High-Copy Number Cloning of Toxic Genes

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DESCRIPTION (provided by applicant): A small but significant fraction of recombinant clones are unstable in E. coli and thus difficult to clone and manipulate using the standard techniques of molecular biology. Many of these cDNAs and genes encode proteins that are toxic to the bacteria even when expressed at very low levels. For instance, only a few molecules of specific proteases, RNAses or ion channels can severely disrupt the growth or survival of a bacterial cell. This proposal is focused on developing a set of new molecular tools to block the functional expression of toxic proteins and allow the propagation of DNA sequences encoding these genes using standard, high-copy plasmid vectors. These tools will function by reducing the expression and activity of toxic gene products. They could replace requirements for special growth conditions, low-copy vectors, special bacterial strains and ether non-standard techniques, and thus simplify the manipulation of toxic genes. The primary objective of this research is to allow simple and efficient cloning and manipulation of genes and cDNAs that are currentlydifficult to grow in E. coli. Blue Heron Biotechnology is a gene synthesis company. If successful, the new technologies will be commercialized in two ways, 1) by improving the reliability of the company s current gene synthesis business, and 2) by selling kits or services using these new technologies