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The investigation of biofilm formation is critical to reducing infection rates as more than 60% of the clinically infectious
diseases are associated with biofilm formation. Here, we used a plasmid pRL27, having an R6Kγ origin of replication,
to generate transposon mutants altered in biofilm development in Escherichia coli 2443 (K-12 strain). The R6Kγ origin of
replication requires the pir gene encoding the π protein, which binds at a 22-bp recognition sequence to initiate DNA replication.
As the Escherichia coli 2443 is a pir negative strain, the plasmid pRL27 cannot replicate in it and this makes the plasmid an
effective suicide vector for genome wide transposon mutagenesis. The plasmid was maintained in MFDpir strain, which is a
diaminopimelate (DAP) auxotrophic mutant (dap?) of Escherichia coli and produces the π protein. The plasmid pRL27 was
conjugatively transferred from MFDpir to Escherichia coli 2443 on minimal medium plates. Transconjugants were selected by
plating the mating mixtures onto LB plates supplemented with dap (300 μg/ml) and kanamycin (50 μg/ml). The transposon
insertion mutants obtained were screened for altered biofilm formation using crystal violet staining. The biofilm formation was
normalized to planktonic growth, thus obtaining the biofilm formation index (BFI). Based on the results two mutants RM 56 and
RM 69, showing significantly lower and higher BFI respectively, were selected. These mutants will be characterized further to
identify the site of the transposon insertions and the underlying mechanism altering biofilm formation. This may be of benefit to
develop a better understanding of biofilm formation.
Akash Kumar is pursuing PhD in Department of Biotechnology, Indian Institute of Technology Kharagpur. He has published one research paper in
Research in Microbiology.
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