Mechanism

Our project design mainly involves three aspects: Chlamydomonas movement and photoreceptor system, red-light controlled polymer protein, split Ranilla Luciferase. The following are detailed explanations of the mechanism of action of each part.

  1.1 Photosensitive and kinesthetic mechanism of chlamydomonas

There are eye spots on the cells of Chlamydomonas for light perception. There are two channels of rhodopsin at the eye spots: ChR1 and ChR2, which can open channels at the maximum absorption wavelength ~500nm and ~470nm respectively, causing proton flux or sodium、potassium and other cation to enter the cell, which will cause cell depolarization. ChR is a light-gated ion channel, and its structure contains a chromophore, the mononucleotide (FMN). After the blue light is irradiated, the phthalate in the structure undergoes a conformational flip and further opens the ion channel by electron transferring. The generation of photocurrent causes a series of reactions such as phosphorylation of the channel rhodopsin and an increase in the intracellular concentration of calcium ions, and the signal is transmitted to the flagella of the Chlamydomonas, causing changes in phototaxis and photopulsation. Although the signaling mechanism of light-induced flagellar movement through rhodopsin has not been elaborated, we have already known that blue light can activate rhodopsin-induced phototropic movement of Chlamydomonas, which is controlled by two flagella of Chlamydomonas, and have connection with increased intracellular calcium concentration, channel rhodopsin phosphorylation, and the concentration of the second messenger molecules such as cAMP.

  1.2 Light-activated protein

We have learned three kinds of protein pairs that can be polymerized under illumination: physicochemical pigment B (PhyB) and its interaction factor Pif under red light; cryptochromic pigments CRY2 and CIBN under blue light; under ultraviolet light Polymerizable UVR8 and COP1. Upon irradiation with a specific wavelength of light, the protein pairs will polymerize, causing combination of the linked protein fragments. Considering that cell micro-robots will play a an important role in the medical field in the future, we take PhyB and Pif controlled by red light as an example. Phytochromes (Phy) is a red and far red light sensitive photoreceptor. In the dark, Phy exists in an inactive form (Pr). At the time of red light stimulation (about 660 nm), in order to perform light exchange, PhyB is isomerized with the tetrapyrrole chromophore of phycocyanin (PCB), and Pr undergoes a conformational change to become an active form (Pfr). Pfr can be heterodimerized with phytochrome interacting factors such as PIF3 or PIF6 to perform multiple functions. The combination of PhyB-PIF and a two-hybrid system is often used to mediate light-dependent protein interactions or to reconstitute the cleavage protein domain.

  1.3 Split Renilla luciferase

Renilla luciferase can catalyze the emission of blue light with a maximum wavelength of about 480 nm. Its molecular weight is around 36 kDa and it is often used as a reporter gene to explore gene expression, while split Renilla luciferase is often used to study protein protein interactions. The Renilla luciferase is split into two subunits at a specific site, and the recombination of N-terminal part (N-hrluc) and the C-terminal part (C-hrluc) of the split Renilla luciferase (hrluc) could restore the activity of the protein. Combining the interaction protein with the split Renilla luciferase, when the interaction protein interacts, the split luciferase to which it is linked also complements to restore activity and catalyze the substrate to emit blue light.

Our Design

In order to broaden the spectrum to control Chlamydomonas movement, we first considered modifying the photosensitive structure of Chlamydomonas, the channel rhodopsin ChR. We have searched for the current method of modifying the wavelength of ChR. It is found that the mutation of the amino acid site of ChR can achieve the red shift of the maximum photosensitive wavelength, and the redshift distance is at most about 100 nm. Therefore, our first step in improving the idea is to express the red-shifted ChR mutant in Chlamydomonas. But the 100nm mutation distance can't satisfy our demands. So we quickly tried to find other ways to make the cell move under different lights.

We first attempted to produce endogenous blue light in Chlamydomonas to verify its activation of Chlamydomonas. However, considering that blue fluorescent protein requires ultraviolet light excitation, it will affect the motion control light source. Therefore, we further express the complete Renilla luciferase in Chlamydomonas for catalytic luminescence to avoid interference from uncontrolled light sources.

After the activation of Chlamydomonas movement, we fused the light-control protein to the split Renilla luciferase to control the endogenous blue light and the control of Chlamydomonas movement activation. Considering that cellular micro-robots will play an important role in the medical field in the future, We used red light-controlled polyproteins for PhyB and Pif3, and split Renilla luciferase. We fused the N-terminal part of PhyB and Renilla luciferase (rluc), the C-terminal part of Pif3 and Renilla luciferase (rluc) in Chlamydomonas cells respectively. Because of red light stimulation (about 660 nm). PhyB and PIF3 will undergo heterodimerization, and the N-hrluc and C-hrluc, which are respectively linked by the two proteins, are close to each other and complement to restore the formation of the active intact Renilla luciferase. In the presence of substrate coelenterazine, the activity-recovered Renilla luciferase catalyzes a blue light (about 470 nm), and intracellular blue light activates the channel rhodopsin at the eye spots of Chlamydomonas, causing ion channels to open and light. The change in current further causes an increase in the concentration of second messenger molecules such as intracellular Ca2+, thereby affecting the movement of Chlamydomonas.

Summary

In our design, we found that by replacing light-controlled polymeric proteins, we can theoretically achieve the polymerization of split renilla luciferase under different light conditions. Therefore, we can theoretically control the movement of Chlamydomonas under different light conditions. Expanding the spectral motion control of Chlamydomonas may have more potential applications in different fields. We have also explored the potential applications of such multi-spectral control Chlamydomonas micro-nanobots in different fields in human practice (see our HP).

In our design, we have innovatively proposed a concept of a "molecular light converter." Different input light is converted to blue light output by fusing different light-controlled polymeric proteins with split luciferase. Furthermore, combining different light-controlled polymeric proteins with different split fluorescent proteins, can achieve selectable light input and light output. Although such molecular light converters are also limited by the types of light-controlled polymeric proteins and fluorescent proteins, they are modular light converters at the molecular level, which is also an extension of the application of split luciferase in addition to the interaction of reporter genes with research proteins. Unfortunately, we don't have enough time to fully verify the concept.

Expectation

Wild Chlamydomonas has a sensitive perception of blue light and no motion response under red light. Our experiments hope to construct Chlamydomonas albicans that responds to red light and further drive the direction of red light on Chlamydomonas.

Due to the time limit of iGEM, we are unable to complete more experimental verification in just one year. However, we still hope to further improve the corresponding performance of our red light control sports algae. The first is to improve the conversion efficiency of red and blue light in Chlamydomonas, to increase the sensitivity of Chlamydomonas to low-intensity red light and the driving force of red light to Chlamydomonas; the second is to achieve the decoupling of red and blue light control. To realize the independent control of Chlamydomonas movement in red and blue light; the third is to further expand the motion control of Chlamydomonas in different light, and complete the experimental verification of the concept of molecular light converter.

In the application of our engineering algae, we hope that the Chlamydomonas cell nano-robot has a wider application potential and expand its application outside the medical field. We also conducted a preliminary investigation (see our HP) to understand the micro-nano size of Chlamydomonas cell robots, which has potential application value in drug carrier platforms, micro-drivers and microfluidic cargo transport. . We have selected several of the most promising application prospects to further expand the downstream design of our project (see our Following Design)