INTRODUCTION. Overview. Biofilm is an aggregate of microorganisms in which cells adhere to each other on a surface.

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2 INTRODUCTION

3 INTRODUCTION Overview Biofilm is an aggregate of microorganisms in which cells adhere to each other on a surface.

4 INTRODUCTION Characteristics Survival in unfavorable growth conditions Strong Resistance to Natural Formation & antibiotics or heavy metal Automatic Chemical Gradients

5 INTRODUCTION Motivation Instead of Destroying Biofilm, Can we make use of it and even make profits out of it?

6 INTRODUCTION Modules BIOFILM Realize Biofilm Formation in Lab standard, cheap and easy RAINBOFILM Construct an oxygen dependent stratified expression system of CFP, YFP and RFP in biofilm SUGARFILM Apply our system to multi-step cellulose degradation

7 BIOFILM

8 BIOFILM Introduction to Biofilm Aggregation of bacteria. Attachment, Maturation and Dispersion. Porous structure

9 BIOFILM Modeling Results Biofilm Formation Modeling Biofilm Growth Biofilm Thickness Oxygen Gradient Meaning to wet-lab experiments

10 BIOFILM DIY Biofilm Reactor Cell Culture Plate Method Silicone Tube Method Bubbling Method Our methods are a lot cheaper than commercialized methods. Also we co-transformed an constitutively expressed device expressing AI2, a quorum sensing molecule to promote biofilm formation (Gonzalez Barrios AF, 2006).

11 BIOFILM Results and Comparison of Methods Silicone Tube sheer force Thickness 100µm However, it is unstable. Cell Culture Plate Method Confocal observation Best maintained structure Very stable.

12 BIOFILM A Closer Look Bacteria cuddling together Ω Ω Ω Ω

13 RAINBOFILM

14 RAINBOFILM Introduction Natural gradients within biofilm Appl Environ Microbiol October; 70(10): Fig. Oxygen concentration profiles for P. aeruginosa colony biofilms. Triplicate data sets are shown for each strain. Depth zero on the x axis corresponds to the aircolony interface.

15 RAINBOFILM Introduction Rainbofilm

16 RAINBOFILM Design

17 RAINBOFILM Aerobic Design

18 RAINBOFILM Micro-aerobic Design

19 RAINBOFILM Anaerobic Design

20 RAINBOFILM Part Characterization

21 RAINBOFILM Modeling

22 RAINBOFILM Modeling

23 RAINBOFILM Frequency Results Fluorescence Intensity

24 RAINBOFILM Results Rainbofilm Fig.4 D Merged fluorescent biofilm picture of CFP (aerobic condition induced) YFP (micro-aerobic condition induced) and RFP (anaerobic condition induced) under Zeiss LSM510 Meta confocal laser scanning microscope, using a 63X oil-immersion objective.

25 RAINBOFILM Results

26 SUGARFILM

27 SUGARFILM A stratified system Adherence Intriguing features of Rainbofilm? Robustness

28 SUGARFILM Into real-world, into practice How can we expand it?

29 SUGARFILM Into real-world, into practice A stratified system Adherence Robustness Better Starting for producing substrate unfavorable as substratum products Multi-step synthesizing pathway

30 SUGARFILM Cellulose Utilization by Sugarfilm

31 SUGARFILM Design Upper Layer: ptetr X pvgb bglx tetr Middle Layer: pfdhf cex cena tetr Bottom Layer: Note: Cex - Cellulomonas fimi exo-glucanase CenA - Cellulomonas fimi endo-glucanase BglX - Cytophaga hutchinsonii beta-glucosidase X as coding sequence of desired enzyme(s) in the following pathway

32 SUGARFILM Modeling Bottom Layer Middle Layer Overall Stimulation

33 SUGARFILM Over Sugarfilm and More Concentrated enzyme distribution Neat and elegant material flow New era of bioreactor design Significant versatility Ready to upgrade: for more complex system United States Patent

34 Human Practice

35 HUMAN PRACTICE Human Practice Outline Popularization Funding Guide Analysis App A Investigation Novel Guidebook Club

36 HUMAN PRACTICE FUNDING GUIDE FUNDING REPORT OF IGEM TEAMS A LIMIT to igem team numbers FUNDS!

37 HUMAN PRACTICE App & SCIENCE FICTION EDUCATION FOR FUN

38 HUMAN PRACTICE CAMPUS ACTIVITY: THE SYNTHETIC BIOLOGY CLUB LECTURES

39 ACHIEVEMENTS

40 ACHIEVEMENTS Realized standard biofilm formation in lab, laying foundation for further engineering. Cheap Biofilm!!! Constructed an oxygen dependent stratified expression system of different fluorescence proteins. Our Design Works!!! Substituted fluorescence proteins with cellulose degrading enzymes and applied our system to real industry. Promising Application Prospect!!! Taken great efforts in Human practice to make synthetic biology fun and accessible to the public and funding easier for igem teams. Long Live igem!!!

41 ACHIEVEMENTS Realized standard biofilm formation in lab, laying foundation for further engineering. Cheap Biofilm!!!

42 ACHIEVEMENTS Constructed an oxygen dependent stratified expression system of different fluorescence proteins. Our Design Works!!!

43 ACHIEVEMENTS Characterized every device we used and developed tools for characterization data analysis. Easy Characterization!!!

44 ACHIEVEMENTS Substituted fluorescence proteins with cellulose degrading enzymes and applied our system to real industry Promising Application Prospect!!!

45 ACHIEVEMENTS Taken great efforts in Human Practice to make synthetic biology fun and accessible to the public and funding easier for igem teams. Long Live igem!!!

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