对AraC进行定向进化以提高阿拉伯糖和乳糖诱导型启动子的兼容性。
Directed evolution of AraC for improved compatibility of arabinose- and lactose-inducible promoters.
作者信息
Lee Sung Kuk, Chou Howard H, Pfleger Brian F, Newman Jack D, Yoshikuni Yasuo, Keasling Jay D
机构信息
Department of Chemical Engineering, University of California, Berkeley, California 94720, USA.
出版信息
Appl Environ Microbiol. 2007 Sep;73(18):5711-5. doi: 10.1128/AEM.00791-07. Epub 2007 Jul 20.
Abstract
Synthetic biological systems often require multiple, independently inducible promoters in order to control the expression levels of several genes; however, cross talk between the promoters limits this ability. Here, we demonstrate the directed evolution of AraC to construct an arabinose-inducible (P(BAD)) system that is more compatible with IPTG (isopropyl-beta-D-1-thiogalactopyranoside) induction of a lactose-inducible (P(lac)) system. The constructed system is 10 times more sensitive to arabinose and tolerates IPTG significantly better than the wild type. Detailed studies indicate that the AraC dimerization domain and C terminus are important for the increased sensitivity of AraC to arabinose.
摘要
合成生物系统通常需要多个独立可诱导的启动子来控制多个基因的表达水平；然而，启动子之间的串扰限制了这种能力。在此，我们展示了AraC的定向进化，以构建一种阿拉伯糖诱导型（P(BAD)）系统，该系统与乳糖诱导型（P(lac)）系统的异丙基-β-D-1-硫代半乳糖苷（IPTG）诱导更兼容。构建的系统对阿拉伯糖的敏感性比野生型高10倍，并且对IPTG的耐受性明显更好。详细研究表明，AraC二聚化结构域和C末端对于AraC对阿拉伯糖敏感性的提高很重要。
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本文引用的文献
1
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Curr Opin Biotechnol. 2006 Oct;17(5):548-57. doi: 10.1016/j.copbio.2006.09.001. Epub 2006 Sep 15.
2
Dual selection enhances the signaling specificity of a variant of the quorum-sensing transcriptional activator LuxR.
Nat Biotechnol. 2006 Jun;24(6):708-12. doi: 10.1038/nbt1209. Epub 2006 May 21.
3
Production of the antimalarial drug precursor artemisinic acid in engineered yeast.
Nature. 2006 Apr 13;440(7086):940-3. doi: 10.1038/nature04640.
4
Environmentally controlled invasion of cancer cells by engineered bacteria.
J Mol Biol. 2006 Jan 27;355(4):619-27. doi: 10.1016/j.jmb.2005.10.076. Epub 2005 Nov 14.
5
A propionate-inducible expression system for enteric bacteria.
Appl Environ Microbiol. 2005 Nov;71(11):6856-62. doi: 10.1128/AEM.71.11.6856-6862.2005.
6
Advances in synthetic biology: on the path from prototypes to applications.
Curr Opin Biotechnol. 2005 Aug;16(4):476-83. doi: 10.1016/j.copbio.2005.07.002.
7
A synthetic multicellular system for programmed pattern formation.
Nature. 2005 Apr 28;434(7037):1130-4. doi: 10.1038/nature03461.
8
Spatiotemporal control of gene expression with pulse-generating networks.
Proc Natl Acad Sci U S A. 2004 Apr 27;101(17):6355-60. doi: 10.1073/pnas.0307571101. Epub 2004 Apr 19.
9
Programmed population control by cell-cell communication and regulated killing.
Nature. 2004 Apr 22;428(6985):868-71. doi: 10.1038/nature02491. Epub 2004 Apr 4.
10
AraC protein: a love-hate relationship.
Bioessays. 2003 Mar;25(3):274-82. doi: 10.1002/bies.10237.
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