times (1, 2, 4 and 6 hours). Numbers indicate average stress granule (Pab1-GFP, green text) or P-body (Edc3-mCh, red text) foci per cell, and the percentage of Edc3-mCh foci co-localized with Pab1-GFP (white text). (C) Quantification of stress granule (SG) and P-body (PB) size in (B). Data is presented as mean ± standard deviation of 3 independent experiments; n.s., not significant. (D) BY4741 cells transformed with pRB1 plasmid and incubated at 42°C for different time were assessed for elevated mutation rates by development of canavanine resistance. Transformed cells were plated on canavanine media (60 mg/L). Simultaneously, 1/200 diluted amount of cells were coated on the YPD plate. Colony number was counted after 2 days incubation at 30°C. Mutation rates were normalized to the 0-hour heat shock. Data is presented as mean ± standard deviation of 3 independent experiments; n.s., not significant.</p

Gate Modulation of Threshold Voltage Instability in Multilayer InSe Field Effect Transistors

We report a modulation of threshold voltage instability of back-gated multilayer InSe FETs by gate bias stress. The performance stability of multilayer InSe FETs is affected by gate bias polar, gate bias stress time and gate bias sweep rate under ambient conditions. The on-current increases and threshold voltage shifts to negative gate bias stress direction with negative bias stress applied, which are opposite to that of positive bias stress. The intensity of gate bias stress effect is influenced by applied gate bias time and the sweep rate of gate bias stress. The behavior can be explained by the surface charge trapping model due to the adsorbing/desorbing oxygen and/or water molecules on the InSe surface. This study offers an opportunity to understand gate bias stress modulation of performance instability of back-gated multilayer InSe FETs and provides a clue for designing desirable InSe nanoelectronic and optoelectronic devices

Solid-State Reaction Synthesis of a InSe/CuInSe₂ Lateral p–n Heterojunction and Application in High Performance Optoelectronic Devices

Graphene-like layered semiconductors are a new class of materials for next generation electronic and optoelectronic devices due to their unique electrical and optical properties. A p–n junction is an elementary building block for electronics and optoelectronics devices. Here, we demonstrate the fabrication of a lateral p–n heterojunction diode of a thin-film InSe/CuInSe₂ nanosheet by simple solid-state reaction. We discover that InSe nanosheets can be easily transformed into CuInSe₂ thin film by reacting with elemental copper at a temperature of 300 °C. Photodetectors and photovoltaic devices based on this lateral heterojunction p–n diode show a large photoresponsivity of 4.2 A W^–1 and a relatively high light-power conversion efficiency of 3.5%, respectively. This work is a giant step forward in practical applications of two-dimensional materials for next generation optoelectronic devices