Intuition Behind Our Circuit Design
How Can We Protect Coral?
In our research, we have identified temperature and light as the two most significant environmental triggers for coral bleaching. Given that it is impractical to reduce the temperature of coral reefs below the ambient levels, our strategy focuses on protecting corals by mitigating the impact of excessive light through the expression of a specialized chromoprotein. Concurrently, we have observed similar mechanisms in natural corals, where the expression of certain chromoproteins under stress conditions contributes to stress tolerance. Our goal is to enhance this inherent mechanism through our genetic circuit, thereby equipping corals with an augmented defence against bleaching events. This approach aims to harness the power of biotechnology to support the resilience of coral ecosystems in the face of environmental challenges.
Is Regulation Necessary?
Due to fluctuations in both environmental temperature and light intensity, coral habitats oscillate between stressful and non-stressful conditions. As a result, continuous chromoprotein expression may not always be necessary. In fact, under normal or low light conditions, chromoprotein expression could reduce photosynthesis efficiency, ultimately harming the coral. Therefore, a regulated expression system is essential to limit chromoprotein production when it’s not needed.
How Do We Regulate It?
To minimize chromoprotein's impact on the symbiotic system when environmental conditions are suitable, we need indicators that reflect stress levels and regulate our expression system accordingly. We identified light and heat as key factors. Due to the complexity of natural environments, we believe that relying solely on either light or heat as a stress indicator is inaccurate. Thus, we propose that chromoprotein expression should only be triggered when both light and heat exceed specific thresholds.
Moreover, extensive literature shows significant variation in coral and zooxanthellae bleaching thresholds for temperature and light intensity across different geographic scales and species. This implies that if we use biological sensors with fixed thresholds, the applicability of our circuit would be greatly reduced. Hence, the sensors in our circuit must be adjustable.
In summary, our project utilizes four key biological components:
- Light sensor: Detects whether light intensity exceeds a certain threshold.
- Thermosensor: Senses if the environmental temperature surpasses a set threshold.
- AND Gate: Integrates downstream signals from the light and thermosensors to achieve the AND gate functionality.
- Chromoprotein: Blocks excess light exposure.
The diagram below illustrates the full circuit design. We will explain the parts used for each step in detail later in the text.
Components of Our Circuit
Light Sensor
The light sensing system we use is called eLightOn, with the key regulator LexRO. The regulator LexRO, derived from the fusion of the LOV domain of Rhodobacter sphaeroides (RsLOV) (Dietler et al., 2021b) with the DNA-binding domain of LexA, represents a significant advancement in the field.
LexRO forms a transcriptionally repressive dimer in the dark, binding to the promoter and repressing the expression of downstream genes; upon exposure to light of the appropriate wavelength, the LOV domain undergoes a photochemical reaction, triggering structural changes that ultimately lead to the dissociation of the dimer, releasing the repression of downstream gene expression. This process is illustrated in the figure below. This unique photoresponsive behavior allows for direct and reversible control of gene expression with high sensitivity and specificity (Li et al., 2020)
In our project, we use the light sensor to drive the expression of a upstream component SupD in the AND Gate, whose role will be stated in the AND Gate section below.
Tunability
Regarding its tunability, researchers have discovered that the sensitivity threshold of a light sensor can be adjusted by manipulating the expression levels of the regulatory protein. This is because the association of two monomers to form a dimer is akin to a reversible reaction, and the equilibrium of such a reaction can be influenced by the concentration of the substrate. By increasing the substrate concentration, we can elevate the level of dimers within the cell, enhance the repression effect, and thereby raise the threshold of the photoreceptor. This approach provides a strategy for fine-tuning the response characteristics of optogenetic tools like LexRO to suit various experimental requirements.
As illustrated in the figure, LexRO exhibits varying responses to light of different wavelengths and intensities, indicating that our photoreceptor has adjustable sensitivity. This feature is crucial for optogenetic applications where precise control over gene expression in response to specific light conditions is required. The ability to modulate the strength of the response allows for fine-tuning the system to match the desired biological or technical outcomes.
