Figure 8 Three macrodroplet assay formats.
Lawn Overlay Assay
nomycete known to produce tetracycline, to generate a cosmid library. This natural pathway library was transferred to S. lividans, and recombinant spores were encapsulated for activity screening. The size of the tetracycline pathway is about 30 kb, and the size of the insert for vector is 40-45 kb, resulting in one producing clone per 2,000 clones screened. Approximately 12,000 clones were screened using the macrodroplets in a plate assay format. Each macrodroplet contained 1 or 2 clones, and about 10,000 macrodroplets were screened. About 150-200 macrodroplets were placed on each plate (9 X 13 X 2 cm), and about 60 of these plates were used. Each plate was incubated at 30°C for 10 days in a humid atmosphere (to prevent the macrodroplets from drying out). After this period of fermentation, the plates of macrodroplets were overlaid with a soft nutrient agar containing a strain of E. coli. After an overnight incubation at 37°C, the plates were examined for the presence of clearing zones, which indicate the presence of a clone, which produces tetracycline (Fig. 9). The plates contained 12 recombinant clones that produced clearing zones. Six were selected for further analysis, which confirmed the heterologous production of tetracycline. The isolation of these six clones demonstrated the power of the macrodroplet approach to screening. The entire screening of 10,000 macrodroplets occupied a portion of a shelf
of a standard laboratory incubator, in contrast to the effort to screen the 10,400 clones from our previous combinatorial biology experiment [27,28].
Alginate encapsulation could also be used to encapsulate photocleavable resins . A resin bound penicillin V was prepared, which upon illumination of light at 365 nm will be cleaved. This wavelength of blue light does not interfere with normal growth of E. coli. The resin bound penicillin V in an alginate matrix was encapsulated, and the macrodroplets were placed on lawns of E. coli prepared in soft nutrient agar. Without illumination the lawns grew luxuriantly around the macrodroplets. With illumination the lawns grew luxuriantly on the plates but showed zones of clearing around the macrodroplets. The zones increased in size as the time of illumination increased. Since the light source was directed from above, approximately half of the resin remained unexposed to the light and retained the bound antibiotics. These macrodroplets were dissolved with sodium citrate and the resin was recovered. The resin was washed and exposed to blue light at 365 nm, which resulted in the liberation of penicillin V as measured by UV absorption and verified by mass spectrometry.
Already formed macrodroplets can be encapsulated in a second, outer layer of alginate. In this way a macrodroplet containing a developed actinomycete colony can be coated with an outer layer that contains an assay organism such as E. coli (Fig. 10). If the actinomycete produces an antibiotic, that kills E. coli, then it will diffuse into this added outer layer and kill the E. coli. In this case, the outer layer will remain clear. If the actinomycete does not produce an antibiotic, then the E. coli in the outer layer will grow and the outer layer will be cloudy. This approach is a further miniaturization of the lawn assay (Fig. 10).
An advantage of this approach is that it allows organisms with very different growth rates to be assayed in the same macrodroplet. Double encapsulation was used to make macrodroplets containing actinomycetes in the core and E. coli, fungi, or mammalian cells in the second layer of alginate. Those actinomycetes in the core of a doubly encapsulated macrodroplet that produced antifungal compounds killed the fungi in the outer layer and it remained clear. In doubly encapsulated macrodroplets with an actinomycete in the core that produced antibiotic compounds, the E. coli in the outer layer was killed and it remained clear. Those actinomycetes in the core of a doubly encapsulated macrodroplet that produced no antibiotics failed to kill fungi or E. coli and their outer layer was filled with fungal or E. coli colonies. In principle, enzymes could also be included in the outer layer as well. The diffusion of the antibiotic out of the macrodroplet during the application of the second layer may limit the usefulness of this approach. Preliminary work measuring the diffusion of tetracycline from macrodroplets en-
Was this article helpful?