Team:Alberta/Project

A full color spectrum may be made by mixing three saturated colors, each reflecting light appropriate for the color sensors in the human eye. For the subtractive color relevant for mixes of bacterial color on a plate, the idea colors are cyan, yellow, and magenta. From part sequences in the Registry, we selected the the blue chromoprotein amiCFP (K592010), the yellow chromoprotein amilGFP (K592009), and the classic red fluorescent protein (RFP, E1010). These parts were selected due to their excellent presentation by the 2011 Uppsala iGEM team. We altered the sequences to remove KpnI sites, for convenience during cloning and assembly, designed custom ribosomal binding sites for each open reading frame using the Salis RBS calculator (https://salis.psu.edu/software/) to give consistent medium-high expression levels (TIR: 50k), ordered the sequences synthesized as gBlocks (IDT), and assembled them. Promoters were selected from the Anderson collection of constitutive sigma 70 promoters of various strengths, with changes to xxx.

Unfortunately, initial assemblies did not produce color. We therefore undertook an optimization program, xxx, change of base strain (TG1 produces larger, more saturated colonies than TOP10, which we interpret as a consequence of more robust growth).
After optimization, xxx
The resulting color cassettes have been submitted as xxx

[photo of color plates]

In order to produce and reproduce a predictable gradient that can be manipulated, a diffusion coefficient must be obtained. Diffusion coefficients come in the form of a unit area over a unit time, and are in relation to both the solvent and solute utilized in the experiment.
One avenue we used for measuring diffusion coefficients was based on published studies of bacterial susceptibility to antibiotics undergoing diffusion in agar plates (Bonev et al, J Antimicr Chemoth, 61:1295 2008).
First, we needed to develop a method to create diffusion plates.

Spatial patterning of gene expression requires, in addition to spatial chemical gradients, genetic elements allowing gene expression to be controlled by those spatial gradients. We opted to work with three common and well-studied repressors: lacI and tetR, which effectively shut down transcription of operons containing the lacO and tetO operator sequences in their promoter, and for which repression can be relieved by addition of the small molecule inducers isopropyl-thio-galactopyranoside (IPTG) and anhydrotetracycline (ATC).

Figure. RFP on IPTG gradient plates