Transformants and cultivation

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

Fluorescence observation of mCerulean3 transformed Chlamydomonas Reinhardtii

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

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.

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.

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.

Measurement of the movement features of Chlamydomonas Reinhardtii

1. The speed of wild C. Reinhardtii under 11.46lx blue light was measured, and the results were shown in table 1.

Control Group：C. Reinhardtii moving without exogenous blue light (480nm, 11.46lx)

Experiment Group：C. Reinhardtii moving under exogenous blue light (480nm, 11.46lx)

The movement of wild C. Reinhardtii under 12.00lx blue light and 12.00lx 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 table 2, and the corresponding illumination-velocity curve was obtained, as shown in figure 1. The illumination-velocity function is obtained:

.The speed of movement of C. Reinhardtii is logarithmically related to illuminance. When the illuminance reaches about 500.00lx, chlamydia will produce light avoidance movement. In this experiment, the maximum illuminance of blue light source is 89.70lx.

Control Group：C. Reinhardtii moving without exogenous orange light (about 590nm)

Experiment Group: C. Reinhardtii moving under exogenous orange light (about 590nm)

Figure 1. Velocity - illumination curve of wild C. Reinhardtii under blue light

Characterization of the mutant channel rhodopsin VchR from the Volvox

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.

Control Group：Without external blue light of 480nm, 25.4lx

Experiment Group：Under external blue light of 480nm, 25.4lx

Control Group：Without external orange light of 590nm

Experiment Group：Under external orange light of 590nm

Renilla luciferase

The effect of incubation time on luminescence intensity is shown in Table 1 and Figure 1.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.

Table 1 relationship between luminescence intensity of Rluc crude enzyme solution and incubation time

Blank control: crude enzyme solution 2900ul+ 100ul water

relative luminescence intensity: crude enzyme solution luminescence/blank control luminescence

Figure 1 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.

Table 2 relationship between luminescence intensity of Rluc crude enzyme solution and substrate concentration

Blank control: crude enzyme solution + water (same volume as substrate)

Relative luminescence intensity: crude enzyme solution luminescence/blank control luminescence

Figure 2 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.

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.

able 1. comparison of nanoLuc catalyzed coelenterazine luminescence at different split sites

Blank contrast：2700 μl mixed crude enzyme +300 μl deionized water

Relative luminous intensity：Crude enzyme luminescence /（Blank contrast luminescence×Protein concentration of crude enzyme solution）