Design

First we inoculated 1% overnight cultured E.coli DH5α in 60ml LB (depends on the volume of tube used). Then we placed a glass slide inside the tube, leaving a short end outside the liquid. With a common air pump for aquarium, an ultrafilter membrane and rubber tubes we constructed a bubble supply system. And air bubbles were pumped into the culture medium. We placed the set in a clean bench to prevent contamination since the tube was not strictly isolated. The formation can take place at about 27℃. Biofilm was generally formed in 72 hours. It was usually small clusters of bacteria clinging to the glass slide. We used a blank slide, placed it in the culture and compared with the other one to see whether the bacteria simply clung to the glass slide or they had formed biofilm on it.

We used a glass ware with two opening at the bottom to attach rubber tube and used rubber tube of 2mm*4mm(inside diameter*outside diameter) and 1mm*2mm to make a circulating flowing incubator. We then sealed all connecting locations with parafilm. The set was inoculated with 1% overnight cultured DH5ɑ in and sealed with parafilm. We then placed it in 37℃ for 3 hours for E.coli to attach. We used a mounted pump to create a liquid flow of culture medium. The flowing speed should be about 60ml per 24 hours. Biofilm was formed in about 48 hours and white substance was visible in the inner tube. We cut them with scissors from the long tube at two ends at the same time and cut open along the long axis. This prevented the sudden flow of water from destroying the structure of biofilm. Then we sliced the tube with frozen tissue slicer to into pieces of 50μm and observed under regular microscopy for vertical structure.

Microbial inoculation
For every experiment, the cells (modified Escherichia.coli, DH5alpha) were cultured in Lysogeny broth (LB) medium overnight (14-18 hours) at 37 C, 200 rpm. The initial culture was 100* diluted into new LB medium next morning and cultured in the same conditions until the cells grew to OD600 0.5-0.7.

Biofilm formation
20 L of the cell culture of OD600 0.5-0.7 were then added into each well of a 6-well plate or 24-well cell culture plate (Costar, Corning Incorporated, USA) containing 2 mL LB medium and small pieces of glass (cover slip), Polyethylene terephthalate (PETE) and PC (poly carbonate). The cell culture plates were placed in the incubator at 37 C for 24 hours, 48 hours and 72 hours. Glass, PETE and PC coupons were taken out from the culture afterwards w/ or w/o washing in distilled water.

Confocal laser scanning microscopy
The CLSM images of biofilm were obtained with Zeiss LSM 710NLO confocal laser scanning microscope using a 20⨉ water-immersion objective, or a 63X oil-immersion objective, or with Zeiss LSM510 Meta confocal laser scanning microscope using a 63X oil-immersion objective. The mono-florescence images, multichannel simulated fluorescence projection images and vertical cross sections of the biofilm were obtained by utilizing the software ZEN.

Using AI-2 as biofilm formation accelerator
Quorum sensing plays an important role in biofilm formation. In many Gram-negative bacteria, quorum-sensing systems respond to N-acyl homoserine lactones (AHLs), which were proved to be essential for the architecture of biofilm (Dickschat JS, 2010; Lopez D, Vlamakis H and Kolter R, 2010). In addition, there has been study indicated the universal quorum sensing signaling molecule autoinducer 2 (AI-2) can increase E.Coli’s biofilm biomass 30-folds (Gonzalez Barrios AF, 2006). Thus we co-transformed our device along with BBa_K091191, a device capable of sending out AI-2 molecules under the control of a pBAD promoter that can be induced with arabinose. We also did co-transformation with our device and another device from the registry consisting of LuxI under the control of a lacI repressible promoter.

Biofilm may be dangerous to humanbeing: click to see our special concerns with biofilm safety

Biofilm thickness is important for further extension of Biofilm. Our promoter library makes it possible for Biofilm stratification to become more concise and sophisticated, while a thicker Biofilm provide more distinct oxygen gradient. If we need more layers of Biofilm to construct a complicated system, then thicker Biofilm will render this task much easier.

Bubbling method

Rubber Tube method

For Cell Culture Plate Method