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where the alcohol can be glycerol, allyl alcohol, D-galactose, galactopyranosides, oligosaccharides, and polysaccharides. This enzyme has been used in biosensors to measure D-galactose and lactose in blood samples, dairy samples and in fermentation process control. The goals of the directed evolution experiment were to increase stability and activity toward nonnatural substrates.

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When the application being monitored is a homegrown one, the developers should be made aware of the monitoring requirement early in the development process, so that the testing can be done in as nonintrusive a way as possible. If the failure is in a hardware component, then the choices are basically the same as for software, but there are some additional options. If the failed component is a network interface card (NIC), it is possible to move the IP addresses that were housed on the failed NIC to a dedicated standby NIC that is in the same host and attached to the same network. In that case, the system can completely recover from the failure in the time it takes to detect the problem, and migrate the IP addresses to the new card, which should take no more than a few seconds. (However, some client applications such as databases, may need manual assistance to reconnect.) When a disk fails, and it has been made properly redundant (that is, mirrored or RAID-5), the only action that is required is notification. The failed disk must be replaced, an activity that is most assuredly a manual one. But since the data on the failed disk is protected, it need not be replaced immediately; the system administrator may choose to wait until a more convenient time to perform the work (keeping in mind that some level of redundancy has been lost). All the while, the system will continue to operate. If the failed disk is further protected with hot spares, then no redundancy will be lost as the system automatically (and logically) replaces the failed disk with the hot spare. If the failed disk sits within an array that supports hot swapping, then the disk can be replaced without any service interruption.

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( Axway, 2006)

So now our automated testing software has determined that a failure has occurred. For our purposes, we have concluded that a failover is the best way to recover from this particular failure. There are two ways that this failover might be initiated: manually and automatically. In a manual failover, all the work to respond to the already identified failure lands squarely on the shoulders of the system administrator. Before anything else can happen, the system administrator must find out about the problem. She could be notified via pagers or email, or her users could start screaming or storm her desk carrying pitchforks and torches. Of course, for this to work, she has to be near her desk or phone (and not using it) or be carrying her pager. What if she is out of the building at lunch or in the bathroom What if the failure occurs late at night or on a weekend

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In the rst step, the GAO gene was isolated from Fusarium sp., and fused to a strong promoter (lacZ). When the promoter is induced, transcription by RNA polymerase begins, and both mRNA and GAO are produced. Error-prone PCR was used to generate genetic diversity, at a 2 3 base substitution rate per gene. Mutated genes were cloned into a vector (plasmid pUC18), and used to transform E. coli cells by electroporation. Transformed cells were plated on agar, and single colonies were picked off. These were transferred to a duplicate plate, grown 12 h in the presence of IPTG to induce the lacZ promoter, and GAO was then produced. The cells were then centrifuged, washed, and lysed. Galactose oxidase activity was measured for each variant culture product before and after 10 min at different temperatures (55 70 C) to determine stability. Selected improved variants were later recombined using a method similar to DNA shuf ing. Three or four rounds of mutation allowed the selection of a mutant enzyme with 15-fold higher activity, and $ 5 C increase in stability. Substrate selectivity was unchanged with respect to the wild-type enzyme from Fusarium.23 This example is one of the many possible applications of directed evolution for the generation of new bioassay reagents with new or improved characteristics. SUGGESTED REFERENCES

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M. Famulok, E.-L. Winnacker, and C.-H. Wong, Eds., Combinatorial Chemistry in Biology , in Current Topics in Microbiology and Immunology, Vol. 243, Spriger-Verlag, Berlin, 1999. F. H. Arnold, Ed., Evolutionary Protein Design , Advances in Protein Chemistry, Vol. 55, Academic Press, San Diego, CA, 2001.

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