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The opportunistic pathogen, Pseudomonas aeruginosa, a flagellated bacterium, is one of the top model organisms for biofilm studies. To elucidate the location of bacterial flagella throughout the biofilm life cycle, we developed a new flagella biotracking tool. Bacterial flagella were site-specifically labeled via genetic code expansion. This enabled us to track bacterial flagella during biofilm maturation. Live flagella imaging revealed the presence and synthesis of flagella throughout the biofilm life cycle. To study the possible role of flagella in a biofilm, we produced a flagella knockout strain and compared its biofilm to that of the wild-type strain. Results showed a one order of magnitude stronger biofilm structure in the wild type in comparison with the flagella knockout strain. This suggests a possible structural role for flagella in a biofilm, conceivably as a scaffold. Our findings suggest a new model for biofilm maturation dynamic which underscores the importance of direct evidence from within the biofilm.