In our application scenarios, since the light intensity in coral's living environment is much higher than the usual threshold of eLightOn, we must find a way to amplify the expression level of LexRO. We then decided to use the amplifier BBa_K3611009 to make the expression level high enough to function well in the conditions of real life cases.
- As for the amplifier, it is a cascade amplifier with a secondary amplifier in series on the basis of the original hrp amplifier. The output of the first amplifier is the input of the secondary amplifier, so the second amplifier can amplify the output signal of the first amplifier.
- For the working mechanism of the amplifier, you can refer to the figure below. For more information, please refer to the part's page to find a detailed description.
The figure below shows a zoom-in version of our full circuit design.
Thermosensor
In order to express the target protein aeBlue specifically during coral bleaching events, we have designed a collection of thermosensor with different activation threshold (see BBa_K5280410 to BBa_K5280431). Here, for the convenience to illustrate our circuit design, we have selected an RNA-based temperature sensor submitted by the Jilin University iGEM team in 2018, for its proper activation threshold. This sensor is designed to activate protein expression at temperatures of 29.3°C or higher, which corresponds to the temperature range associated with coral bleaching. The sensor functions as a thermal switch, allowing for the controlled expression of aeBlue in response to temperature changes that indicate the onset of stressful conditions for corals.
The design principle of BBa_K2541203 is based on Escherichia coli's RNase III. Under low-temperature conditions, a stem-loop structure forms, which allows RNase III to cleave the double-stranded mRNA, leading to mRNA degradation and the shutting down of gene expression. At high temperatures, the stem-loop structure becomes unstable, and RNase III cannot recognize the single-stranded mRNA, thus allowing ribosomes to bind to the RBS (ribosome binding site) and initiate gene expression. This temperature-sensitive mechanism makes BBa_K2541203 an effective genetic switch for controlling gene expression in response to thermal changes.
For various application scenarios, the coral symbiosis system exhibits different bleaching temperatures. When applying our circuit to different real-world situations, it is only necessary to modify the specific sensor used (we have designed a collection, and users can also use other thermosensor with proper thresholds, like BBa_K2541203). This adaptability allows for the customization of the genetic circuit to suit the temperature thresholds associated with coral bleaching in different environments, ensuring the targeted expression of protective or adaptive genes in response to thermal stress.
AND Gate
We use an AND Gate developed by Anderson et al in 2007 (Environmental signal integration by a modular AND gate). This AND Gate design utilise two basic parts: K228000(T7ptag) and K228001(SupD). This ANG Gate requires two upstream promoters as 2 different input, and eventually will drive the expression of a downstream gene as the output of the AND Gate. To sum up, it integrates two different biological signals into one output signal.
The AND Gate needs two upstream promoters as two inputs. The first promoter controls the transcription of a T7 RNA polymerase gene with two internal amber stop codons blocking translation. The second promoter controls the amber suppressor tRNA supD. When both components are transcribed, T7 RNA polymerase is synthesized and this in turn activates a T7 promoter.
Chromoprotein
Given the unclear characteristics of chromoproteins expressed by corals under stress and their underutilization in bioengineering, we decided to search the Registry for a chromoprotein with similar functions that has been more extensively developed.
We identified such a protein in the Registry, specifically aeBlue (BBaK864401). Originally discovered in the sea anemone _Actinia equina, this protein exhibits favorable light absorption properties. Its relatively strong absorbance characteristics, coupled with the fact that it originates from a sea anemone, which is evolutionarily close to corals, lead us to believe that this protein is less likely to cause tissue rejection or abnormal tissue reactions when introduced into the coral system. This makes aeBlue a promising candidate for applications in coral symbiosis and stress response studies.
