The pKSM710 vector cassette provides tightly regulated lac and T7lac promoters and strategies for manipulating N-terminal protein sequences.
We describe a set of plasmid vectors that are very useful for cloning, expressing, mutagenizing, deleting, and sequencing DNA fragments. A strategy for using one (pKSM717) to obtain mutant protein products that contain deletions of N-terminal amino acids is also presented. Desirable sequences were first combined in plasmid pKSM710 in a manner that facilitates construction of similar vectors carrying alternative selectable markers or replication origins: a cassette that includes LacI-regulated T7 (T7lac) and lacUV5 promoters, a multiple cloning site (MCS)/lacZ alpha sequence, a set of transcription terminators (T phi, rrnBT1, rrnBT2, and Tfd), and an fd origin of replication can be moved as a single unit. Alternative restriction sites permit a lambda PL promoter and/or the sequence of the pMB1 replicon to be included in this unit when desired. With vectors containing the cassette, inserts in the MCS can be identified by their lack of lacZ alpha peptide complementing activity and expressed from the dually regulated T7 (T7lac) and/or lacUV5 promoter. We found expression from this pair of promoters to be very tightly regulated in appropriate hosts; the degree of repression obtainable in the absence of inducer (IPTG) should allow these constructs to be useful for engineering and expressing gene products that are potentially toxic to the cell. Using the pKSM710 cassette, we made derivatives carrying kan (KmR) (pKSM711, pKSM712), kan and lacI (pKSM715), kan and lacIq (pKSM713, pKSM714), and amp (pKSM717, pKSM718). One can use pKSM717 to obtain deletion derivatives that lack the original amino-terminal coding region of a cloned gene sequence but express the polypeptide encoded by the portion of the gene that remains.
Maneewannakul
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Department of Medical Microbiology and Immunology, Texas A & M University, College Station 77483.
Maneewannakul
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Ippen-Ihler
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D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular / Q000379:methods*; D004247:DNA / Q000737:chemistry / Q000235:genetics*; D015870:Gene Expression; D005798:Genes, Bacterial; D005822:Genetic Vectors; D007700:Kinetics; D008969:Molecular Sequence Data; D016296:Mutagenesis; D010957:Plasmids; D011401:Promoter Regions, Genetic; D011994:Recombinant Proteins / Q000096:biosynthesis*; D015183:Restriction Mapping; D017384:Sequence Deletion; D001616:beta-Galactosidase / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism
D011994:Recombinant Proteins; D004247:DNA; D001616:beta-Galactosidase