Difference between revisions of "Team:DUT China B/Demonstrate"

Every important moment

To transform Chlamydomonas into a cellular micro-robot, our mission is to broaden the spectrum light to control the movement of Chlamydomonas. The primary work is to demonstrate the activation of intracellular endogenous blue light to Chlamydomonas. We express blue fluorescent protein and intact Renilla luciferase in Chlamydomonas. After intracellular blue light is generated, we observe the movement of algae under UV and red light conditions
Secondly, we constructed a red-controlled molecular switch, and conducted preliminary functional verification at the molecular level;. We then examined the expression of this switch in Chlamydomonas. In order to reduce the inevitable steric hindrance when the split renilla luciferase bind together, we found a smaller molecular weight Gauss luciferase to replace Renilla luciferase to optimize the original molecular design. We used a protein splitting model to calculate an optimal protein cleavage site for Gauss luciferase and compared it with the Nanoluc with different cleavage sites in the biobrick library, confirming the cleavage site of split Gauss luciferase we predicted has a better recovery activity.
Next, we will continue to optimize design of molecular light converters, trying to increase the expression level of molecular light converters in Chlamydomonas and improve the combination and luminous efficiency of molecular light converters.

Characteristics of wild type Chlamydomonas
Before the start of the experiment, we thoroughly explored the kinetic characteristics of wild type Chlamydomonas. Wild type Chlamydomonas has a phototaxis movement under blue light, while it has no motion response under red light. We measured the movement speed of wild type Chlamydomonas under blue light of different intensity, and obtained the kinematic characteristics of Chlamydomonas, which provided a reference for the subsequent measurements of algae movement. (Check out our experiment)

● Movement stimulation of Chlamydomonas by Endogenous blue light to Chlamydomonas
The effect of intracellular blue fluorescent protein (BFP) on the movement of Chlamydomonas
We first attempted to transfer the blue fluorescent protein gene into Chlamydomonas to observe whether the blue fluorescence produced by UV excitation would affect the movement of Chlamydomonas. Our experimental results show that the algae has a tendency to move under ultraviolet light, and the movement speed is higher than previous movement under blue light and the movement of wild algae under blue light, which proves the endogenous blue light produced by BFP has an effect on its movement.

In order to let BFP generates endogenous blue light in Chlamydomonas, it is necessary to use exogenous light to simulate the BFP, which may cause interference or spectral limitation to the externally controlled light source. Therefore, we further constructed an engineered algae to express intact Renilla luciferase. The renilla luciferase expressed in Chlamydomonas catalyzes the progesterone , which enters the cell through the plasma membrane, to generate the blue light, and the endogenous blue light activates the movement of Chlamydomonas to realize the motor response under external red light.

We completed the construction of the Rluc engineered algae and measured its kinematic properties. Unfortunately, we have not observed the motor response of Chlamydomonas under red light conditions. We intend to observe blue light in the engineered algae under a fluorescence confocal microscope but the result turned out negative. We hypothesized that the substrate coelenterazine could not enter the Chlamydomonas due to the blocking effect of the cell wall. Therefore, we performed a transient electroporation to the cell and then add coelenterazine, and observed the results under the fluorescence confocal microscopy, but there is still no generation of blue light. We hypothesize that the absence of codon-optimized Renilla luciferase gene, may cause a lower expression in Chlamydomonas, which cause a Bad catalytic effect. Secondly, it may be that coelenterazine still fails to enter the Chlamydomonas or oxidizes before it works.

● Construction of molecular light converter system
After preliminarily verificating the effect of endogenous blue light on Chlamydomonas movement, we constructed a molecular light converter of red light-controlled polymeric protein PhyB and Pif3 fusion with split Renilla luciferase, characterized it in vitro and constructed engineered Chlamydomonas.

We first expressed them in E.coli, and the luminescence intensity was measured by protocols after collecting the crude enzyme solution. As for the results, the red light conversion system can work normally, generating four times than blue light. However, the luminescence could not be detected with intact C. reinhardtii. We concluded that it is because the coelenterazine has poor permeability to cell walls of C. reinhardtii or E. coli, thus We can consider other illuminating systems with strong permeability.

We have obtained double-converted engineered Chlamydomonas, but the algae were not characterized for their movement properties. But the results support the function of light converter system and we would carry on our experiment on alga motion measurement.

Considering that the closer the endogenous blue light is generated to the eye spot area, the easier it is to activate the channel rhodopsin to be activated, we added a signal peptide targeting the eye spot on the molecular light converter, and characterized this signal peptide. We have transformed the signal peptide with green fluorescent protein into Chlamydomonas and observed it under the laser scanning confocal microscope. We can see the green light spots in the eye spot area, confirming that the signal peptide we selected has successfully located in the eye spot area of Chlamydomonas.

● Optimization
In the design of molecular light converter, the splitting Renilla luciferase (Weight about 39 kDa) which have steric hindrance when polymerization occurs under red light control, may affects the binding rate and the activity of recombination, which in turn affects the intensity of the blue light generated. To further optimize our design, we found a luciferase with a lower molecular weight, Gaussia luciferase (weight about 18kDa) and hope to use it instead of Renilla luciferase. To determine the optimal cleavage site, we constructed a protein splitting model and calculated a good cleavage site. We selected the Spytag/SpyCatcher interaction protein pair with smaller molecular weight contributed by 2013 TU_Munich in the Biobrick library, and separately expressed it with splitting Guassia luciferase to measure the recovery intensity of the enzyme activity in vitro. We also compared it with the existing Gaussia luciferase with a different cleavage site in the Biobrick library. The results showed that the splitting Gaussia luciferase at the site we selected had higher recovery activity. This also proves the validity of our protein splitting model.

● Following experiments and Application prospects
Due to the limitation of time, we were unable to complete all the experimental verification work. But we are still carrying out the construction of engineered algae and molecular verification experiments. Next we hope to continue to verify the engineered algae and further optimize our design;

1. Completing the molecular characterization and engineered algae motion verification of red light-controlled Renilla luciferase molecular light converter; 2. Constructing a molecular light converter with splitting Gaussia luciferase and completing the corresponding molecular experiments and engineered algae characterization; 3.Constructing molecular light converters different light-controlled polymeric protein pairs.

For the future application of Chlamydomonas cell micro-robots, we conducted a feasibility study of its application in the medical field at the beginning of the project (see our HP). In the later research in different fields, we also found that the Chlamydomonas micro-robot has most applications of targeted therapy in precision medicine. For this direction, we constructed a model to explore the minimum light intensity threshold required to control microalgae swimming at different blood flow velocities. Our model provides valuable data and rules for future clinical trials of Chlamydomonas micro-robots as a targeted therapeutic vector.