Genetic optimization of a bacteriophage-delivered alkaline phosphatase reporter to detect Escherichia coli
Genetic optimization of a bacteriophage-delivered alkaline phosphatase reporter to detect Escherichia coli
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DOI：
10.1039/c6an00479b
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发表时间：
2016-01-01
期刊：
影响因子：
4.2
通讯作者：
Sela, David A.
中科院分区：
文献类型：
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作者：
Jackson, Angelyca A.;Hinkley, Troy C.;Sela, David A.
很大一部分食源性疾病与（约46%）被农业用水中的病原体污染的多叶绿色蔬菜有关。为了防止这种情况，需要精确的生产点检测工具来识别和量化产品中的细菌污染物，以免消费者受到影响。在这项研究中，一个概念验证模型工程的噬菌体为基础的大肠杆菌检测系统。我们设计了大肠杆菌噬菌体T7，使其表达碱性磷酸酶（ALP）作为大肠杆菌的信号。大肠杆菌检测将野生型phoA（T7（ALP））和显性活性等位基因phoA D153G D330N（T7（ALP*））插入T7基因组中，通过CRISPR介导的未改变染色体的切割选择工程化构建体并通过PCR确认。工程菌E.将大肠杆菌靶细胞共孵育16小时以产生具有与输入细胞浓度相关的释放的ALP的裂解物。使用对硝基苯基磷酸盐（pNPP）的比色测定证明与载体对照（T7（EV））相比，T7（ALP）和T7（ALP*）产生显著的ALP（p
A large fraction of foodborne illnesses are linked to (similar to 46%) leafy green vegetables contaminated by pathogens harbored in agricultural water. To prevent this, accurate point-of-production detection tools are required to identify and quantify bacterial contaminants in produce before consumers are impacted. In this study, a proof-of-concept model was engineered for a phage-based Escherichia coli detection system. We engineered the coliphage T7 to express alkaline phosphatase (ALP) to serve as the signal for E. coli detection. Wild type phoA (T7(ALP)) and a dominant-active allele, phoA D153G D330N (T7(ALP*)) was inserted into the T7 genome, with engineered constructs selected by CRISPR-mediated cleavage of unaltered chromosomes and confirmed by PCR. Engineered phages and E. coli target cells were co-incubated for 16 hours to produce lysates with liberated ALP correlated with input cell concentrations. A colorimetric assay used p-nitrophenyl phosphate (pNPP) to demonstrate significant ALP production by T7(ALP) and T7(ALP*) compared to the vector control (T7(EV)) (p