(567ac) Microfluidic Cell Sorter for Screening Bacterial Libraries

Bacteria are capable of sampling the levels of different molecules or signals in the environment and use it for directing their motion. By comparing the current concentration against ones taken in the previous seconds, bacteria orient their movement either towards attractants or away from repellents. This migration follows a biased random walk and is termed chemotaxis. While chemotaxis has been classically investigated from a fundamental perspective, it has the potential to be utilized for biotechnology applications. For example, bacteria being used in bioremediation applications for breaking down harmful substances into nontoxic byproducts are often repelled by the toxic molecule and migrate away from it; thus, reducing the bacteria's usefulness in remediation purposes. Engineering bacteria with an attractant chemotactic response to the pollutant would be of great benefit as the bacteria could seek out more as they deplete the current location. A major limitation in engineering bacteria for biotechnology applications such as bioremediation is that a large set of colonies need to be screened to identify a population having the desired traits. Typically, the number of mutants that have the desired properties is less than 5% of the total population. The process of screening tens of thousands of mutants is time and resource intensive. Here we present the development of a new bacterial sorter that can be used to sort and enrich populations based on chemotaxis. The sorter is conceptually based on microfluidic chemotaxis model systems developed in our laboratory (Appl. Environ. Microbiol. 75: 4557-64, 2009) and utilizes migration up or down a gradient to isolate bacteria with the desired response. Different gradient strengths ? linear, non-linear ? are generated through diffusive mixing and used to screen the mutant library. Mutants that either migrate toward or away from a molecule are collected based on the distance migrated by the bacteria. Depending on the strength of the response and the extent of purification desired, the collection ports can be modified or the time of exposure changed. The screened mutants can also be enriched by passing through the device for further refinement or processed with more traditional screening; thereby, significantly facilitating the screening process. We will present results where this device is used to isolate E. coli RP437 mutants that show altered responsiveness to amino acids and quorum sensing molecules.