Team:Alberta/Project

The end goal of our project is to create spatial color patterns using bacteria, such as a color wheel and a rainbow. Not only would this give an attractive end result, it would also demonstrate a fine level of control over gene expression, as it requires spatial control over several colors simultaneously. Patterned through spatial gradients of the inducers IPTG and ATC, using established red, yellow, and blue proteins as outputs, we needed to turn on and off expression of these genes as a function of inducer level. Induction of color expression as the inducer level increases is achieved via a typical inducible promoter system, with the repressor expressed elsewhere on the plasmid under a weak promoter. To expand the dynamic range of this system, we developed a means of adjusting gene expression through plasmid copy number control, achieved by placing the RNA II gene in the pMB1 replication origin under inducible control. At low levels of inducer, copy number increases with inducer, as greater RNAII expression leads to increased plasmid replication. Note that this mechanism makes plasmid replication dependent on the presence of inducer, effectively creating a ne

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).

Fig ###. Colour quality.Shown is the range of colour development for our submitted parts, BBa_K879222 containing amilCP (blue), BBa_K879313 containing amilGFP (yellow under visible light/green under UV light) and BBa_K879311 RFP (red) under different growth and light conditions. Each pigment gene is driven from a constitutive promoter. Complete descriptions can be found under Parts. The pellet colours shown in the bottom-right panel are derived from the overnight cultures shown above.

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Fig ###. Colour quality.Shown is the range of colour development for our submitted parts, BBa_K879222 containing amilCP (blue), BBa_K879313 containing amilGFP (yellow under visible light/green under UV light) and BBa_K879311 RFP (red) under different growth and light conditions. Each pigment gene is driven from a constitutive promoter. Complete descriptions can be found under Parts. The pellet colours shown in the bottom-right panel are derived from the overnight cultures shown above.