Team:Peking/Project/Luminesensor/Characterization

Sensitivity

Why high sensitivity & our luminesensor sensitivity
We developed a light-switchable system that provides a robust and convenient way to control gene expression, and can be used to manipulate many biological processes in living systems with minimal perturbation on the basis of our innovative fusion protein.

Our light-switchable system has incomparable advantages and potentials not only because of the features we have discussed in the ‘motivation’ section of our project overview, but also because the system presents a higher level of sensitivity that researchers have never put into effect before. By taking advantage of this high level of sensitivity, we can easily accomplish the goal of controlling gene expression using light without the need for an extra energy source.

There are two critical reasons for why we endeavored ourselves into establishing such a highly sensitive Luminesensor. Firstly, sensitivity is one of the most critical evaluation criteria in optogenetics for the reason that the modularized systems can function under low intensity light, like natural light, as the sensor possess a high sensitivity with the purpose of minimizing the injury to the cells which possibly will be seriously affected by high intensity light exposure in the meantime maximizing the exploiting of natural light. According to information collected in the past decade, only about 25% of experimental results have achieved the goal of controlling gene expression by optogenetic approaches with the light intensity under 1W/m², which is the exact equivalent of natural light, and is our greatest concern due to the availability and user-friendly characteristic of natural light (Figure 1:Optogenetic Approaches to Control Gene Expression under different light sensitivity):

Figure 1. Optogenetic Approaches to Control Gene Expression under different light sensitivity.

Secondly, most accessible methods used to trigger or block gene expression require an extremely complicated setup, sometimes even an invasive device, or a specialized laboratory technician to maneuver subjects to the rough Luminesensor modules. Although numerous efforts have been devoted to improving the experimental conditions, they still retain great handicaps. For example, the use of continuous laser irradiation is required in every measurement and cannot afford reversible monitoring.

Hence, based on the current situation talking above that the light sensors constructed previously always regrettably fail to achieve the goal, Peking iGEM 2012 spares no effort to assemble a high sensitivity light sensor and finally makes it come true. Incessant gene depression from our system using natural-light-intensity sensor optimized by amino acid sequence mutation has been considered a suitable option by means of the natural light intensity, which is very low in comparison with the laser intensity level and is easy to obtain without requiring toxic chemicals. The sensor we assembled solves the sensitivity problem in a new way: it is flexible and highly tunable, as well as modularized, and is constructed onto simple material with a user friendly setup.

To achieve such sensitivity while maintaining minor cell perturbation that is proper for experimental control, we had to verify our light-dependent impact on the transcriptional activity of a GFP-ssrA reporter which has the advantages of visualization and high-throughput driven by LexA binding sites fused with VVD after transformation in E.coli and incubator in our user-friendly setup. According to the literature we searched, we may safely draw a conclusion that our luminesensor possess a high sensitivity as a result of VVD itself owning a high sensitivity. Fully malleable and modularized components were realized utilizing tolerable intensity, natural-light-intensity irradiation to facilitate regulation of gene expression with diverse intentions. What’s more, with the intention of accelerating the degradation of GFP to observe the light-switchable phenomenon and enhance the stability of the light-induced fusion protein, we tagged our GFP reporter gene with an ssrA-tag that is known as a central feature of protein-quality control in all bacteria when the target protein is tagged with. Based on the consideration of guaranteeing accuracy and precision, our setup(Figure 2):

Figure 2. Setup

consists of three central parts: light source, incubator and 48-well plate. On account of high sensitivity, protecting the system form the preventable light exposure with the purpose of acquiring the accurate results which is the true reflection of our sensitivity is necessary. In order to solve the problems, we focus on two foremost aspects: utilizing attenuators to weaken the light intensity and using tin foil to avoid light leakage. For more details about how we conducting the experiment, see experiment procedures. In our experiments, illumination with different light intensity conditions at 460nm peak light from blue LED arrays for 16 hours show marked light-depressed reporter gene transcription, which indicates that under different blue light exposure conditions, there was hardly any light-induced reporter gene transcriptional activity. But when in the dark environment (packaged with three layers of aluminum foil), our systems showed extremely high GFP expression (Figure 3)

Figure 3. Expression of GFP.

There are a couple of plausible and conceivable reasons for why our system possesses such high sensitivity that can sense unbelievably weak light intensity. First, it includes the unusually stable, photo-activated state of VVD, which causes the system to be extremely sensitive to light. Secondly, without relying on the chromophores, our system can directly regulate gene expression without time delay.

Results and conclusions
Our system exposed to different light intensity containing the LexA408 depression domain showed manifest light-depressed reporter gene transcription. As shown in the figure, all of the systems with dissimilar attenuators showed incredible depression efficiency. In the first four sets of data, which are completely exposed to blue light and covered by attenuators No. 2, 4, 7 respectively (see more details by clicking on the protocol, figure 4: luminance measurement of different attenuator, we choose the 48-well plates that covered with No. 2, 4, 7 attenuator as our experimental groups.)

Figure 4. luminance measurement of different attenuator, we choose the 48-well plates that covered with No. 2, 4, 7 attenuator as our experimental groups.

It proves that once the system is exposed to natural light, reporter gene transcription will be absolutely depressed regardless of the attenuators we attached to it. In other words, all of the systems protected from equal to or lower than natural-light-intensity light blocked light-depressed gene expression as expected. Besides, in the fifth sets of data, which was entirely in the dark state, the expression of GFP ran up to a high degree of 50,000. Taking everything into account, our luminesensor does possess high sensitivity that across several orders of magnitude. Our next work plan is to improve our devices and attenuators in order to find out the threshold point in which the light-depressed gene expression will present linear results.

We are impartially proud of accomplishing the goal of controlling gene expression using light without excessive or unnecessary energy or substrates, and we have respectable and appropriate reasons to have complete faith in our system; that it has imposed specific innovative challenges of light-dependent process for the future researchers.