As for its absorption spectrum, we know that for coral, blue light enhances the thermal bleaching tolerance of coral by modulating the symbiotic relationship between the coral host and its algal symbionts, Symbiodiniaceae. This modulation decreases photosynthesis, respiration, and ROS release under heat stress, thereby protecting the coral from bleaching. Meanwhile, it is deduced that orange-yellow light's damage to coral symbionts is one of the most obvious ones, similar to higher plants. So aeBlue also has a proper absorption peak.
Full Circuit Design
The aforementioned figure illustrates our comprehensive circuit design. Located in the upper left corner of the diagram is our light sensor, which is responsible for regulating the expression of SupD. Adjacent to it, in the upper right corner, the thermosensor is depicted controlling the mRNA of T7. Upon fulfilment of both light and heat conditions, the expression of SupD and the mRNA with a premature mutation will be unleashed, culminating in the production of the functional T7 polymerase. This polymerase then acts as a functional effector, driving the expression of aeBlue, which is situated at the most downstream position in the circuit. This orchestrated response ensures that the expression of aeBlue is tightly controlled and occurs only under the specific environmental cues of light and temperature that mimic the conditions of coral bleaching.
Chassis
Following discussions with coral symbiosis experts from the South China Sea Institute of Oceanology, Chinese Academy of Sciences, we decided to transfer our designed genetic circuit into Endozoicomonas after completing the prototype verification in E. coli.
Figure 1. Endozoicomonas montiporae (Hsu, 2015).
Endozoicomonas, which maintains an endosymbiotic relationship with corals, is widely recognized as the most prevalent bacterial genus symbiotic with corals globally. We aim to leverage this symbiotic relationship to selectively express and secrete the chromoprotein aeBlue under high light intensity and elevated temperatures. By doing so, Endozoicomonas could effectively absorb excess light, thereby providing a protective mechanism against light-induced stress for coral holobiont.
**_<Need a figure to show the endosymbiosis of corals and _endozoicomonas*>*__
The reasons for selecting Endozoicomonas as our chassis are as follows:
- It is found across various coral species, allowing us to adapt it to multiple coral types with minimal modification once we have successfully enabled aeBlue expression in a specific coral species.
- Endozoicomonas has been extensively studied by researchers over time, providing a wealth of knowledge to facilitate a safer and more efficient gene transfer process.For instance, Ding et al. (2016) conducted a detailed genomic study on Endozoicomonas montiporae, demonstrating its potential as a facultative endosymbiont capable of thriving both within coral tissues and the surrounding marine environment. Additionally, Yang et al. (2009) provided crucial insights into the specific habitat requirements of Endozoicomonas montiporae, which aids in replicating the necessary conditions for culturing this bacterium in laboratory settings, thereby facilitating our experimental work.
- Its natural cohabitation with corals minimizes the potential for adverse effects, making it a more compatible choice for coral protection. Lastly, current research does not indicate any inherent ability of Endozoicomonas to secrete chromoproteins, ensuring that our modifications would be an innovative addition to its functional repertoire.
References
Ding, J., Shiu, J., Chen, W., Chiang, Y., & Tang, S. (2016). Genomic Insight into the Host–Endosymbiont Relationship of Endozoicomonas montiporae CL-33T with its Coral Host. Frontiers in Microbiology, 7. https://doi.org/10.3389/fmicb.2016.00251
Hsu, J.-H. (2015). Endozoicomonas montiporae [Photograph]. Okinawa Institute of Science and Technology Graduate University. https://groups.oist.jp/ja/grad/event/seminar-searching-health-associated-bacteria-corals-story-about-endozoicomonas-prof-sen
Yang, C., Chen, M., Arun, A. B., Chen, C. A., Wang, J., & Chen, W. (2009). Endozoicomonas montiporae sp. nov., isolated from the encrusting pore coral Montipora aequituberculata. INTERNATIONAL JOURNAL OF SYSTEMATIC AND EVOLUTIONARY MICROBIOLOGY, 60(5), 1158–1162. https://doi.org/10.1099/ijs.0.014357-0
