The experiments presented in this workshop are part of a semester-long laboratory course in molecular biology, which is offered to third-year undergraduate students in Biological Sciences- Applied Bioscience specialization. This intensive lab course is designed as a mini research project. This format provides students with laboratory research experience, allowing them to develop excellent lab skills and the ability to interpret results using critical thinking and originality. Other learning outcomes include maintaining a laboratory notebook, performing oral presentations, and writing a scientific manuscript on their research. Even though the lab course is taught over an entire semester, parts of the course can easily be adapted as standalone experiments. The first part of the course has already been presented as a major workshop at ABLE (Bardin and Mortaji, 2020). This included the creation of E. coli transposon mutants, the screening for auxotroph mutants and the phenotypic characterization of the mutants. This workshop focuses on the genetic characterization of selected auxotroph mutants using rescue cloning. Rescue cloning is a technique that allows for the isolation of the mutant’s genomic DNA fragment that contains the transposon, so that the gene disrupted by the transposon insertion can be identified using sequencing. Rescuing the fragment of genomic DNA that contains the transposon is possible because the transposon used, Tn5-RL27, possesses an origin of replication, making the rescue clones behave like plasmids when the fragments are re-ligated and transformed into cells.
Participants in the workshops will perform the various steps involved in the creation of the rescue clones, design restriction analysis experiments of the rescue clones to confirm they are the correct clones, determine the best primer to use for sequencing and perform sequence analysis using student’s data. The workshop will end by describing potential next steps for the experiment.
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