Start of funding 01.07.2016
Microfluidics for directed evolution of magnetotactic bacteria

Prof. Dr. Dirk Schüler
University of Bayreuth
Department of Microbiology

Prof. Dr. Dino Di Carlo
University of California, Los Angeles (UCLA)
Department of Bioengineering

Magnetosomes produced by magnetotactic bacteria have various promising biomedical applications such as magnetic imaging and hyperthermia. Combining targeted genetic engineering of magnetosome biosynthesis developed by the Schüler lab (UBT) and expertise in rare cell sorting from the Di Carlo lab (UCLA), this project aims to make use of microfluidics to sort and quantify magnetic contents in single cells of M. gryphiswaldense in a semi-automated and high throughput fashion. Furthermore, we expect to build a magnetic microfluidic platform for directed evolution of M. gryphiswaldense overproducer mutants for mass production of industrially important magnetic nanoparticles. Lastly, genetic analysis of the mutants will also reveal novel gene functions that are undiscovered but crucial to enhance magnetosomes production.

Magnetic nanoparticles (MNPs) are utilized in numerous biomedical applications, but it remains a challenge to synthetically produce them in large numbers with highly uniform properties and surface functionalization. Magnetotactic bacteria (MTB) produce MNPs with homogenous size, shape and magnetic properties, and encapsulate particles in a well-defined protein-studded membrane to form magnetosomes. Although targeted genetic manipulation has been shown to generate magnetosome overproducers, there are currently limited technologies to facilitate the selection of MTB with enhanced magnetic content. Our aim was to develop a magnetic microfluidic platform that facilitates a fast enrichment of MTB based on their magnetic content in a semi-automated and high throughput fashion. Therefore, a magnetic microfluidic device consisting of 2 inlets (for media and MTB suspension, respectively) and 2 outlets (selection and waste, respectively) was constructed. A neodymium magnet was placed adjacent to the main micro-channel to generate a local magnetic field. MTB experience different magnitudes of magnetic forces based on their magnetic contents, which determines whether they exit through the selection or waste outlet. To understand the initial streamlines occupied by MTB under flow in micro-channels and optimize flow rate and magnetic field strength, we first characterized the motion of 3 µm non-magnetic and magnetic beads (which is about the average length of MTB). As a proof of principle, we then attempted to sort cells form individual populations and mixtures of fluorescently labeled Magnetospirillum gryphiswaldense wildtype and of a non-magnetic ΔmamAB strain (both strains either labeled with GFP and/or mCherry). Magnetic wild-type cells were isolated at the selection outlet with 71% (WT-GFP) and 80% (WT-mCherry) efficiency while ΔmamAB cells which do not have magnetic nanoparticles were largely absent from the selection outlet. When we mixed equal numbers of cells of WT and ΔmamAB of different fluorescent colors together and sorted them, the isolation purity was high, yielding ~95% of the WT populations at the selection outlet. In a second set of experiments, we did characterize the isolation efficiency of previously generated Magnetospirillum mutants with different numbers of magnetosomes/bacterium, demonstrating that our device is also capable to sort mutant strains with altered numbers of magnetosomes per cell. In summary, our data supports the utility of our magnetic microfluidic platform to quantitatively sort MTB based on their magnetic contents (i.e. number of magnetosome/bacterium) in a fast, cheap and convenient manner, which e.g. might in particular be useful for directed evolution of MTB overproducer mutants for mass production of industrially important magnetic nanoparticles. The device can also be useful as a means for single cell analysis, by using a slow flow rate in the micro-channel and a diluted MTB suspension, single MTB of interest may be isolated for subsequent culture and downstream assays like genome sequencing. A common manuscript authored by Andy Tay and Dino DiCarlo (UCLA) and Daniel Pfeiffer and Dirk Schüler (University of Bayreuth) titled “Microfluidic Magnetic Separation for Estimating the Magnetic Contents of Magnetotactic Bacteria” describing these results is currently under review.