Computer-Aided High-Throughput Cloning of Bacteria in Liquid Medium

Abstract

Bacterial cloning was first introduced over a century ago and has since become one of the most useful procedures in biological research, perhaps paralleled in its ubiquity only by PCR and DNA sequencing. However, unlike PCR and sequencing, cloning has generally remained a manual, labor-intensive, low-throughput procedure. Here we address this issue by developing an automated, computer-aided bacterial cloning method using liquid medium that is based on the principles of (i) limiting dilution of bacteria, (ii) inference of colony forming units (CFUs) based on optical density (OD) readings, and (iii) verification of monoclonality using a mixture of differently colored fluorescently labeled bacteria for transformation. We demonstrate the high-throughput utility of this method by employing it as a cloning platform for a DNA synthesis process.

Catalytically-active inclusion bodies for biotechnology—general concepts, optimization, and application

“…However, this can be easily performed by lab automation technology, which could be seamlessly hyphenated with the next steps like the transformation of an expression host with the Golden Gate products, CatIB production, and CatIB purification and analysis. The general automation of such molecular biology workflows have been successfully demonstrated for E. coli (Ben Yehezkel et al 2011 ; Billeci et al 2016 ; Olieric et al 2010 ), which is the current major producer of IBs (Carrio et al 1998 ; Ventura and Villaverde 2006 ) as well as CatIBs in literature. The automation of all these processes would be desirable to enable fast provision of suitable CatIBs for new catalytic enzymes.…”

Automation aided optimization of cloning, expression and purification of enzymes of the bacterial sialic acid catabolic and sialylation pathways enzymes for structural studies

“…Yehezkel et al . () automated cloning until the step of transformation, whereas expression and purification steps were not automated. Here, we have developed automated cloning, expression and purification yielding in a fast and economic method with minimal manual intervention.…”

The effectiveness of glass beads for plating cell cultures

“…We have analysed the properties of cell spread using digitized images and mathematical modelling. Optical density (OD) readings have been used to evaluate the number of CFU based on the time it takes to reach the predetermined OD of 0.1 when compared with a reference standard curve (Ben Yehezkel et al 2011). However, this technique would not provide the necessary details for the spatial analysis we performed.…”