Low copy plasmids

Jones, K.L., Kim, S.W., and Keasling, J.D., Low-copy plasmids can perform as well as or better than high-copy plasmids for metahohc engineering of bacteria, Metab. Eng. 2, 328, 2000. 

In principle, directed evolution procedures could be repeated indefinitely until any desired activity has been attained. At some point, however, the catalyst will be sufficiently active that the host cell grows like the wild-type strain, making selection for further improvement difficult. This is true for the modified hexamer even though it is still an order of magnitude less efficient than the homodimeric MjCM [37]. Since total activity depends on the catalyst concentration as well as specific activity, reducing the available catalyst concentration can further increase selection pressure. In practice, intracellular protein concentrations can be lowered in a variety of ways, including the use of low copy plasmids [101], weak promoters [102] and inefficient ribosome binding sites [103]. 

Evidence to support the hypothesis that MsbA may be involved in lipid A transport is growing. htrB mutants accumulate tetraacylated lipid A in the inner membrane at the non-permissive temperature of 42 °C [41, 63). However, when mshA is provided in trans on a low copy plasmid, the tetraacylated lipid A is transported to the outer membrane [41]. In cells lacking mshA, hexaacylated lipid A accumulates in the inner membrane indicating that the transport of lipid A has been affected by the lack of mshA [41]. Taken together this strongly suggests that mshA plays a role in the movement of lipid A from the inner to the outer membrane, but direct biochemical assays remain to be developed. 

Plasmid DNAs. Plasmids are nucleic acid molecules capable of intracellular extrachromosomal repHcation. Usually plasmids are circular DNA species, but linear and RNA plasmids are known. In nature, plasmids can assume a variety of lifestyles. Plasmids can recombine into the host chromosome, be packaged into vims particles, and repHcate at high or low copy number relative to the host chromosome. Additionally, their information can affect the host phenotype. Whereas no single plasmid is usually capable of all these behaviors, the properties of various plasmids have been used to constmct vectors for a variety of purposes. 

Bacterial cells may harbor one or many copies of a particular plasmid, depending on the nature of the plasmid replicator. That is, plasmids are classified as high copy number or low copy number. The copy number of most genetically engineered plasmids is high (200 or so), but some are lower. 

Careful empirical selection of the expression platform for carotenogenesis has included selection of the best strains for stability and degree of accumulation and the selection of compatible drug-resistance combinations and low copy number polycistronic plasmids to enhance product accumulation by decrease of metabolic burden." 5 Matthews and Wurtzel discussed culture and induction conditions - that have been explored in most studies. Most efforts to engineer carotenoid biosynthesis in E. coli focused on the genes and enzymes of the pathway and had a modest effect on improved accumulation. For example, substitution and over-expression of a GGPPS that uses IPP directly (discussed in... 

Some plasmids are present in cells in low-copy number - one or a few per cell, since the plasmid DNA replicates only once or twice before cell division. However, other plasmids exist in large numbers - from... 

High-copy plasmids might also express large amounts of products that are toxic to the host. Without selective pressure and if the product is not toxic, the losses are at low frequency (lO -KT6 per generation) but can increase tremendously when the expressed insert is toxic (Baneyx, 1999). This can even occur under selective pressure because the plasmid population contains a mixture of plasmids with and... 

All genes cloned by PCR must be sequenced. Even proofreading enzymes will occasionally make errors. Because the pET vectors have low-copy, pBR322 origins of replication, plasmid yield from a standard miniprep is low. Typically, DNA from several minipreps is pooled or a larger-scale DNA preparation method is used to obtain sufficient DNA for sequencing. Alternatively, PCR with vector specific primers may be used to amplify and sequence the final insert. 

Origin of replication (determines in what host the plasmid may be replicated) also determines whether the plasmid is high copy or low copy ... 

Stoker, N. G., Fairweather, N. F., and Spratt, B. G. (1982). Versatile Low-Copy-Number Plasmid Vectors for Cloning in Escherichia coli. Gene 18 335. 

The accuracy of real-time PCR quantification depends not only on the method chosen to analyze the curves, but also on the quality of calibrators used. Purified PCR products quantified by spectrophotometry are easily obtained. When serially diluted, these calibrators can accurately quantify the amount of target in human genomic DNA. Alternatively, purified plasmids or genomic DNA can be used as calibrators. Limiting dilution analysis to determine the amount of ampMable DNA is seldom necessary. The precision of quantitative real-time PCR depends on the copy number. When the initial target concentration is low, imprecision is high. Part of the variance comes from stochastic limitations as defined by the Poisson distribution as described earlier. In addition, the PCR efficiency may be more variable at low copy numbers. 

One way of alleviating a number of these problems is actually to make several parallel clone banks using different expression vectors and strains, regulatable expression systems, and low copy-number vectors. Vectors choices [28] such as plasmids vs phagemids, high vs low copy number vectors, all can affect what is cloned, and there is not one solution which will satisfy every project. Furthermore, the cloning host also makes a difference. E. coli is usually the host of choice, but Bacillus can be preferred for secretable enzymes, and yeast for eukaryotic or post-transitionally modified enzymes. 

---