Design

• Our Aim
• Chassis selection
• Genetic Circuit Design
• Plasmids and strains

Our Aim

We endeavor our strains to grow into patterns by arranging themselves in a synchronous, self-organised manner, “just as in organisms in nature which all are able to develop shapes and patterns.” Implementing such idea on bacteria sheds light to a mechanism involving cell-cell communication that would produce a key response, predominately a respond affecting cell motility. The characteristics of the response logically should be critical towards the formation overall pattern.

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Chassis selection

Past chemotaxis studies have provided the molecular basis of cellular motility regulation, Escherichia coli and Bacillus subtilis are notably the well-understood strains which are ideal to be the chassis of our designed genetic circuit. We chose E.coli as our chassis for the project reasoning that cell-cell communications will require the use of a signaling molecule, which are often density related. E.coli is known to be less motile than Bacillus in terms of speed, thus would ease the accumulation of the signaling molecule. We hope the subsequent pattern generated by using E.coli as chassis would be finer and more interesting.

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Genetic Circuit Design

An On-Off motility design is desired as the response to cell-cell communication. Based on the pioneer work [3] shows the motility can be abolished by knocking out the cheZ gene, and can be restored by subsequent re-introduction of the cheZ gene under a controllable promoter back into the host.
We designed two DNA constructs whose cheZ expression level would be sensitive to the concentration of AHL (Acetyl homoserine lactone). One would become motile in the presence of AHL, while the other one be become immotile under the same condition. Since concentration of AHL is proportional to cell density, the motility of our strains would be dependent on local cell density.

Predicted Pattern:

According to the results of our model, we got the patterns with ring-like low cell density regions, if initially we dropped a small volume of cell onto the center of the plate. (Detials to chick here)

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Plasmids and strains

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Reference

Design

1. Topp S, Gallivan JP. Guiding bacteria with small molecules and RNA. J Am Chem Soc. 2007, 129: 6807-11
2. You L, Cox RS 3rd, Weiss R, Arnold FH. Programmed population control by cell-cell communication and regulated killing. Nature. 2004, 428: 868-71
3. Basu S, Gerchman Y, Collins CH, Arnold FH, Weiss R. A synthetic multicellular system for programmed pattern formation. Nature. 2005, 434: 1130-4

Protocol

1. Watt RM, Wang J, Leong M, Kung HF, Cheah KS, Liu D, Danchin A, Huang JD. Visualizing the proteome of Escherichia coli: an efficient and versatile method for labeling chromosomal coding DNA sequences (CDSs) with fluorescent protein genes. Nucleic Acids Res. 2007, 35(6):e37.

Results

1. Datta S, Costantino N, Court DL. A set of recombineering plasmids for gram-negative bacteria. Gene. 2006, 379: 109-15.
2. Dunlap PV, Kuo A. Cell density-dependent modulation of the Vibrio fischeri luminescence system in the absence of autoinducer and LuxR protein. J Bacteriol. 1992, 174: 2440-8.
3. Haseltine EL, Arnold FH. Implications of rewiring bacterial quorum sensing. Appl Environ Microbiol. 2008, 74: 437-45.