Team:Alberta/Project

A full color spectrum may be made by mixing red, yellow, and blue in varying proportions. 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 (designed transcription initiation rate 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. These promoters were trimmed outside the -10 and -35 boxes, and the bases in the interior region have been varied to limit recombination when multiple promoters are used in the same system. For details on the promoters, see our Parts page

Unfortunately, initial assemblies did not produce color. We therefore undertook a multifactorial optimization program, targeting as follows: for RFP and amilGFP, the engineered RBSes were replaced with B0034, and also a change of base strain. TG1 produces larger, but less saturated colonies than TOP10, however the color develops with time. Factors which lead to additional color expression lead to diminished cell growth, thus best color was achieved with moderate promoter strength.

Since we were uncertain if the promoter and RBS regions were too strong (leading to toxicity, poor cell growth and poor color) or too weak (leading to little protein generation and poor color), we adopted a library approach, cloning in

The resulting color cassettes have been submitted as BBa_K879311 (red), BBa_K879313 (yellow), and BBa_K879222 (blue).

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).

Fig#.Ways to make gradient plate

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