Markerless gene deletion by floxed cassette allelic exchange mutagenesis in chlamydia trachomatis

Gabrielle Keb, Kenneth A. Fields

Research output: Contribution to journalArticlepeer-review

4 Scopus citations


Chlamydia trachomatis is an obligate intracellular pathogen that has been historically difficult to genetically manipulate. Definitive progress in elucidating the mechanisms that C. trachomatis use to create and maintain a privileged intracellular niche has been limited due to a lack of genetic tools. Fortunately, there have recently been several new advances in genetic manipulation techniques. Among these is the development of fluorescence-reported allelic exchange mutagenesis (FRAEM). This method allows targeted gene deletion coupled with insertion of a selection cassette encoding antibiotic resistance and green fluorescent protein (GFP). Reliance on this strategy can be complicated when targeting genes within polycistronic operons due to the potential of polar effects on downstream genes. Floxed cassette allelic exchange mutagenesis (FLAEM), the protocol for which is described here, was developed to alleviate cassette-induced polar effects. FLAEM utilizes Cre-loxP genome editing to remove the selection cassette after targeted deletion by allelic exchange. The resulting strains contain markerless gene deletions of one or more coding sequences. This technique facilitates direct assessment of gene function and expands the repertoire of tools for genetic manipulation in C. trachomatis.

Original languageEnglish
Article numbere60848
JournalJournal of Visualized Experiments
Issue number155
StatePublished - Jan 2019

Bibliographical note

Funding Information:
This work was supported by Public Health Service grants from the National Institute of Health, NIAID (grants A1065530 and Al124649), to K.A. Fields.

Publisher Copyright:
© 2020 Journal of Visualized Experiments.


  • Allelic exchange
  • Allelic recombination
  • Chlamydia
  • Cre recombinase
  • Immunology and Infection
  • Issue 155
  • Mutagenesis

ASJC Scopus subject areas

  • Neuroscience (all)
  • Chemical Engineering (all)
  • Biochemistry, Genetics and Molecular Biology (all)
  • Immunology and Microbiology (all)


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