| 摘要 |
Bacterial plasmid vectors based on alpha -amylase, are new. Bacterial plasmid vectors based on alpha -amylase, with a wide host spectrum and multiple uses, particularly amongst gram-positive bacteria, may support: cloning of foreign DNA with positive selection; isolation of any constitutive promoter; isolation of inducible/repressible promoters, where a single regulatory agent is known; quantitative and qualitative analysis of promoter activities, operator sequence, ribosome binding sites, start codons, signal peptides or display signals for cell wall proteins; constitutive expression of foreign proteins; constitutive expression of fusion proteins containing alpha -amylase, where it is possible to choose, whether the product is cytoplasmic or extracellular, and whether the fusion protein will be at the 5' or 3' end; cell surface display of fusion proteins in gram-positive bacteria; alpha -amylase activity test without cell information; alpha -amylase activity test in organisms with their own alpha -amylase, where this is not heat stable; purification of fusion proteins with strong adsorption; detection of alpha -amylase, or fusion derivatives, through color activity in a native or denatured polyacrylamide gel; availability for Escherichia coli with two alternative replication origins and two alternative resistance markers; and insertion in other bacteria through three alternate methods. Independent claims are also included for: pre-made medium that makes it possible for positive selection of alpha -amylase activity without use of I2/I<-> solution; a method of cloning with help of a pre-linearized vector to faultlessly construct a shuttle-vector in accordance with (m) for the insertion of vectors in gram-positive bacteria, choice between three differing copy numbers in target organisms; a helper vector and degenerate oligonucleotide for the possible direct use amongst vectors from above in gram-positive bacteria; and vector and selection method to isolate regulated promoters in accordance with 1.c) by selective cell sorting.
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