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| - | Project Title
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| + | Selection, design and optimization of color expression cassettes |
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| - | <div class="underline"><font size=5>Overall Project</font></div> | + | <div class="underline"> |
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| + | Selection and Design |
| - | A microbial color wheel
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| - | This year, the University of Alberta team is developing a biological sensor circuit with three pigment colours to generate a multiple coloured output. We developed this idea because traditional biological reporters have been limited to a small set of fluorescent proteins and colour genes, which produce only an all-or-none output. Furthermore, having only a single channel of output limits the application of other sensors in a single biological device. Therefore, it is clear that the traditional sensor system requires easy-to-use bioreporters that are capable of responding to chemical gradients and mixing independent output channels. To construct the biosensor circuit, we used existing genetic parts pioneered by the 2009 and 2010 iGEM teams, which will result in the circuit being capable of responding to chemical gradients and producing a multi-coloured output in the form of a colour wheel. This biosensor will become the new age of easy–to-read reporters that are incorporated into new versions of the Genomikon kit, which directly impact both research and education usage.
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| - | |<font size=2>[[https://2012.igem.org/Team:Alberta/Team Top page]]</font>
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| + | 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. |
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| - | |<font size=2>[[https://2012.igem.org/Team:Alberta/Team Top page]]</font>
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| + | 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). |
| - | <div class="underline"><font size=5>The experiment</font></div>
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| - | <div class="underline"><font size=5>Step wise Result | + | <div class="underline"> |
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| | + | Experiment Result |
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| - | Step one (June.20.2012)
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| - | We finished the first part of our biological circuit construction (promoter switching) and discovered that the red fluorescent protein (rfp) gene was only expressed from the two strongest promoters, 4 (second strongest) and 5 (strongest), out of nine different promoters tested. The blue pigment protein (bpp) gene was only expressed from promoter 2, which is the second weakest of the tested promoters..Expression of the green fluorescent protein (gfp) gene was not detected using any promoter, unexpectedly. These results indicate that the recruitment of the RNA polymerase to initiate transcription and expression of these pigment genes can be accomplished using promoter 2 or higher. However, the strength of the promoter does affect visible color development in E. coli colonies. A larger amount of RFP is required as expression was found to only be driven by the strongest promoter while BPP was produced at sufficient levels using a weaker promoter. This may also indicate that BPP is toxic at higher levels driven from stronger promoters. The lack of GFP production may be a result of cloning errors or secondary structure of mRNA blocking RBS from ribosome binding. Therefore, We have decided to construct plasmids to test the different strengths of RBS and investigate the toxicity of colour proteins. We have also begun cloning transcriptional repressors in order to modulate level of pigment gene expression.
| + | After optimization, xxx |
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| + | The resulting color cassettes have been submitted as xxx |
| - | Step Two (June.20.2012)
| + | [photo of color plates] |
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| - | Using theold Ribosome binding site (RBS) generated by a computer program and unique foreach colour gene, we achieved tiny, colorless or dull colonies. Therefore, weswitched the lacking RBS with a new known RBS, which was acquired from adifferent iGEM group.
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| - | The two differencesbetween the old and new plasmids are that all the colour genes now share anidentical RBS, and the novel RBS has been previously confirmed as working. Weattached the new RBS by performing PCR and transformed it in Top10 E.coli cells. As a result, the yellowcolonies expressed a vivid colour but still grew quickly, indicating that theoverexpression experienced with the blue gene’s original RBS was not a problemwith the yellow gene’s modified RBS. The blue gene with the new RBS, however,is not expressed. Overexpression, resulting in toxicity because of highconcentrations of pigments, and underexpression, resulting in death from lowconcentration of antibiotic resistance may be possible explanations. The redgene shows the same expression compared to the old RBS. | + | |
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| - | Step three
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| | + | Site map |
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| - | After completing the pieces that will produce the actual output we moved on to the more complicated process of constructing a regulatable promoter system that will produce the outputs that we are aiming for. After assembling the different pieces relevant, we started testing our regulation of the basic color gene RFP with the Lac operator. These experiments paved the way for the creation of a plasmid which had its copy number under control of an inducible repressor. We tested plasmids with the standard puc19 origin of replication promoter, and also a plasmid which was identical with the exception of this plasmid containing a much stronger promoter. This second plasmid was much more difficult to test, as a cell containing this plasmid is not viable without also having the LacI gene present; a feature not on our initial construct. The promoters were tested again once we had added the LacI gene to the plasmid, and the results were surprising. The cells grew in a large range of IPTG concentrations (1/20th to 1.6 times the recommended concentration), where we had been expecting a much more narrow window.
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Selection, design and optimization of color expression cassettes
Selection and Design
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).
Experiment Result
After optimization, xxx
The resulting color cassettes have been submitted as xxx
[photo of color plates]
Site map
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