iGEM Tsinghua

Result Demonstration

Result Part I

I. Demonstration of multivalent phase separation elements

Some protein domains are prone to liquid-liquid phase separation (LLPS) based on multivalent interactions, including multiple modular interaction domains or intrinsically disordered domains (IDR)[1]. In our project, we exploited the GCN(4)-FUSLCD, SIM/SUMO as basic elements to design phase separation systems. .

1. GCN(4)-FUSLCD phase separation system We cloned the low complexity domain (LCD) of the fused in sarcoma (FUS) protein (FUSLCD), which was reported to phase separate spontaneously at high concentrations in vitro due to their multiple weakly adhesive sequence elements[2-4] (figure 1a). We fused GFP (for visualization) and GCN(4) (for spontaneous formation of tetramers) with FUSLCD and expressed GFP-GCN(4)-FUSLCD in E.coli (figure 1a). Spherical green droplets indicating phase separation were observed (figure 1b) at two ends of E.coli. The fast fluorescent recovery after photobleaching (FRAP) (figure 1c) indicated the high fluidity of puncta and validated the feasibility and effectiveness of GCN(4)-FUSLCD phase separation system.

We also tried other variations of GCN(4)-FUSLCD phase separation system, including FUSLCD alone, and GCN(3)-FUSLCD (for spontaneous formation of trimers), 1i4k-FUSLCD (for spontaneous formation of hexamers). As GCN(4)-FUSLCD phase separation system had the best performance among them, we finally chose to use this design.

Figure 1. Phase separation element GCN(4)-FUSLCD (a).The domain structure of FUS. The QGSY-rich region (aa. 1-165) and part of the G-rich region (aa. 165-212) constitute the FUSLCD (aa. 1-212), which is prone to phase separation. (b). The domain structure of GCN(4)-GFP-FUSLCD. (c). Confocal images of E.coli expressing GCN(4)-GFP-FUSLCD. Green spherical puncta formed in the cell. (d). Plot of normalized GFP fluorescence intensity of GCN(4)-GFP-FUSLCD puncta versus time after total photobleaching. The green fluorescence signal started to recover immediately after it was bleached by 488 nm wavelength laser, and recovered up to 100% of original fluorescence intensity within 500 seconds.

2. (SIM-SUMO)3 phase separation system We cloned a series of tandem repeats of SIM-SUMO into a reading frame and fused with GFP for visualization. Phase separation was observed in three repeats of SIM-SUMO expressed in E.coli (figure 2a). We exploited FRAP to validate the phase separation (figure 2b, 2c and video 2). The fast recovery of fluorescent intensity indicated its high fluidity

3. (SIM)3&(SUMO)3 phase separation system For the purpose of light induction of phase separation, we cloned three tandem repeats of SIM and SUMO, together with light sensitive elements CIB1 and Cry2 into two expression vectors respectively (figure 2a). The LLPS droplets appeared after light stimulation (figure 2b). The fluidity of one of the newly formed droplets were validated by FRAP (figure 2c and video 2).

According to our data, the multivalent phase separation systems had relatively better fluidity but less light inducibility or less stability in comparison with the IDR phase separation systems. This fact is consistent to previous report [5].

Figure 2. Tandem repeats phase separation element (SIM-SUMO)3. (a). Illustration of the phase separation element. (b). FRAP image of GFP-(SIM-SUMO)3. Left panel: before photobleaching. Middle panel: after photobleaching. Right panel: after the fluorescent recovery. (c). Plot of normalized GFP fluorescence intensity of GFP-(SIM-SUMO)3 puncta versus time after total photobleaching. The green fluorescence signal started to recover immediately after it was bleached by 488 nm wavelength laser, and recovered up to 80% of original fluorescence intensity within 150 seconds.

Video1

Video2

Result Part I

II. Demonstration of LLPS in morphologically engineered E.coli cells

We found a ubiquitous phenomenon in our experiment: the LLPS droplets mainly appeared at the two polar regions of a rod-like bacterium. This is a counterintuitive result, for we hypothesized that the LLPS droplets will randomly distribute inside the bacterial cells. To determine the relationship between the cell shape and the distribution of LLPS droplet, we overexpressed prokaryotic cytoskeleton protein ftsZ or mreB in E.coli, which will cause the elongation or the spherical shape of the bacteria.

As shown in figure 4, in elongated bacterium, the LLPS droplets distributed in random distance along the axis; in spherical bacterium, the LLPS droplets distributed in two poles of the ellipse. All the droplets showed a common feature: they tended to be plasma membrane-bound, not randomly distributed inside the cells, which implies the hydrophobic property of the LLPS droplet. These results partly explain what we observed. In the polar regions of bacteria, the droplets can bind to the plasma membrane most efficiently, making the LLPS droplets most stable.

