Inspiration

Our project design mainly involves three aspects: Chlamydomonas movement and photoreceptor system, red light control polymer protein, split sea kidney luciferase. The following is a detailed explanation of the mechanism of action of each part.

OPhotosensing and movement mechanism of ChlamydomonasThere 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 wavelengths ~500nm and ~470nm, respectively, causing proton flux or sodium. A cation stream such as potassium enters the cell, causing cell depolarization. ChR is a light-gated ion channel, and its structure contains chromophore 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 transfer. The generation of photocurrent causes a series of reactions such as phosphorylation of the channel rhodopsin and an increase in the concentration of internal 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 with Chlamydomonas cytoplasm Increased intracellular calcium concentration, channel rhodopsin phosphorylation, and elevated second messenger molecules such as cAMP.

Light control proteinWe have learned three kinds of protein pairs that can be polymerized under illumination control: 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. 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-protein interactions or to reconstitute a cleavage protein domain.

nanorobot

Background

When synthetic materials are difficult to meet the needs of control and loading, scientists have long thought of loading and modifying biological cells or molecules, using their own characteristics to operate on a small scale. Whether it is micro-nano manufacturing, precision medicine, single cell sorting or targeted drug loading, easy to manipulate micro-nano-scale robots have irreplaceable advantages. Depending on the invasiveness of the bacteria, the ability of the virus to transduce and self-replicate, the membrane encapsulation and drug loading capacity of the cells, different cells or biomolecules have been developed for micro-nano robots for specific application scenarios for targeted drug delivery. Or gene, cell therapy. The tiny size and control system of micro-nano robots make it a powerful tool for precision medical applications.

Rigid micro-robots developed in the field of machine engineering have the best accuracy and control under programmable and automated operation, but are limited to the composition of mechanical control systems covering sensors, actuators and control circuits. The size of rigid robots is difficult to control in millimeters. Energy supply below the level and difficult to obtain wireless and reasonable output; poor biocompatibility of rigid mechanical materials also limits the development of mechanical micro-robots in the medical field. Compared with mechanical micro-nano robots, biological cells with micro-nano size and self-sufficient growth have a complete control system at the micro-nano scale, and the cell's own energy conversion system solves the micro-nano robot energy supply problem, which is also easier to carry out. Expression modification of drug proteins.

However, due to the uncertainty of the living body, the precise control of the cell micro-nano robot has become a major problem in the development of cell micro-nano robots. The control of cell mobility is one of the difficulties. One is limited to the weaker mobility of the cells themselves, and the other is limited to the sensing and motion control methods of the cells. At present, the commonly used methods are control of light, magnetism, material wrapping, etc. However, magnetic control requires more complicated external equipment and computer algorithms. The material wrapping needs to make more modifications to the cells, which may affect the activity of the cells, and may cause in vivo. Problems such as residual material modification, potential damage to the human body. Light control is a relatively convenient method of control, but still requires a more transparent application environment.

Chlamydomonas reinhardtii

Chlamydomonas reinhardtii is a single-cell photoautotrophic eukaryote with the ability to accurately fold and assemble complex proteins. It can be used to express various complex proteins and high-value products. It is known as “green yeast”. It is said that the complete sequencing of the nuclear genome, chloroplast genome and mitochondrial genome and genetic transformation under three genomes are the most clear photosynthetic autotrophic eukaryotic substrates. Chlamydomonas cells have two flagellae, which are highly mobile and have a blue-light sensing system. They have the advantages of carrying protein-loading drugs and the potential for transformation using light control. They are characterized by the use of micro-nano robots. Good chassis creatures. Therefore, we hope to use Chlamydomonas as a chassis and modify it with its own strong mobility to solve the problem of mobility control of cellular nano-robots in applications.

Chlamydomonas reinhardtii with strong mobility

The optical control system has the advantages of simple equipment, wireless control, good penetrability, etc., and Chlamydomonas itself has a blue light sensing system, so that the control of the movement of Chlamydomonas cells can be more fully utilized. Since cell micro-nano robots are mainly used in the medical field for targeted therapy, red light is more penetrating than other light tissues and is the most commonly used optical means in the medical field. Therefore, we hope to achieve red color in Chlamydomonas cells. The engineering of light control movement.

To achieve the kinetic control of Chlamydomonas, how to transform its endogenous motion control and light perception system is the most effective means to achieve our transformation goals. However, because the movement of Chlamydomonas cells is controlled by two flagella, there are three different movement modes of swimming, fluctuation, and sliding under different conditions, and the movement mechanism is complicated. The specific molecular regulation network of two flagella in Chlamydomonas has not been obtained yet. Clear interpretation. Therefore, we cannot start from the molecular mechanism of the Chlamydomonasis movement. In the blue light sensing system of Chlamydomonas, we have learned that the eye spots of Chlamydomonas are used for blue light perception, and then the light signal is transmitted to the flagella to regulate the different movements of the two flagella under the action of the second messenger molecule. Therefore, we try to activate the Chlamydomonas light perception system from the light-gated ion channel at the eye spot to achieve the motion control of Chlamydomonas. But unfortunately, the mutants of the light-gated ion channels have limited redshift range, and we have not been able to find other substances that specifically activate or inhibit the rhodopsin of the Chlamydomonas channel, but only the general purpose of the cells. The second messenger molecule has a regulatory effect on it. We are unable to control the transformation of the universal messenger molecules in the cell, as this can distort the growth regulation of Chlamydomonas cells. So we have to give up on this idea.

After encountering a bottleneck in the molecular mechanism transformation, we tried to find a simpler way to control the algae.We have considered that since the channel of rhodopsin is excited by blue light, in the literature search, we have learned the research method of split protein and found the work of splitting luciferase for protein interaction. It is noted that the catalytic reaction of luciferase can produce blue light. We associate it with the possibility of combining red-controlled polymerized proteins with split luciferase. This enables the generation of blue light under red light control. We call this a molecular light converter. By expressing this molecular light converter in Chlamydomonas cells, we can achieve the excitation of endogenous blue light in Chlamydomonas cells, thus realizing the activation and motion control of Chlamydomonas light perception system. Our solution is thus generated.

Molecular light converter