Hts Using Alginate Macrodroplets

Sodium alginate is the water-soluble salt of alginic acid, a copolymer of 1-4 linked a-D-mannuronic and P-L-guluronic acids, which are extracted from various seaweeds. Sodium alginate solutions have the desirable property of gelling upon contact with divalent cations, such as Ca2+ [26,29]. Ca-alginate has been used as a convenient means of encapsulating cells and enzymes. The physical properties, such as gel strength and porosity, can be controlled by appropriate formulation of the starting alginate solution and the choice and concentration of Ca2+.

Ca-alginate gel spheres were used in the screening program. The spheres, called macrodroplets, are formulated by adding sodium alginate to a solution containing growth medium and then adding this formulation dropwise to a solution containing Ca ions. The slightly viscous Na-alginate/medium solution becomes a solid gel upon contact with Ca2+ (Fig. 6). The macrodroplets are quite firm, yet they are permeable to atmospheric oxygen, small molecules, and proteins according to the pore size of the gel. Permeability is primarily determined by the alginate concentration. In practice, a 1% Na-alginate solution is permeable to all essential medium components.

In order to encapsulate actinomycete cells, actinomycete cells are added in the form of spores, protoplasts (cells enzymatically treated to remove cell walls), or mycelial fragments to an alginate solution containing growth medium [30]. The alginate solution and cells are thoroughly mixed to ensure a uniform cell suspension. This cell suspension is placed in a reservoir and extruded through an appropriately sized orifice. The cellular suspension leaves the orifice in the shape of a teardrop and becomes spherical as it falls. Upon contact with the solution of calcium ions, the suspension instantly gels to form a rigid sphere that gently encapsulates the cells and retains their viability.

Figure 6 Formation of macrodroplets.

A. Development of Macrodroplet Bioassays

A wide variety of cells, among them mammalian, fungal, bacterial, and actinomy-cete, were encapsulated. All remain viable after encapsulation, so that this approach is useful for a wide range of bioassay applications. The precise formulation, diameter, and configuration of macrodroplets vary and are dependent upon the desired bioassay. The number of cells encapsulated within an individual ma-crodroplet is determined by the initial density of the cell suspension and the volume of the generated macrodroplet. Though various size macrodroplets can be generated, those in the range of 2 to 4 mm in diameter are found to be the most useful. After sieving to remove them from the CaCl2 solution, the macro-droplets are placed on a grid in a plastic tray to separate them from one another. The tray is placed in a humid incubator at an appropriate temperature for an empirically determined length of time (Fig. 7). For actinomycete combinatorial biology library clones, a 10 to 14 day incubation period at 30°C is typically used. During this time, the actinomycetes grow into fully developed colonies and produce natural products. Importantly, natural products produced by the encapsulated colonies accumulate and are retained within the matrix of the macrodroplet.

The macrodroplet screening system is a remarkably flexible assay format, and three specific formats were tested. The plate assay format involves placing a number of macrodroplets, which encapsulate 1 or 2 actinomycete clones, on a plate. The clones are allowed to ferment and grow for a period of 7 to 14 days. After the fermentation period, the entire plate is overlaid with soft nutrient agar that contains the desired assay organism (Fig. 8). In the double encapsulation format, macrodroplets containing actinomycete clones are fermented in the same way as the plate assay (Fig. 8). Once the fermentation period has passed, the macrodroplets are encapsulated in a second layer of Ca-alginate that contains the desired assay organism. In this way the assay lawn is wrapped around the macrodroplet. The third format is coencapsulation, where we take the actinomy-cete clone and encapsulate it in a macrodroplet with the assay organism (Fig. 8). In this way the macrodroplet contains the assay lawn. Examples of each of these formats will be discussed below.

B. Uses of the Plate Assay Format

Recently we isolated the heterologously expressed genes for the tetracycline pathway [31,32]. Genomic DNA was prepared from Streptomyces avellaneus, an acti-

Was this article helpful?

0 0

Post a comment