Caveat Mutator. Escherichia coli

The use of whole genome microarrays for monitoring mutagenised or otherwise engineered genetic derivatives is a potentially powerful tool for checking genomic integrity. Using comparative genomic hybridization of a number of unrelated, directed deletion mutants in Escherichia coli K-12 MG1655 we identified unintended secondary genomic deletions in the flhDC region in deltafnr, deltacrp, and deltacreB mutants. These deletions were confirmed by PCR and phenotypic tests. Our findings show that non-motile progeny are found in some populations of MG1655 directed deletion mutants, and studies on the effects of gene knock-outs should be viewed with caution when the mutants have not been screened for the presence of secondary deletions, or confirmed by other methods. Keywords: comparative genomic hybridisation Overall design: We used the CGH method to genetically characterize a series of regulatory gene deletion mutants we had made in MG1655 using the lamda-Red method of Datsenko and Wanner. A number of strains were tested using CGH, and each strain was tested only once. Genomic DNA isolated from wt MG1655 was used as a reference in all hybridisations.

利用全基因组微阵列监测诱变株或其他工程化遗传衍生株，是核查基因组完整性的潜在有力工具。本研究采用比较基因组杂交（comparative genomic hybridization，CGH）技术，对大肠杆菌K-12 MG1655中多株无关的定向缺失突变株开展分析，在Δfnr、Δcrp及ΔcreB突变株的flhDC区域中鉴定出非预期的次级基因组缺失。上述缺失经聚合酶链式反应（PCR）与表型检测得以验证。研究结果显示，部分MG1655定向缺失突变株的种群中存在非运动性子代；若未对突变株进行次级缺失筛查或通过其他方法验证，则开展基因敲除效应相关研究时需保持谨慎。关键词：比较基因组杂交（comparative genomic hybridization）。实验整体设计：我们采用Datsenko与Wanner提出的λ-Red重组系统，在MG1655中构建一系列调控基因缺失突变株，并通过CGH技术对其进行遗传鉴定。共测试多株菌株，且每株仅测试一次。所有杂交实验均以野生型MG1655分离得到的基因组DNA作为参照样本。