Highlights：

A.We constructed mCerulean3 transformed C. reinhardtii and proved endogenous blue light also can induce the directed movement of C. reinhardtii successfully.
B.We split NanoLuc at a new site by modelling and it has high luminescence.

DUT_China_B--results_final.pdf

1 Transformants and cultivation

There are some representative generated transformants and cultivation of engineered Chlamydomonas Reinhardtii

Figure 1. Representative generated transformants and cultivation

2 Fluorescence observation of mCerulean3 transformed Chlamydomonas Reinhardtii

In order to explore whether endogenous blue light can make C. reinhardtii moving directional, we used the plasmid pOpt-mCerulean3-Hyg (donated from professor Kong) for our project.

Figure 2. 1% Agarose gel electrophoresis of DNA extracted from the positive clones (Marker: λ-EcoT14 Ⅰ digest DNA marker;lane 1,2,3 : plasmid pOpt-mCerulean3-Hyg)

Detect the intracellular fluorescence of mCerulean3 accumulated in the cytoplasm by Olympus FV-1000 laser confocal microscope.

Figure 3. Detection of the intracellular fluorescence of mCerulean3 accumulated in the cytoplasm by confocal laser microscopy

3 Motion characteristics of mCerulean3 transformed Chlamydomonas Reinhardtii

1) The ultraviolet lamp is mixed with certain blue light. The wild type Chlamydomonas Reinhardtii has the characteristic that only blue light phototaxis. According to this characteristic, we tested the proportion of blue light in the ultraviolet lamps used in the experiment. Irradiate wild algae with 120.3 lx and 46.7 lx ultraviolet radiation to obtain the corresponding speed：0.0999 mm/s, 0.0647 mm/s；Substitute them into the model fitting illumination-velocity formula

(A1, A2: amount of blue light in ultraviolet lamps under different illumination;B1, B2: ultraviolet lamp illumination)

It is calculated that the proportion of blue light in the ultraviolet lamp used in the experiment is 14.6 %.

2) Measure the velocity of mCerulean3 transformed Chlamydomonas Reinhardtii movement under ultraviolet light. And measure the time that engineering algae move 1 mm under different illuminance.

Figure 4. Detection of the intracellular fluorescence of mCerulean3 accumulated in the cytoplasm by confocal laser microscopy

Take the movement of engineering algae with illumination of 62.3 lx as an example (speed 0.124mm /s). According to equation (2), the illumination of blue light in ultraviolet light at this time is about 9.1 lx.

3) Illuminate the engineered algae with 9.1 lx pure blue light and calculate its movement speed.

Table 1 The movement of engineering algae under blue light irradiation

When the blue illuminance is 9.1lx, the mCerulean3 transformed Chlamydomonas Reinhardtii move faster under UV light than that under blue light. So we believe that endogenous blue light also can induce the directed movement of Chlamydomonas.

4 Measurement of the movement features of Chlamydomonas Reinhardtii

1 The speed of wild C. reinhardtii under 11.46 lx blue light was measured, and the results were 0.1018±0.0105 mm/s. The movement of wild C. reinhardtii under 12.00 lx blue light and 12.00 lx red light is shown in video. It can be seen that wild C. reinhardtii showed obvious phototropism under blue light, but none under red light. Our project will focus on broadening the photosensitive spectrum of C. reinhardtii.

2. Measurement of illuminance influence on the experiments

It is known that illuminance affects the movement characteristics of C. reinhardtii. A light too strong will cause light avoidance movement. In a certain range, illuminance has a certain relationship with the movement speed. The movement speed of wild C. reinhardtii was measured under different illuminance, and the results were shown in Figure 4, and the corresponding illumination-velocity curve was obtained.

The illumination-velocity function is obtained:

Figure 5. Velocity - illumination curve of wild C. reinhardtii under blue lightt

5 Characterization of the mutant channel rhodopsin VchR from the Volvox

Figure 6. pGSⅠ-VchR EcoR I, Bgl Ⅱ Double digestion (M: λ-EcoT14 Ⅰ digest DNA marker ; lane 1,2 : plasmid pGSⅠ-VchR)

Figure 7. pOpt_VchR_paro EcoR I, Bgl Ⅱ Double digestion (M: λ-EcoT14 Ⅰ digest DNA marker ; lane 1,2,3: plasmid pOpt_VchR_paro)

In the video, VchR-engineered C. Reinhardtii were seen to move under orange light( 590nm, 25.4lx) while the wild type C. Reinhardtii showed no apparent movement.

Under standard protocols of C. Reinhardtii movement measuring, the movement data of VchR -engineered C. Reinhardtii is showed in table 1,2. As we can see from the table, VchR -engineered C. Reinhardtii is able to move under light of 590nm.The moving speed of C. Reinhardtii declines with light intensity, until showing random movement pattern at 32.6lx with almost none phototaxis pattern. Compared with the data of 480nm light, we can learn that VCHR responses in light of 590nm more weakly than 480nm.

Figure 7. Speed of VchR-engineered C. reinhardtii under 590 nm,orange light

6 Renilla luciferase

In order to verify the function of Renilla luciferase at the protein molecule level and Chlamydomonas reinhardtii level, a pOpt_Rluc_paro expression vector for C. reinhardtii and a pET-28a_Rluc expression vector for E. coli were constructed. The results of the construction are shown in Figure 8,9.

