High level expression of porcine growth hormone in Escherichia coli from an expression vector containing bacteriophage lambda PL and N gene untranslated region

An Escherichia coli expression vector, pG408N containing a PL promoter and the upstream untranslated region of the N gene of bacteriophage lambda has been constructed. We have designed a PvuII site immediately behind the untranslated region. A DNA fragment starting with an initiation codon ATG could be inserted into this site for expression. This vector also contains 7 additional cloning sites downstream from the PvuII site. A gene could be cloned into one of these sites and the 5' sequence of this gene could be modified with synthetic oligonucleotides and ligated to the PvuII for the purpose of increasing gene expression. We have also cloned the lambda cl gene into a p15A plasmid. Cotransformation of this plasmid with the expression vector allows the cloning vector pG408N to be used in any E. coli strain. Using this system, we were able to express porcine growth hormone to approximately 35% of total proteins in E. coli DH5 alpha.     

Sequence tolerance of the phage lambda PRM promoter: implications for evolution of gene regulatory circuitry

Much of the gene regulatory circuitry of phage lambda centers on a complex region called the O(R) region. This approximately 100-bp region is densely packed with regulatory sites, including two promoters and three repressor-binding sites. The dense packing of this region is likely to impose severe constraints on its ability to change during evolution, raising the question of how the specific arrangement of sites and their exact sequences could evolve to their present form. Here we ask whether the sequence of a cis-acting site can be widely varied while retaining its function; if it can, evolution could proceed by a larger number of paths. To help address this question, we developed a lambda cloning vector that allowed us to clone fragments spanning the O(R) region. By using this vector, we carried out intensive mutagenesis of the P(RM) promoter, which drives expression of CI repressor and is activated by CI itself. We made a pool of fragments in which 8 of the 12 positions in the -35 and -10 regions were randomized and cloned this pool into the vector, making a pool of P(RM) variant phage. About 10% of the P(RM) variants were able to lysogenize, suggesting that the lambda regulatory circuitry is compatible with a wide range of P(RM) sequences. Analysis of several of these phages indicated a range of behaviors in prophage induction. Several isolates had induction properties similar to those of the wild type, and their promoters resembled the wild type in their responses to CI. We term this property of different sequences allowing roughly equivalent function "sequence tolerance " and discuss its role in the evolution of gene regulatory circuitry.     

A new selective phage cloning vector, lambda 2001, with sites for XbaI, BamHI, HindIII, EcoRI, SstI and XhoI

An improved bacteriophage lambda cloning vector, lambda 2001, has been constructed. The phage includes a 34-bp polylinker oligonucleotide which introduces cleavage sites for XbaI, SstI, XhoI, EcoRI, HindIII and BamHI, and can accommodate 10-kb to 23-kb fragments. Inserts that destroy the BamHI or XhoI cloning sites may be recovered by excision at flanking sites in the polylinker sequence. Insertion of foreign DNA into lambda 2001 generates phage with a Spi- phenotype. The recombinant phage are able to grow on P2 lysogens but the parental vector phages are not. In the course of this work, the polylinker sequence was also introduced into M13mp8. This produced a new vector, M13mp12, with cloning sites for EcoRI, SmaI, XbaI, SstI, XhoI, BamHI, and HindIII.     

A small mobilizable IncP group plasmid vector packageable into bacteriophage lambda capsids in vitro

A mobilizable cosmid derivative of an IncP group plasmid was constructed by cloning the oriT region of RK2, a wide host-range plasmid, and the minimal DNA sequence of bacteriophage lambda required for efficient packaging in vitro. This cosmid is 13 kb in size and has unique restriction sites for EcoRI, XhoI, HindIII, and SalI. The XhoI and HindIII sites are within the kanamycin-resistance gene and the SalI site is in the tetracycline-resistance gene. This plasmid was mobilizable from an Escherichia coli donor to a number of diverse gram-negative bacteria at a frequency of 0.8 to 10 per 100 donors. This vector is one of the smallest of all wide host-range cosmids described in the literature. As part of this study, another mobilizable IncP group plasmid vector has also been constructed which, in addition to the sites listed above, has a unique BglII site, but which lacks the packager sequence.     

Construction of a newEscherichia coli — Saccharomyces cerevisiae shuttle plasmid cloning vector allowing positive selection for cloned fragments

A new E. coli-S. cerevisiae shuttle plasmid cloning vector (pPW263) with a positive type of selection, was constructed. The selection system, based on the regulatory region of lambda phage controlling the expression of tetracycline resistance, was derived from the cloning vector pUN121 (Nilsson et al. 1983). There are three cloning sites in the cI gene, EcoRI, HindIII and BglII, and, in addition, two unique sites in the neighborhood, BamHI and SalI. The size of the vector is 7.8 kb. The maintenance of the vector and the selection in yeast was ensured by the replication region of the 2 mu plasmid and by the URA3 marker gene, respectively.