Essential validation methods for E. coli strains created by chromosome engineering
Essential validation methods for E. coli strains created by chromosome engineering
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DOI：
10.1186/s13036-015-0008-x
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发表时间：
2015-07-01
影响因子：
5.6
通讯作者：
Dekker, Nynke H.
中科院分区：
文献类型：
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作者：
Krishnan, Sriram Tiruvadi;Moolman, M. Charl;Dekker, Nynke H.
背景资料：染色体工程包括一系列基于同源重组的技术，这些技术用于以受控的方式修饰模式生物的基因组。这些技术广泛用于基础研究和工业研究，以在同一大肠杆菌菌株中引入多个插入。迄今为止，在大肠杆菌中最成功实施的染色体工程技术是Aprioda-Red重组（也称为重组工程）和P1噬菌体转导。杆菌然而，由于在菌株创建过程中可能发生的错误，可靠的验证方法是必不可少的菌株的chromosome.Results和讨论改变后：聚合酶链反应（PCR）为基础的方法和DNA序列分析是快速和强大的方法来验证成功整合到染色体的DNA序列。尽管这些验证方法是必要的，但它们可能不足以检测所有错误，因此需要额外的验证方法。例如，由于外源插入可能发生在重组过程中，我们强调使用Southern印迹检测它们的存在。这些不需要的突变可以通过使用P1 β将感兴趣的区域转导到野生型染色体中来去除。然而，在这样做时，必须验证所用的P1裂解物和菌株均不受温杆菌污染，因为这些菌株可以在细胞内溶原化为大质粒。因此，我们举例说明了各种方法来探测温和的噬菌体污染，包括交叉划线琼脂和伊文思蓝铀（EBU）平板测定，其中后者是一个新报道的技术，用于这一目的，在大肠杆菌。杆菌最后，我们讨论的方法检测缺陷的细胞生长和形状特征，这应该是作为一个额外的check.Conclusion：简单，但关键的验证技术，这里讨论的可以用来可靠地验证任何染色体工程E。大肠杆菌菌株的错误，如染色体中的非特异性插入，温和的噬菌体污染，以及生长和细胞形状的缺陷。虽然PCR和DNA序列验证等技术应继续进行，但我们说明了进行这些额外测定的必要性。所讨论的技术是高度通用的，可以很容易地应用于任何类型的染色体工程。
Background: Chromosome engineering encompasses a collection of homologous recombination-based techniques that are employed to modify the genome of a model organism in a controlled fashion. Such techniques are widely used in both fundamental and industrial research to introduce multiple insertions in the same Escherichia coli strain. To date, lambda-Red recombination (also known as recombineering) and P1 phage transduction are the most successfully implemented chromosome engineering techniques in E. coli. However, due to errors that can occur during the strain creation process, reliable validation methods are essential upon alteration of a strain's chromosome.Results and discussion: Polymerase chain reaction (PCR)-based methods and DNA sequence analysis are rapid and powerful methods to verify successful integration of DNA sequences into a chromosome. Even though these verification methods are necessary, they may not be sufficient in detecting all errors, imposing the requirement of additional validation methods. For example, as extraneous insertions may occur during recombineering, we highlight the use of Southern blotting to detect their presence. These unwanted mutations can be removed via transducing the region of interest into the wild type chromosome using P1 phages. However, in doing so one must verify that both the P1 lysate and the strains utilized are free from contamination with temperate phages, as these can lysogenize inside a cell as a large plasmid. Thus, we illustrate various methods to probe for temperate phage contamination, including cross-streak agar and Evans Blue-Uranine (EBU) plate assays, whereby the latter is a newly reported technique for this purpose in E. coli. Lastly, we discuss methodologies for detecting defects in cell growth and shape characteristics, which should be employed as an additional check.Conclusion: The simple, yet crucial validation techniques discussed here can be used to reliably verify any chromosomally engineered E. coli strains for errors such as non-specific insertions in the chromosome, temperate phage contamination, and defects in growth and cell shape. While techniques such as PCR and DNA sequence verification should standardly be performed, we illustrate the necessity of performing these additional assays. The discussed techniques are highly generic and can be easily applied to any type of chromosome engineering.