In the rainbofilm, one kind of bacteria forms a multicellular system, differentiating into three different layers. Each layer performs one function by expressing one fluorescent protein. Not only is this system aesthetically beautiful, but also is pretty capable to realize different purposes, such as making biosensor and building bioreactor. To cater different application needs, we extend the current primitive stratification system into a more precise, more controllable, and more versatile device. We start with improving the input signals of the rainbofilm system by
1. Fine-tuning the stratification pattern
2. Changing the differentiation signal.

1. Fine-tuning the stratification pattern
In the rainbofilm, we use vgb promoter to regulate the behavior of middle layer. The characterization result of vgb promoter shows the highest PoPS around 2.5% oxygen concentration. In attempt to change the distribution of the middle layer vertically, we decide to construct promoter library to screen for mutated vgb promoters that have expression peaks in different oxygen concentrations. The detailed regulation mechanism of vgb promoter still remains unclear. The current understanding is that three transcription factors, Crp, ArcA and Fnr coordinate the vgb promoter regulation together. Each of them exerts some degree of influence on the expression. The presumptive binding sites of vgb promoter are illustrated below.

We use degenerative primer PCR to mutate all the binding regions in the vgb promoter. GFP is put behind the promoter for testing. By measuring the fluorescence intensity of the device in different oxygen concentration conditions, we screen for the desired promoter which transcript only in 5%, 7.5%, and 10% oxygen concentration respectively.

2. Changing the differentiation signal
The differentiation of rainbofilm occurs spontaneously since the input signal is oxygen concentration. In the extension of rainbofilm, we want to use different input signal such as metal ion. We are interested to build the rainbofilm system into a biosensor and we choose Hg2+ and Pb2+ as our candidate signal.

MerTPCAD promoter, regulated by MerR, senses Hg2+,; PbrA promoter, regulated by PbrR, senses Pb2+. Last year Peking University engineered merTPCAD promoter, this year we did similar work in both merTPCAD promoter and pbrA promoter. We make random mutation in the promoter region by degenerative primer PCR, and construct a library for screening ideal metal sensing promoter. For the merTPCAD promoter, we mutate the same four sites in the promoter, the semi-conservative region, as Peking did last year. For the pbrA promoter, we choose four homologous sites and expand the mutation region to eight sites.