Figure 3. (SIM)3&(SUMO)3 phase separation system. (a). Illustration of the phase separation elements. (b). Light induction of the LLPS droplets. (c). FRAP of a newly formed LLPS droplets induced by light.

Figure 4. The distribution pattern of LLPS droplets in elongated (a) and spherical (b) E.coli.

References

[1]Nakamura, H., DeRose, R., & Inoue, T. (2019). Harnessing biomolecular condensates in living cells. The Journal of Biochemistry, 166(1), 13-27.

[2]Patel, A., Lee, H. O., Jawerth, L., Maharana, S., Jahnel, M., Hein, M. Y., ... & Pozniakovski, A. (2015). A liquid-to-solid phase transition of the ALS protein FUS accelerated by disease mutation. Cell, 162(5), 1066-1077.

[3]Molliex, A., Temirov, J., Lee, J., Coughlin, M., Kanagaraj, A. P., Kim, H. J., ... & Taylor, J. P. (2015). Phase separation by low complexity domains promotes stress granule assembly and drives pathological fibrillization. Cell, 163(1), 123-133.

[4]Lin, Y., Protter, D. S., Rosen, M. K., & Parker, R. (2015). Formation and maturation of phase-separated liquid droplets by RNA-binding proteins. Molecular cell, 60(2), 208-219.

[5]Zhou, H. X., Nguemaha, V., Mazarakos, K., & Qin, S. (2018). Why do disordered and structured proteins behave differently in phase separation?. Trends in biochemical sciences, 43(7), 499-516.

[6]Banani, S. F., Rice, A. M., Peeples, W. B., Lin, Y., Jain, S., Parker, R., & Rosen, M. K. (2016). Compositional control of phase-separated cellular bodies. Cell, 166(3), 651-663.

Result Part II Photo-activated switch

I. Design

In order to control the distribution of protein in a cell with high spatial-temporal specificity, we decided to use laser as a switch for our system. To achieve the response to light stimulation, light-sensitive proteins, CIB1 and Cry2, are fused to our phase separation elements and application elements, respectively (the collocation relationships can be changed). We designed two photo-activated phase separation switch systems, namely PhASE #1 and PhASE #2. In PhASE #1, phase separation element is FUSLCD which is fused with CIB1, and Cry2 is fused with mCherry (Figure 1A); In PhASE #2, phase separation element is (SIM-SUMO)3-SUMO3 which is fused with Cry2, and CIB1 is fused with mCherry (Figure 1B). When exposed to 488nm laser, CIB1 and Cry2 will bind to each other. Take PhASE #1 for instance, Since CIB1 is primarily amplified in the compartment formed by FUSLCD, Cry2 would be recruited to phase, as the switch turns on. This is also suitable to PhASE #2. Therefore, the distribution of enzyme or other proteins fused to mCherry would be altered by light stimulation. (Figure 1C)

Figure 1. Illustration of elements used in our switch phase separation system. A. Domains of two fused proteins, element 1 and element 2 in short, are shown here. B. Domains of another version of two fused proteins, element 3 and element 4 in short. The differences are the position exchange of CIB1 and Cry2, and the change of phase separation protein to (SIM-SUMO)3-SUMO3. C. Function of both elements are shown here. Element 1 is used to create a new compartment in dark, since when local concentration of FUSLCD goes beyond a threshold, it will result in the creation of a phase in cell. Element 2 can be recruited into the phase when this system is exposed to 488 nm laser, due to the strong interaction between CIB1 and Cry2.

Result Part II Photo-activated switch

II. Demonstration

PhASE #1

E. coli transformed with element 1 and element 2 are observed using confocal microscope. Only mCherry channel and TD channel are shown in the video below (Figure 2A), since 488 nm laser, which is used to stimulate GFP, can also lead to bound of CIB1 and Cry2. At 0 second, it is almost smear in the cell. (Figure 2B) Yet, after stimulation, it is recruited to the ends of the cell and form two sphere-like droplets. The screen shots below show the distribution of mCherry before and after 488 nm laser stimulation. (Figure 2C) In order to reflect the recruitment of Cry2-mCherry into phase, we use the ratio of light intensity in phase to the rest of the cell as a standard. As presented in the plot, this ratio quickly increases after stimulation and can stay at a rather static level for a long time. (Figure 2D 2E)

