A Plasmid Cloning Vector with Precisely Regulatable Copy Number in Escherichia coli

Abstract

We have developed a genetic system allowing for precise regulation of plasmid copy number in Escherichia coli cells. A cloning vector based on this system is described in this article. The pTC lambda 3 plasmid is a lambda replicon, but transcription controlling initiation of plasmid DNA replication starts from the PtetA promoter instead of phage lambda PR promoter. Additionally, activity of PtetA promoter is negatively controlled by the TetR repressor whose gene is located on the same plasmid vector and is induced by an analog of tetracycline, autoclaved chlortetracycline (aCT). Using different concentrations of the inducer it is possible to strictly regulate the copy number of pTC lambda 3 and thus the copy number of a cloned gene. The usefulness of the system for the regulatable production of a protein encoded by a gene inserted into pTC lambda 3 plasmid is demonstrated by dependence of beta-galactosidase activity on the lacZ gene dosage.

Bacteriophage replication modules

“…It was shown in numerous subsequent studies that the cII and the cro regulatory loops are not essential for λ plasmid replication; transcription from the p o promoter seems important but not the transcript, oop RNA (for details, see Taylor & Wegrzyn, 1995). Finally it was shown that p R can be replaced by a different (inducible) promoter (Herman‐Antosiewicz et al , 2001), which relieves λ plasmid replication from the intricate control by host DnaA (Glinkowska et al , 2003). These results led to the functional definition of the λ‐type replication module being composed of the O ( ori λ) and P genes (Wrobel & Wegrzyn, 2002).…”

A genetic toolkit for co-expression of multiple proteins of diverse physiological implication

“…Differences in the copy number of the plasmids because of their unique origin of replication will have implications to the expression of the gene [18] . One would like to believe that increasing the copy number of a plasmid would lead to increased gene dosage [ 4 , 19 ].…”

SeqA, the Escherichia coli origin sequestration protein, is also a specific transcription factor

“…Plasmids pKB2 (Kur et al ., 1987) and pCB104 (Boyd and Sherratt, 1995) are standard λ plasmids bearing a replication region of bacteriophage λ and a kanamycin resistance and a chloramphenicol resistance gene respectively. Plasmid pTCλ3 is a λ replicon bearing the p tet promoter instead of p R (Herman‐Antosiewicz et al ., 2001). For its replication in host cells, pTCλ3 requires stimulation of the p tet promoter by the addition of an inductor, autoclaved chlortetracycline, to a culture medium (for details, see Herman‐Antosiewicz et al ., 1998a,b; 2001; Herman‐Antosiewicz and Wegrzyn, 1999).…”

“…Plasmid pTCλ3 is a λ replicon bearing the p tet promoter instead of p R (Herman‐Antosiewicz et al ., 2001). For its replication in host cells, pTCλ3 requires stimulation of the p tet promoter by the addition of an inductor, autoclaved chlortetracycline, to a culture medium (for details, see Herman‐Antosiewicz et al ., 1998a,b; 2001; Herman‐Antosiewicz and Wegrzyn, 1999). Plasmid pR‐HG (Szalewska‐Palasz et al.…”