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Team:Alberta/Project
From 2012.igem.org
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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.
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 favored 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 favored 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.
- Tested three great protein colors into our lab: RFP (red), amilGFP (yellow in visible light), and blue chromoprotein amilCP.
- Designed and tested versions of the common repressor proteins lambda CI, LacI, and TetR which lack the LVA degradation tag
- Designed tested new RBSes for all of these proteins, using the Salis RBS calculator. For amilCP, the new RBS is substantially stronger than the existing Registry part (K592009); for RFP, color expression is substantially worse (E1010).
- Experienced some of the benefits and weaknesses of current in silico design tools (ie RBS calculator fail)
- Developed and tested methods for measuring diffusion constants of small molecules in agar plates, to aid engineering inducer gradient plates
- Tuned control system for inducible gene expression with repressor being generated on same high copy plasmid as controlled gene
- Currently have three separate colors under multiple constitutive and inducible promoters.
- Developed new system for controllable copy number, based on the use of repressible promoter for the RNA II gene in the pMB1 replication origin. We have validated this system with both LacI repression and TetR repression through assessment of colony size and color output intensity.
- All developed parts have been sequenced, validated, and submitted to the Registry.
- We have supplied strains to the UBC, Calgary, and CINVESTAV-IPN-UNAM_MX iGEM Teams.
