Biofilm is a multi-cellular bacteria community, which is prevalent in diverse natural and artificial settings. It is best known for its robust adaptability and strong resistance to environmental stress. We intended to utilize and modify this natural multicellular structure to develop a new kind of extracellular scaffold, constructing a stratified protein expression system using the naturally formed graded oxygen concentration within the bacteria biofilm.

Biofilm formation process can be roughly divided into four stages: bacteria attachment, micro colony formation, biofilm maturation and biofilm dispersion. The best sample for the observation is the matured biofilm, which, according to previous studies, was formed after 48 hours after inoculation with only exception in bubbling method (Bandara HM et al. 2009; Klausen M. et al. 2003.).

Based on the references (Almeida C. et al. 2011; Rani SA et al. 2007), we’ve developed three different biofilm formation methods: Bubbling method, Rubber tube method and cell culture plate method. The biofilm formed in the two former ways were observed through cryo-sectioning coupled with normal fluorescence microscopy while confocal laser scanning microscope was employed to obtain the biofilm images of the third method. In addition to the above, we’ve tested different material’s applicability in biofilm formation experiments and introduced autoinducer AI-2 as an accelerator molecule.