e49 Nucleic Acids Research, 2005, Vol. 33, No. 5 PAGE 6 OF 7 Figure 4. Gel image showing PCR amplifications of inserts from 46 cultures grown from single sorted GFP+/DsRed cells containing members of a S.purpuratus shotgun library. All 46 cultures yielded a PCR product consistent with a cloned insert. The majority of these inserts were in the 1.5–3 kb size range selected during initial library construction. reaction chambers. This transport has been achieved previ- ously (22) and could occur at very fast rates. Cell lysis, DNA ampliﬁcation and even DNA sequencing reactions can all be performed in individual chambers. Microfabricated chips have been previously constructed with the ability to per- form thermocycling and sequencing using micro-capillary electrophoresis (23). Beyond sequencing, the advantages offered by dual- ﬂuorescence reporter systems make them attractive platforms for diverse biosensor based assays. For example, motifs responsive to trans-acting regulatory proteins can be inserted into the linker region between GFP and DsRed to serve as detectors for speciﬁc protein–nucleic acid interactions. Dual ﬂuorescence can also be used to measure the frequency of mutational events. For example, repetitive DNA elements can be cloned between the ﬂuorescent reporters to study DNA polymerase replication ﬁdelity. After in vitro or in vivo rep- lication of the reporter, the frequency of slippage events can be quantiﬁed by either loss or gain of ﬂuorescence signal. The pGRFP vectors can be reconﬁgured for other applica- tions by putting the ﬂuorescence proteins under the control of separate response elements and promoters that respond to environmental cues. For example, Axtell and Beattie (24) linked GFP under the control of the E.coli proU promoter, which is sensitive to solute concentration in environment sur- rounding the cell. Modiﬁed bacteria then provided rapid detec- tion of water availability on plant leaves. Using in-ﬁeld ﬂow cytometers, dual or multiple ﬂuorescent proteins could serve as efﬁcient reporters of environmental conditions. The dual-ﬂuorescence approach can be further enhanced by the ability of ﬂow cytometers to isolate rare sub-populations from a vast background of negatives, making cell-sorting a powerful technique for isolating rare events. For example, Koo et al. (25) were able to encode daa mRNA processing activity in E.coli as a loss of GFP signal. High-speed cell-sorters were used to examine 500 million cells before successfully isolating one rare E.coli mutant that had reduced daa mRNA processing ability. Dual-ﬂuorescence reporting lays the foundation for a large number of novel high-throughput processes based on ﬂow cytometers. 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