Abstract: We have developed a new gene transfer system for extreme thermophiles of the genus Thermus, including Thermus flavus., using a chromosomal gene, and a thermostable derivative of the kanamycin-resistance gene (kantr2). A plasmid mediated gene-replacement process is used to insert it into the chromosome resulting in the production of Leu−Kmr transformants. This system not only allows stable, single-copy gene insertion into the chromosome of an extreme thermophile, but can be used in the thermo-genetic process described here to generate thermo-stabilized enzymes and proteins for industrial processes. This host-vector environment makes it possible to generate further thermo-stabilizing mutations in the kan gene beyond those levels previously reported.

Abstract: The present invention relates to a system for identifying, isolating and utilizing promoter elements useful for expression of nucleotide sequences and the proteins encoded thereby in a thermophile. In one embodiment, a recombinant DNA molecule is provided, and comprises a reporter sequence, a putative thermophile promoter, a selectable marker sequence, and a 3′ and a 5′ DNA targeting sequence that are together capable of causing integration of at least a portion of said DNA molecule into the genome of a thermophile. Further, within the recombinant DNA, the reporter sequence is under the transcriptional control of a promoter which functions in a thermophile to form a promoter/reporter cassette, the promote/reporter cassette is flanked by said 3′ and said 5′ DNA targeting sequences, and the promoter/reporter cassette is positioned in the opposite orientation of the DNA targeting sequences.

Abstract: The instant disclosure teaches transposons and methods of the instant invention for making Protein Fusions by rapid, random shuffling of protein domains to produce novel protein fusions. This system is generally applicable to production of multifunctional chimeric proteins in vivo and in vitro. The methods and constructs of the instant invention can be used to randomly create both carboxy- and amino-terminal protein fusions in vivo. The methods and contructs of the instant invention are useful in the development of a protein domain library, in the construction of multifunctional enzymes, and in the accelerated evolution of new enzymatic activities.

Abstract: We have developed a new gene transfer system for extreme thermophiles of the genus Thermus, including Thermus flavus., using a chromosomal gene, and a thermostable derivative of the kanamycin-resistance gene (kan.sup.tr2). A plasmid mediated gene-replacement process is used to insert it into the chromosome resulting in the production of Leu.sup.- Km.sup.r transformants. This system not only allows stable, single-copy gene insertion into the chromosome of an extreme thermophile, but can be used in the thermo-genetic process described here to generate thermo-stabilized enzymes and proteins for industrial processes. This host-vector environment makes it possible to generate further thermo-stabilizing mutations in the kan gene beyond those levels previously reported.

Abstract: We have developed a new gene transfer system for extreme thermophiles of the genus Thermus, including Thermus flavus., using a chromosomal gene, and a thermostable derivative of the kanamycin-resistance gene (kan.sup.tr2). A plasmid mediated gene-replacement process is used to insert it into the chromosome resulting in the production of Leu.sup.- Km.sup.r transformants. This system not only allows stable, single-copy gene insertion into the chromosome of an extreme thermophile, but can be used in the thermo-genetic process described here to generate thermo-stabilized enzymes and proteins for industrial processes. This host-vector environment makes it possible to generate further thermo-stabilizing mutations in the kan gene beyond those levels previously reported.