High-Copy Number Cloning of Toxic Genes

有毒基因的高拷贝数克隆

• 批准号: 6993443
• 负责人: John Thomas Mulligan
• 金额: $ 11.82万
• 依托单位: BLUE HERON BIOTECHNOLOGY, INC.
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-08-01 至 2007-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6993443
• 关键词: DNA damage; Escherichia coli; complementary DNA; cytotoxicity; genetic manipulation; molecular cloning; nucleic acid chemical synthesis; nucleic acid sequence; plasmids; posttranslational modifications; technology /technique development; transfection /expression vector

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

描述(由申请人提供)： 一小部分但很大一部分重组克隆在大肠杆菌中不稳定，因此很难使用标准的分子生物学技术进行克隆和操作。其中许多cDNA和基因编码的蛋白质对细菌有毒，即使在非常低的水平表达时也是如此。例如，只有几个特定的蛋白水解酶、核糖核酸酶或离子通道分子就能严重干扰细菌细胞的生长或存活。 这项提议的重点是开发一套新的分子工具来阻止有毒蛋白质的功能性表达，并允许使用标准的高拷贝质粒载体来传播编码这些基因的DNA序列。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。它们可以取代特殊的生长条件、低拷贝载体、特殊的细菌菌株和非标准技术的要求，从而简化有毒基因的操作。 这项研究的主要目标是允许简单有效地克隆和操作目前难以在大肠杆菌中生长的基因和cDNA。蓝鹭生物科技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)通过提高S公司目前基因合成业务的可靠性，2)通过销售使用这些新技术的工具包或服务