Optimized compatible set of BioBrick™ vectors for metabolic pathway engineering
Optimized compatible set of BioBrick™ vectors for metabolic pathway engineering
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DOI：
10.1007/s00253-011-3633-4
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发表时间：
2011-10
影响因子：
5
通讯作者：
J. E. Vick;Ethan T. Johnson;Swati Choudhary;Sarah E. Bloch;F. López‐Gallego;P. Srivastava;Ilya B. Tikh;G. Wawrzyn;C. Schmidt-Dannert
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文献类型：
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作者：
J. E. Vick;Ethan T. Johnson;Swati Choudhary;Sarah E. Bloch;F. López‐Gallego;P. Srivastava;Ilya B. Tikh;G. Wawrzyn;C. Schmidt-Dannert
用于酶途径组装的BioBrick™范例正在被采用，并成为微生物工程的标准实践。我们提出了一种策略，以适应BioBrick™范例，以允许多基因途径快速组装到许多载体中，以及快速动员任何克隆的基因到具有不同特征的载体中，用于基因表达和蛋白质纯化。开发了初级BioBrick™（BB-eGFP），其中启动子/RBS、多克隆位点、任选的蛋白质纯化亲和标签和报告基因都通过另外的限制性酶被分离成离散区域。然后，该初级BB-eGFP用作具有不同复制起点、抗生素抗性、诱导型启动子（阿拉伯糖、IPTG或脱水四环素）、具有凝血酶切割的N-或C-末端组氨酸标签、aLacZα报告基因和另外的移动起点（oriT）的另外的BioBrick™载体的模板。通过测量报告基因表达，所有开发的BioBricks™和BioBrick™相容载体均显示出功能性。最后，组装C30类胡萝卜素途径作为模型酶途径，以证明该工程载体系统的体内功能性和相容性。
The BioBrick™ paradigm for the assembly of enzymatic pathways is being adopted and becoming a standard practice in microbial engineering. We present a strategy to adapt the BioBrick™ paradigm to allow the quick assembly of multi-gene pathways into a number of vectors as well as for the quick mobilization of any cloned gene into vectors with different features for gene expression and protein purification. A primary BioBrick™ (BB-eGFP) was developed where the promoter/RBS, multiple cloning sites, optional protein purification affinity tags and reporter gene were all separated into discrete regions by additional restriction enzymes. This primary BB-eGFP then served as the template for additional BioBrick™ vectors with different origins of replication, antibiotic resistances, inducible promoters (arabinose, IPTG or anhydrotetracycline), N- or C-terminal Histidine tags with thrombin cleavage, aLacZαreporter gene and an additional origin of mobility (oriT). All developed BioBricks™ and BioBrick™ compatible vectors were shown to be functional by measuring reporter gene expression. Lastly, a C30carotenoid pathway was assembled as a model enzymatic pathway to demonstrate in vivo functionality and compatibility of this engineered vector system.