Isolation of monoclonal microcarriers colonized by fluorescent E. coli

Abstract

Microencapsulation gains increasing importance for processing of bacterial libraries and especially in high-throughput (HT) environments where [10 6 samples per day are studied. As a rule, a one-to-one relationship between an individual cell and analytical results is of key importance. Ideally, each microcarrier would therefore contain exactly one cell or colony. However, synthesis of larger numbers of capsules containing exactly one cell is not feasible as cells are randomly distributed during carrier-production. The dilemma is that high dilution conditions will yield a satisfactory degree of monoclonality, but also a very large fraction of empty compartments, whereas distribution under low dilution generates unacceptable numbers of polyclonal compartments for whose removal no satisfactory technologies exist. Hydrogel carriers with a volume of 35 nL were used as growth compartments for individual microbial colonies. E. coli cells expressing green fluorescent protein (GFP) were encapsulated at low dilution thereby intentionally producing a considerable amount of polyclonal microcarrieres. Empty and polyclonal microcarriers were then removed from the desired monoclonal fraction by a COPAS Plus particle analyzer. The results were compared with model predictions in order to investigate possible limitations in the analysis and sorting of monoclonal microcarriers by COPAS. Fluorescent E. coli cells (GFP) distributed randomly throughout the microcarrier population. Cells were successfully propagated to colonies in the microcarriers and enriched to 95% monoclonality by a COAPS sorter. Enrichment-efficiency was found to mainly depend on the colony diameter. With increasing colony size two contrary effects were observed: First, improved sorting efficiency due to increased fluorescence intensity and therefore higher detection efficiency, and second, deterioration of sorting efficiency due to occlusion occurring in polyclonal carriers. The combination of microencapsulation under low dilution conditions followed by HT sorting procedures is an efficient way for isolating larger amount of monoclonal carriers from bacterial libraries while concomitantly keeping the amounts of empty carriers at a moderate level. ' International Society for Advancement of CytometryKey terms microencapsulation; monoclonality; microcarrier; in vitro compartmentalization (IVC); fluorescent E. coli; COPAS plus particle analyzer and sorter; ultrahigh throughput screening THE enormous complexity of living systems makes high-throughput (HT) experimenting and analyses an indispensable tool for research and development in the lifeand biosciences. Sample numbers exceeding 10 6 per day (1-4) require new technologies for sample miniaturization and highly parallelized processing (5). Naturally, most experimental strategies rely on formation of spatially separated monoclonal cell assemblies at the beginning. If microbial species are studied, an approach that is frequently employed for generating larger numbers of monoseptic compartments is to first ...

Identification and Characterisation of a pH-stable GFP

“…Each nLR was seeded with 2–3 recombinant cells and the cells were allowed to grow until the generated microcolonies consisted of about 1,000 cells. The nLRs were then incubated in acetate buffer (pH 5) to lower the intracellular pH and sorted based on fluorescence intensity (ex 488 nm, em 515(20) nm) using a particle sorter (COPAS)9. The 0.1% of nLRs containing the brightest microcolonies were selected for further analysis.…”

“…In a typical nLR experiment (“selection”), 50,000 nLRs were analysed at a rate of 20 nLRs per second and dispensed into glass-bottom 96-well plates containing 50  of 10 mM calcium chloride. The sorting was performed in the “pure” mode of COPAS (ensuring only one particle per selected droplet) and all nLRs containing more than one colony were excluded using the COPAS profiler software9. The monoclonality of sorted nLRs was also confirmed using an inverted fluorescence microscope (Axio Observer, Zeiss, GFP filter set).…”

Novel method for high-throughput colony PCR screening in nanoliter-reactors

“…Overview over sample streams during HTS for novel SSRs (short sequence repeats) in a cassava genomic library. a The subset of the 660 000 empty and colonized nl-reactors that was analyzed and sorted in order to isolate 20 000 monoseptic nl-reactors; colony diameter of 32 µm ± 7 µm. b Sorted and monoseptically enriched nl-reactors (still containing ∼1 to 2% polyclonal reactors and roughly 2% multiplets) (21). …”

New Insights into Cell Encapsulation and the Role of Proteins During Flow Cytometry

“…Conversely, distribution under low dilution conditions will generate unacceptable numbers of polyclonal capsules for whose removal no satisfactory technologies exists to date. Recent finding demonstrated (Walser et al, 2008(Walser et al, , 2009) that hydrogel micro-carriers can be applied as growth milieu for individual cell colonies. Escherichia coli cells expressing green fluorescent protein (GFP) were encapsulated at low dilution thereby intentionally producing a considerable amount of polyclonal micro-carriers.…”