BglBrick vectors and datasheets: A synthetic biology platform for gene expression.
BglBrick vectors and datasheets: A synthetic biology platform for gene expression.
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DOI：
10.1186/1754-1611-5-12
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发表时间：
2011-09-20
影响因子：
5.6
通讯作者：
Keasling JD
中科院分区：
文献类型：
--
作者：
Lee TS;Krupa RA;Zhang F;Hajimorad M;Holtz WJ;Prasad N;Lee SK;Keasling JD
随着工程化生物系统变得更加复杂，在单个宿主细胞内表达来自不同质粒和诱导型表达系统的多个操纵子越来越常见。优化这样的系统通常需要筛选复制起点、表达系统和抗生素标记的组合。当同时使用一个以上的复制起点或表达系统时，缺乏关于这些组分如何表现的定量数据阻碍了这一过程。此外，这个过程可能很耗时，因为它通常需要创建新的载体或克隆到现有但不同的载体中。在这里，我们报告的发展和表征的表达载体库兼容的BglBrick标准（BBF RFC 21）。我们已经设计并构建了96个BglBrick相容质粒，其具有复制起点、抗生素抗性基因和诱导型启动子的组合。在存在非同源诱导剂分子的情况下，在一系列诱导剂浓度范围内对这些质粒进行了表征，并使用几种生长培养基对其特征进行了描述，并以标准格式记录了其特征。使用三质粒系统来研究多个复制起点对质粒拷贝数的影响。这里提供的标准化载体集合允许用户快速构建和测试具有启动子强度、诱导型表达系统、拷贝数和抗生素抗性的各种组合的基因的表达。为这些载体创建的定量图表将增加基因表达的可预测性，特别是当使用多种质粒和诱导剂时。
As engineered biological systems become more complex, it is increasingly common to express multiple operons from different plasmids and inducible expression systems within a single host cell. Optimizing such systems often requires screening combinations of origins of replication, expression systems, and antibiotic markers. This procedure is hampered by a lack of quantitative data on how these components behave when more than one origin of replication or expression system are used simultaneously. Additionally, this process can be time consuming as it often requires the creation of new vectors or cloning into existing but disparate vectors. Here, we report the development and characterization of a library of expression vectors compatible with the BglBrick standard (BBF RFC 21). We have designed and constructed 96 BglBrick-compatible plasmids with a combination of replication origins, antibiotic resistance genes, and inducible promoters. These plasmids were characterized over a range of inducer concentrations, in the presence of non-cognate inducer molecules, and with several growth media, and their characteristics were documented in a standard format datasheet. A three plasmid system was used to investigate the impact of multiple origins of replication on plasmid copy number. The standardized collection of vectors presented here allows the user to rapidly construct and test the expression of genes with various combinations of promoter strength, inducible expression system, copy number, and antibiotic resistance. The quantitative datasheets created for these vectors will increase the predictability of gene expression, especially when multiple plasmids and inducers are utilized.
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DOI：
10.1126/science.1191652
发表时间：
2010-10-01
期刊：
Science (New York, N.Y.)
影响因子：
--
作者：
Ajikumar PK;Xiao WH;Tyo KE;Wang Y;Simeon F;Leonard E;Mucha O;Phon TH;Pfeifer B;Stephanopoulos G
通讯作者：
Stephanopoulos G
影响因子：
4.4
作者：
Lee, Sung Kuk;Chou, Howard H.;Keasling, Jay D.
通讯作者：
Keasling, Jay D.
影响因子：
48
作者：
Gibson, Daniel G.;Young, Lei;Smith, Hamilton O.
通讯作者：
Smith, Hamilton O.
影响因子：
4.1
作者：
Lee, Changsoo;Kim, Jaai;Hwang, Seokhwan
通讯作者：
Hwang, Seokhwan
影响因子：
46.9
作者：
Canton, Barry;Labno, Anna;Endy, Drew
通讯作者：
Endy, Drew