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Team:Peking/Project
From 2012.igem.org
New Era for Synthetic Biology
Viewing cells as programmable entities, efforts in synthetic biology have focused on the creation and perfection of biological modules and systems in order to achieve specific functions. Since first demonstrated by the bacterial toggle switch and the oscillator in 2000, synthetic biologists now own the ability to rewire genetic circuits, metabolic pathways, even entire genomes. Due to the huge potential reservoir and molecule-receptor specificity, synthetic chemicals, e.g. Isopropyl β-D-1-thiogalactopyranoside (IPTG) and Anhydrotetracycline (ATc), are frequently employed as signals to control molecular and cellular behavior.
However, because chemicals deliver information only by diffusion (Figure 1), chemical signals are seriously limited by the short-range interaction. Another serious issue regarding chemical signals is their tendency to be easily saturated, which makes it difficult for the system to reset. Additionally, the cytotoxicity, high cost and pollution of chemicals urge synthetic biologists to find a more spatiotemporally specific and environmental friendly alternative.
Figure 1. The diffusion of chemicals.
Revolution of Optogenetics
During the last decade, optogenetics has made significant impacts on life sciences and other practices. Literally, optogenetics refers to the genetic strategies which employ light as the input signal in the place of chemical signals. The spatiotemporal specificity of light signals allows for precise manipulation and long range interaction without physical contact. Compared with chemicals, light resources are cheaper, more sustainable and environmentally friendly.
Due to the advantages mentioned above, the rapid revolution of optogenetics has shed light on life sciences: light-switchable promoters, light-induced protein interaction, light-activated neurons and light-controlled animal behavior (Figure 2).
Figure 2. Revolution of Optogenetics.
However, not satisfied with the achievements of optogenetics tools, 2012 Peking iGEM endeavored to analyze the current optogenetics methods systematically and raise a new and even greater generation of optogenetics.
Potential Problems of Current Optogenetic
Methods
In order to evaluate the current optogenetics methods in a systematic manner, this year’s Peking iGEM team has carefully selected 15 optogenetics methods, ranging from applications in prokaryotes to potential uses in eukaryotes. By statistically evaluating the modularity, sensitivity, and dynamic range of these optogenetics methods, we have come to a conclusion to address four potential problems of current optogenetics approaches:
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1. Low sensitivity
To minimize the influence on cellular metabolism, strong light needs to be avoided for practical applications. Almost 50% of the 15 optogenetics tools employ the use of lasers as the light source. We demonstrated the effect of lasers on the organisms by illuminating a lawn of E.coli with a laser pointer. As clearly shown in Figure 3, no bacterial growth could be found on the spot that was illuminated. Only about 25% of the 15 optogenetics tools are able to sense natural light (~1W/m2), an obviously cheaper and much more sustainable source of light (Figure 4).Figure 3. The effect of lasers on bacterial growth.
Figure 4. The sensitivity of the 15 optogeneticsmethods.
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Narrow dynamic range
Many cross-species optogenetics methods require the addition of exogenous chromophores, e.g. phycocyanobilin (PCB) and caged amino acids. This leads to two major problems: (1) the exogenous chromophores may be incompatible with the endogenous components, and observed in some cases, may even have some influence on the bacterial growth; (2) the inhomogeneity caused by the diffusion of chromophores, an undesired AND gate, is constructed in these methods (Figure 6), which makes the output unpredictable.Figure 5. The dynamic range of the 15 optogenetics methods.
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Dependency on exogenous chromophores
Many cross-species optogenetics methods require the addition of exogenous chromophores, e.g. phycocyanobilin (PCB) and caged amino acids. This leads to two major problems: (1) the exogenous chromophores may be incompatible with the endogenous components, and observed in some cases, may even have some influence on the bacterial growth; (2) the inhomogeneity caused by the diffusion of chromophores, an undesired AND gate, is constructed in these methods (Figure 6), which makes the output unpredictable.Figure 2. Both chromophores AND light are required to activate/inactivate the system.
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Limited application in prokaryotes
Another serious issue concerns the limited application of optogenetics methods in prokaryotes. The rapidly reproducing prokaryotes play a very significant role in both fundamental research and industrial application. In many methods, only proof of concept is provided, without practical application concerned. The low sensitivity, narrow dynamic range, and need for exogenous chromophores, as mentioned above, are the main blocks of a broader and more practical application of these methods.
A New Generation of Optogenetics
By gaining insight into the current optogenetics methods, the Peking iGEM team speculates that rationally designing a robust and ultrasensitive transcription factor may circumvent all the potential problems mentioned above: high sensitivity allows dimmer, cheaper and safer light as the source of signals; because the light sensitive transcription factor functions directly on the gene expression, the basal level may be low enough to allow a high dynamic range.
Now that such an ultrasensitive and efficient optogenetics module has been successfully designed, the Peking iGEM endeavors to explore the full proficiency of optogenetics and raise a new generation of optogenetics with broader applications on the cellular behavior level, the intercellular communication level, and the industrial level.
