Application of BRED technology to construct recombinant D29 reporter phage expressing EGFP
Application of BRED technology to construct recombinant D29 reporter phage expressing EGFP
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DOI：
10.1111/1574-6968.12171
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发表时间：
2013-07-01
影响因子：
2.1
通讯作者：
Hirata, Mario H.
中科院分区：
文献类型：
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作者：
da Silva, Joas L.;Piuri, Mariana;Hirata, Mario H.
噬菌体重组的电穿孔DNA（繁殖）已被描述为基因缺失和点突变的分枝杆菌噬菌体的建设。利用BRED将Phsp60-egfp片段（1143bp）插入到分枝杆菌噬菌体D29基因组中，构建新的报告噬菌体，用于分枝杆菌细胞的检测。成功插入重组噬菌体，并纯化重组噬菌体。即使经过几代噬菌体，磁带的DNA测序也没有显示任何突变。用D29::Phsp60-egfp （MOI为10）感染耻垢分枝杆菌mc2155细胞，镜检EGFP表达情况。在感染后2h左右观察到荧光，但随后由于细胞裂解而消失。我们试图通过删除lysA基因来构建一个裂解缺陷突变体，尽管我们无法纯化突变体，即使通过互补也无法达到均匀性。这些观察结果表明，BRED能够将c. 1kbp大小的DNA片段插入到分枝噬菌体基因组中，作为构建新的诊断报告噬菌体的策略。
Bacteriophage Recombineering of Electroporated DNA (BRED) has been described for construction of gene deletion and point mutations in mycobacteriophages. Using BRED, we inserted a Phsp60-egfp cassette (1143bp) into the mycobacteriophage D29 genome to construct a new reporter phage, which was used for detection of mycobacterial cells. The cassette was successfully inserted and recombinant mycobacteriophage purified. DNA sequencing of the cassette did not show any mutations even after several phage generations. Mycobacterium smegmatis mc2155 cells were infected with D29::Phsp60-egfp (MOI of 10) and evaluated for EGFP expression by microscopy. Fluorescence was observed at around 2h after infection, but dissipated in later times because of cell lysis. We attempted to construct a lysis-defective mutant by deleting the lysA gene, although we were unable to purify the mutant to homogeneity even with complementation. These observations demonstrate the ability of BRED to insert c. 1kbp-sized DNA segments into mycobacteriophage genomes as a strategy for constructing new diagnostic reporter phages.