High-Copy Number Cloning of Toxic Genes

描述（由申请人提供）： 一小部分但很重要的重组克隆在E.因此难以使用分子生物学的标准技术进行克隆和操作。这些cDNA和基因中的许多编码即使在非常低的水平下表达也对细菌有毒的蛋白质。例如，只有几个特定的蛋白酶，RNA酶或离子通道分子可以严重破坏细菌细胞的生长或存活。 该提案的重点是开发一套新的分子工具，以阻止有毒蛋白质的功能表达，并允许使用标准的高拷贝质粒载体的DNA序列编码这些基因的繁殖。这些工具将通过减少有毒基因产物的表达和活性来发挥作用。它们可以取代对特殊生长条件、低拷贝载体、特殊细菌菌株和其他非标准技术的要求，从而简化有毒基因的操作。 本研究的主要目的是简单有效地克隆和操作目前在大肠杆菌中难以生长的基因和cDNA。杆菌Blue Heron Biotechnology是一家基因合成公司。如果成功，新技术将通过两种方式商业化：1）提高公司目前基因合成业务的可靠性; 2）销售使用这些新技术的试剂盒或服务。

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