Figure 9. pOpt_Rluc_paro EcoR I, Bgl Ⅱ Double digestion

Figure 10. pET-28a_Rluc EcoR I, Bgl Ⅱ Double digestion

The effect of incubation time on luminescence intensity is shown in Table 2 and Figure 10.As can be seen from the figure and table, the relative luminescence intensity increased with the increase of incubation time, and the growth rate was faster in the first 10 minutes, so the incubation time of subsequent experiments was determined to be 10 minutes.

Figure 11. the relationship between luminescence intensity of Rluc crude enzyme solution and incubation time

The effect of coelenterazine concentration on luminescence intensity is shown in Figure 2, Table 2. As can be seen from the figure and table, the maximum luminescence intensity appeared at the substrate of 10 μM, so the subsequent experimental coelenterin substrate concentration was determined to be 10 μM.

Figure 12. relationship between luminescence intensity of Rluc crude enzyme solution and substrate concentration

In Rluc-engineered C. Reinhardtii ,the luminescence intensity at 480 nm was measured as 2.263, 2.372, 2.341 and 2.380. Compared with the blank control (2.213) with no substrate and only water, there is no significant difference. We concluded that the membrane permeability of C. Reinhardtii cells was poor. It is necessary to increase incubation time or adopt other measures to increase membrane permeability.

7 Nanoluc

According to the above experimental protocol, we measured the enzyme-catalyzed luminescence intensity of the predicted split site and the split site in old part. The results are shown in table 1. The relative luminescence intensity of st-1 and sc-1 is significantly different from which of st-2 and sc-2. It could be inferred that the split site predicted by the model is better. Our subsequent experiments can be guided by the model to cut luciferase more precisely.

Figure 13. pET -21a_N-NanoLuc_Spytag-1 EcoR I, Bgl Ⅱ Double digestion

Figure 14. pET -21a_N-NanoLuc_Spytag-2 EcoR I, Bgl Ⅱ Double digestion

Figure 15. pET-28a_SpyCatcher_C-NanoLuc-1

Figure 16. pET-28a_SpyCatcher_C-Nanoluc-2

We measured the enzyme-catalyzed luminescence intensity of the protein which is split in new site and the old one. The results are shown in figure 16. The relative luminescence intensity of st-1 and sc-1 is significantly higher than o st-2 and sc-2. It could be inferred that the two new split protein can combine easier and the split site predicted by our model is better.

Figure 17. Comparison of NanoLuc catalyzed coelenterazine luminescence at different split sites

Control：2700 μL mixed crude enzyme +300 μL deionized water
Relative luminous intensity: Crude enzyme luminescence/(Control luminescence× Total quality of protein in crude enzyme solution）
Abbreviation:
st-1: Fusion protein of new N terminal of Guassia luciferase and Spytag
st-2: Fusion protein of N terminal of Guassia luciferase and Spytag
sc-1: Fusion protein of new C terminal of Guassia luciferase and SpyCatcher
sc-2: Fusion protein of C terminal of Guassia luciferase and SpyCatcher

8 PhyB-Pif3

In order to construct a red-blue transformation system, we constructed the pOpt_ N-hrluc_Pif3 _Paro, pOpt_SP_PhyB_C-hrluc _Paro expression system in C. reinhardtii. At the same time, in order to prove the feasibility of the system at the protein level, pET-28a_N-hrluc_Pif3, pET-28a_SP_PhyB_C-hrluc E. coli expression vector was constructed. The vector construction result is shown in Figures 17, 18 and 19.

Figure 18. pOpt_ N-hrluc_PIF3 _Paro EcoR I, Bgl Ⅱ Double digestion

Figure 19. pOpt_SP_PhyB_C-hrluc _Paro EcoR I, Bgl Ⅱ Double digestion

Figure 20. pET-28a_N-hrluc_Pif3, pET-28a_SP_PhyB_C-hrluc EcoR I, Bgl Ⅱ Double digestion (M: λ-EcoT14 Ⅰ digest DNA marker ; lane 1,2: plasmid pET-28a_SP_PhyB_C-hrluc; lane 3,4: plasmid pET-28a_N-hrluc_Pif3)

Two kinds of light-controlled proteins were respectively expressed in E.coli, and the luminescence intensity was measured by the above measurement protocol after collecting the crude enzyme solution, and the results are shown in Figure. 20. As can be seen from Figure. 20, the red light conversion system can work normally, generating four times blue light. However, when the luminescence intensity was tested with intact C. reinhardtii, the results were similar to the previous ones, and luminescence could not be detected. We concluded that coelenterazine has poor permeability to cell walls of C. reinhardtii or E. coli. We can consider other illuminating systems with strong permeability.

Figure 21. PhyB-Pif3 protein solution luminescence intensity detection

To determine whether the signal peptide ChR2 is working,we detect the intracellular fluorescence of Clover. The fluorescence is concentrated around the cell membrane, especially on the eyespot, which will help locate our fusion protein PhyB-C-hrluc to the eyespot.

Figure 22. Detection of the intracellular fluorescence of Clover accumulated in the cytoplasm by confocal laser microscopy