"
Team:Alberta/Project
From 2012.igem.org
|
|
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).
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
We were disappointed with the results of testing red gradients under central IPTG control. While the results demonstrated the functionality of control, even when completely repressed the colonies would slowly turn pink over the course of a couple of days.
[xxx describe concept. include figure]
Bacterial cultures were grown at 37OC in LB broth (xxx) overnight under selective conditions that favoured plasmid maintenance [chloramphenicol (xxx/mL) and either IPTG (xxx) or ATC (xxx)]. As a starting point, the number of viable cells in each culture was determined by spotting 5uL of 10-fold serial dilutions ranging from undiluted to 10-9 (xxx spots/culture) onto LB-agar plates containing Chloramphenicol and either IPTG or ATC at the concentrations cited above. Cell count/mL of culture was determined by counting (or estimating) the number of colonies at the highest resolvable dilution and multiplying by the dilution factor.
Fresh cultures were then made by inoculating one uL of the original cultures into 5 mLs of LB broth under conditions that favoured plasmid loss (no antibiotic or inducer) and grown as above. The number of viable cells remaining under non-selective conditions were determined as described above.
- Making the plates. LB agar plates were made using 25 mL of LB agar. A well in each plate was made using two cylindrical neodymium magnets (dimensions xxx) per well that sandwich the lid at the preferred location, as illustrated below. When solidified the magnet is removed by gently lifting off the lid.
- Making the gradients. 40uL of either 100x IPTG (10mM) or 100x ATC (100mM) are added to each well and allowed to diffuse for 8 hours at room temperature prior to plating.
- Plating bacterial lawn. Overnight cultures are diluted to an approximate cell concentration of between 0.5-1.0x105 cells/ml. 1-2x104 cells in 200 uL are plated by swirling 6 ball-bearings (dimensions xxx) on the plate surface until completely wetted. The plates are then dried under a tissue culture cabinet and incubated at 37OC overnight.
- Important! Cell densities that exceed those specified above produce thin transparent lawns with poor colour development.
