Team:DUT China B/Description

BALDR

• Project
  • Inspiration
  • Description
  • Design
  • Demonstrate
  • Expectation
  • Following Design
• Wet_lab
  • Experiments
  • Results
  • Protocols
  • Notebook
  • Safety
• Parts
  • Parts Overview
• Model
• Outreach
  • Human_Practices
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• Team
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• Inspiration


• Background


• Chlamydomonas reinhardtii

Inspiration

Have you ever seen the Superhero movie Antman? Imagine if the ant man's suit in the Marvel movie really exists in life，we can become incredibly small, even to the quantum scale, like the Antman who can get into Ironman’s suit and disable it in a minute, or sneak into a machine and fix it from the inside! We will have the opportunity to observe and even manipulate the world from an extremely microscopic perspective! Although the current technological development has not reached that level, it does not prevent us from exerting such bold imagination and trying to transform anything that might be used as a micro-robot. The cell is no doubt the finest complete living body and the most delicate control system we know. It is not an exaggeration to compare it to a delicate life robot. But if we want natural cells to perform the functions we expect, the most viable way is to transform cells just like the way we transform mechanical robots. Cellular micro-robots have greatly attracted our attention, and we are excited to imagine that we could manipulate such a controllable micro-robot. Synthetic biology has provided us a way to build gene circuits and transform cells according to our wishes. This year, we just can't wait to use synthetic biology tool to build such a controllable cell micro-robot!

nanorobot

Background

When it is difficult for synthetic materials to meet the needs of control and loading, scientists have long thought of loading cells and modifying biological molecules, using their own characteristics to operate on a small scale. Whether in nano-manufacturing, precision medicine, single cell sorting or targeted drug delivering, microscale robots that are easy to manipulate have irreplaceable advantages. Depending on different characteristics of cells and biomolecules, bacteria with invasiveness, virus with the ability of transducing and self-replicating, and cells with membrane encapsulation and drug loading capacity have been developed for micro-robots in specific application scenarios such as targeted drug delivery and gene therapy, etc. The tiny size and delicate control system of micro-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 they are only limited to the compositional parts of mechanical control systems such as sensors, actuators and control circuits. The size of rigid robots is difficult to control in millimeters. Wireless and reasonable output energy supply are difficult to obtain below this level; poor biocompatibility of rigid mechanical materials also limits the development of mechanical micro-robots in the medical field. Compared with them, biological cells with micro or 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 energy supply problem, which meanwhile helps the cell to express and carry modified drug proteins much more easily.

However, due to the uncertainty of living organism, the precise control of the cell micro-robot has become a major problem in its development. The control of cell mobility is one of the difficulties. One reason is that the cells themselves has limitations of weak mobility, and another is that the sensing and motion control methods of the cells are limited. 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, etc. 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, which is known as “green yeast”. The complete sequencing of the nuclear genome, chloroplast genome and mitochondrial genome and genetic transformation under the three genomes have been completed, which gives Chlamydomonas Reinhardtii the clearest research background as a photoautotrophic eukaryotic chassis organism. Chlamydomonas cells have two flagellum, which are highly mobile and have a blue-light sensing system. They have the advantages of carrying protein-loading drugs and the potential of light control transformation, which are good chassis creatures with matched characteristic to be transformed into a micro-robot. Therefore, we hope to use Chlamydomonas as a chassis and modify it on the basis of its own strong mobility as well as to solve the problem of mobility control in applications.

Chlamydomonas reinhardtii with strong mobility

The optical control system has the advantages of requiring simpler equipments, wireless control, good penetrability. Chlamydomonas itself has a blue light sensing system, we can utilize this characteristic to control its movement better. We hope to modify Chlamydomonas cells to achieve its controllable movement under different lights.

To achieve the movement control of Chlamydomonas, how to change 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, they have three different movement modes of swimming, fluctuating, and sliding under different conditions, and the movement mechanism is very complicated. The specific molecular regulation network of two flagella in Chlamydomonas has not been thoroughly researched yet. 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 wavelength range, and we have not been able to find other substances that specifically activate or inhibit the rhodopsin of the Chlamydomonas channel, but only knowing that 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 obstacles 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 can be triggered by blue light. If blue light can be produced in Chlamydomonas cells, can it also activate the channel rhodopsin of Chlamydomonas? We considered the use of blue fluorescent protein for the production of endogenous blue light in Chlamydomonas. However, since the fluorescent protein needs to be excited by specific wavelength light, which may interfere with the light of control, so we use luciferase for catalytic luminescence. In the literature search, we learned about the protein split method and discovered the work of splitting luciferase for protein interaction study. This inspired our thoughts: What about combining light-controlled polymerized proteins with split luciferase to generate blue light under other light control? We call this a molecular light converter. By constructing this molecular light converter in Chlamydomonas cells, we can achieve the excitation of endogenous blue light in Chlamydomonas cells, thus realizing the activation of Chlamydomonas light perception system and motion control. That is how our solution is generated.

Molecular light converter

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