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Synthetic Biology: What's Hindering Us?

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 repressilator in 2000, synthetic biologists now own the ability to engineer genetic circuits, metabolic pathways, and even genomes.

During genetic engineering, chemicals are frequently employed as signals to control molecular and cellular behavior. However, because chemicals deliver information only by diffusion, chemical signals are seriously limited by the short-range interaction. Another serious issue regarding chemical signals is the difficulty to erase them, which makes it difficult for the system to reset. Additionally, the cytotoxicity, high cost and narrow dynamic range of chemicals urge synthetic biologists to find more spatiotemporally specific and environmental friendly alternatives.

Optogenetics: Inspire Synthetic Biology

Literally, optogenetics refers to the genetic strategies which employ light as the input signal in the place of chemical signals. During the last decade, optogenetics has made significant impacts on life sciences and other practices. It has evolved rapidly: light-switchable promoters, light-induced protein-protein interaction, light-activated ion channels and eventually, light-controlled animal behavior. The spatiotemporal specificity of light signals allows for precise manipulation and long range interaction without physical contact. Besides, light resources are cheaper, more sustainable and environmentally friendly. These properties together make optogenetic tools attractive for synthetic biologists.

[Fig 1.]

Figure 1. Evolution of Optogenetics.

However, current optogenetic tools are far from satisfactory for synthetic biology application. In next section, we will systematically analyze the current optogenetic methods and raise issues that our Luminesensor needs to circumvent.

Issues of Current Optogenetic Methods

We carefully selected 15 representative optogenetic methods, ranging from those in prokaryotes to those in eukaryotes. By statistically evaluating the modularity, sensitivity, and dynamic range of these optogenetics methods, we have come to a conclusion to address four issues of current optogenetics approaches:

  • 1. Low sensitivity
    To minimize the influence on cell growth, strong light needs to be avoided for practical applications. Near 50% of current 15 optogenetic tools employ lasers as the light source. We demonstrated the lethal effect of lasers by illuminating a lawn of E.coli with a laser pointer. As clearly shown in Figure 2, 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), a biologically friendly and much more sustainable source of light (Figure 3a).

    [Fig 2.]

    Figure 2. The inhibitory effect of lasers on bacterial growth.

  • 2. Narrow dynamic range
    For a control system, the dynamic range (fold change of output in response to input signal) is an essential parameter used to evaluate the control efficiency. Among the 15 optogenetic methods, few have a dynamic range that reaches 100-fold (Figure 3b). In application, systems with narrow dynamic ranges will be easily interfered by the intrinsic and extrinsic noises. The main motive for having narrow dynamic range is because most of the methods utilize a complex signal cascade where high basal level is inevitable.

    [Fig 3b.]

    Figure 3. The dynamic range of the 15 optogenetic methods.

  • 3. Dependency on exogenous chromophores
    Many optogenetic methods require the supplement 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, even have cytotoxity;
    (2) the uneven diffusion of chromophores, in many cases, makes the output unpredictable.

  • 4. Limited application in prokaryotes
    Another serious issue concerns the limited application of optogenetic methods in prokaryotes. The rapidly reproducing prokaryotes play a very significant role in both fundamental research and industrial application of synthetic biology. 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 optogenetics in synthetic biology.

A New Generation of Optogenetic Tool:
The Luminesensor

By gaining insight into the current optogenetic methods, Peking iGEM team speculates that rationally designing a robust and ultra-sensitive transcription regulator that works in prokaryotes will circumvent all the issues mentioned above: high sensitivity allows dimmer, cheaper and safer light as the source of signals; because the light sensitive transcription regulator functions directly on the gene expression, the basal level will be low enough to guarantee a high dynamic range.

Indeed, as you will see later, such an ultrasensitive and efficient optogenetic tool has been successfully designed. Moreover, we endeavored to explore the full proficiency of our Luminesensor and raised a new generation of optogenetic tool with broader applications on the manipulation of biochemical process, cellular behavior, and even, inter-cellular communication.