Development of an inducible three colour bacterial water colour system T. Brown, C. Chang, B. Heinze, P. Hollinger, J. Kittleson, K. MacDow, D. Reavis, J. Curry and M. Riley Abstract: Here we present the work of the 2006 University of Arizona team in the iGEM (inter- national Genetically Engineered Machine) competition sponsored by MIT. Our aim was to develop an inducible water colour system for painting bacteria in three colours. Using BioBricks, a plasmid was designed for insertion into E. coli which would allow the bacterium to respond to each of three different chemical inducers by producing yellow, cyan or red fluorescent proteins. In principle the inducers could be deposited on a bacterial lawn using a high-resolution printer so that three colour images could be produced with a spatial resolution matching the size of the bacterium. This could be the first step towards producing a bacterial lawn that behaves like a canvas for watercolour paint- ing or eventually a three-colour television set. The application connects with current interest in precise control of cellular response desired in biosensors and bioengineered materials. The University of Arizona iGEM team called the ‘Cell Raisers’ was assembled in May 2006 and worked enthusiastically throughout the summer. The team was comprised of six undergraduates (Tyler Brown, Brian Heinze, Patrick Hollinger, Josh Kittleson, Kevin MacDow, and Dan Reavis), one graduate student (Carlos Chang), and two faculty members (Joan Curry and Mark Riley). This proved to be an ambitious project, and while the final goal was not fully realised, first steps were made in terms of design, plasmid construction and bacterial deposition with an inkjet printer. Patrick Hollinger and Brian Heinze gave the technical presentation to judges and competing teams on Saturday, November 4, 2006 in MIT’s Stata Center. The team also presented a poster that was viewed in the evening after all the presentations. For their efforts, the University of Arizona team received honourable mention with special consideration: ‘For progress toward synthetic biology in three colours’. 1 Aims of the project Our overall aim was to develop an inducible water colour system for painting on bacteria in three colours. Specific goals were (1) to create a colour image utilising bacterial fluorescence and (2) to develop a control scheme that will allow for selective activation of bacteria by precise stamp- ing or introduction of inducers or through precise placement of bacteria to produce complex images. Substantial progress was achieved towards both goals by the University of Arizona Cell Raisers. The team logo is presented in Fig. 1. 2 Description of the work A number of project ideas were considered and substantial time was invested in project design. The initial focus was on developing a plasmid which would allow for the activation of fluorescence by the placement of different chemical indu- cers. As detailed in Table 1, the team identified and selected three independent operons to control expression of three different fluorescent proteins. A plasmid was constructed from BioBricks available from the Registry of Standard Biological Parts (http:// parts.mit.edu/registry/index.php/Main_Page) provided by MIT as a component of the iGEM competition. Using the standardised restriction enzyme sites and preconstructed bricks, the Lac repressor (LacI), Tet repressor (TetR), and HSL inducer (LuxR) were attached to constitutive promo- ters and high output ribosome binding sites. Each control element was then ligated to the associated fluorescent expression element. The expression elements were pre- viously ligated to promoters under the control of the repres- sors and inducers as designated in Table 1. The assembly process was carried out using a parallel assembly to gener- ate composite parts as presented in Table 2. The plasmid was transferred into E. coli using standard methods. DNA sequencing was performed to confirm that the plasmid was constructed correctly. Simultaneously with the plasmid construction exper- iments, team members worked to develop methods for the application of inducers via a controlled mechanism to achieve maximum image resolution. Several inducer intro- duction methods were evaluated for their ability to deliver a precise spatial arrangement on bacterial lawns grown in a standard Petri dish. The first, and least precise approach, was to apply inducers by pipetting by hand and allowing the inducers to seep into the agar. Initially, the bacterial cul- tures were grown on LB þ ampicillin in inducer concen- trations ranging from no inducer to 10 000  K m . All three chemical inducers were applied to the cells. However, no quantifiable difference was found between induced and non-induced bacteria. The initial assessment was: strong expression of yellow, weak expression of red, little to no expression of cyan. Specific colonies grown in the initial cultures were selected for strong expression and grown individually. # The Institution of Engineering and Technology 2007 doi:10.1049/iet-stb:20070015 Paper first received 21st February 2007 The authors are with the University of Arizona, Tucson, AZ 85721, USA E-mail: riley@ag.arizona.edu IET Synth. Biol., 2007, 1, (1–2), pp. 21–24 21