Figure 2. “Turn-on” our switch of PhASE #1. This group of figures shows both observation and analysis of light stimulation process of PhASE #1. A. This video shows the recruitment of element 2 into phase formed by element 1. (Since 488 nm laser is used to stimulate the switch, GFP channel is not shown) B. This image represents the state of element 2 before stimulation by laser, which is smear in E.coli. C. This image stands for the state of element 2 after exposure to 488 nm laser for 6 seconds. (3% laser intensity) D. This image shows the distribution of mcherry signal along the bacteria. Two peaks reflect the enrichment of Cry2 at both ends of this bacterium. Though weak, the signal between two peaks still higher than that of extracellular space, indicating the second phase of Cry2 in this cell. (For detail and protocol of intensity profile, please see measurement Figure 1) E. Quantified result is shown in this plot. We use the ratio of light intensity in phase to cytosol to reflect the distribution difference before and after stimulation. The result turns out that the this ratio is significantly increased after stimulation at 0 second, which means most elements 2 get into phase after stimulation. (For detail and protocol of intensity profile, please see measurement Figure 2) F. Our result has significance in statistic level. Most bacteria incubated in dark for a few hours can behave like above, so that the regulation of enzymatic reactions can be achieved later.

What is more, we validated the ability of reversible manner of our switch. Since bacteria on a single slide cannot be observed for a long time, we acquired sample from a culture dish at different time point. After incubated bacteria in dark for a few hours, we got the first sample, which shows smear distribution in the cell. (Figure 3A) Then, we exposed the whole dish to 488 nm laser for about 20 seconds. Immediately after that, we acquired another sample, with element 2 aggregates at the end of the cell. (Figure 3B) After that, the dish was placed in dark again for half an hour. Next, another sample was acquired, in which element 2 reversed to smear state. Finally, we tried to turn on the switch again, which worked as well as previous attempts. (Figure 3C, 3D)

Figure 3. A reversible switch that can be turned off in dark and turned on again using 488 nm laser. A. After the first time stimulation, most element 2 redistributed into phase. B. Then, the system was incubated in dark for half an hour, most element 2 reverse to a rather smear state. C. A zoom 3 field of bacterium of Figure 3B. D. The reversed element can be stimulated again, just like the first time stimulation. (For detail and protocol of intensity profile, please see measurement Figure 4)

PhASE #2

Similar to PhASE #1, E. coli transformed with element 3 and element 4 are observed using confocal microscope. Since 488 nm laser, which is used to stimulate GFP, can also lead to bound of CIB1 and Cry2. At 0 second, Cry2-mCherry is almost smear in the cell. Yet, after stimulation, it is recruited to the spherical droplets formed by (SIM-SUMO)3-SUMO3-GFP-Cry2. The screen shots show the distribution of mCherry before and after 488 nm laser stimulation as well as the location of GFP signal on the right. (Figure 4)

Figure 4. “Turn-on” our switch of PhASE #2. This group of figures shows both observation and analysis of light stimulation process of PhASE #2. A. This video shows the recruitment of element 4 into phase formed by element 3, stimulated by 488 nm laser. (Since 488 nm laser is used to stimulate the switch, GFP channel is not shown) B. This intensity profile shows the mCherry signal distribution after laser stimulation. (For detail and protocol of intensity profile, please see measurement Figure 4) C,D. The two groups of figures show the screenshots of the videos above, with the GFP channel showing the location of phase separation droplets on the right. Results are similar to PhASE #1.

Result Part II Photo-activated switch

III. More attempts

In order to provide a broader application for our system, we also tried to stimulate another light response system, PixELL. (Figure 5A) Since pixE and pixD form complex in dark and dissociate after stimulation, it can be used in pathways whose regulation are designed to be opposite to CIB1-Cry2 system, distributing more smear after stimulation. Indeed, we successfully controlled the dissociation process of this system. (Figure 5B, 5C)

Figure 5. Illustration of PixELL system. A. Domains of two fused proteins, Fuse PixD and Fuse PixE in short, are shown here. B. After incubated in dark for a few hours, most bacteria had small phases around their cell walls. C. Then, sample was exposed to day light for half an hour. Most aggregates dissociated in cells.

Result Part III Rluc Assay

Background

In order to prove our idea that phase separation has the ability of regulating biochemical reactions, we carried out luciferase assay to reflect the reaction efficiency before and after phase separation. Generating luminescence while catalyzing the oxidation of coelenterazine (clz), renilla luciferase can be used to quantify reaction rate in and out of phase. In addition, according to our model, we selected this specific luciferase because of the poor solubility of clz.

Design

We linked Rluc to the C terminal of cry2-mcherry and co-transformed with CIB1-G4-GFP-FUS to make it possible for Rluc to be recruited into phase. Then, as a control, we eliminated every component in both plasmid that may cause phase separation. Also, in order not to disturb the enzymatic activity, Rluc in both experimental group and control group were linked to mcherry. In detail, control group contained CIB1-GFP and mcherry-Rluc. (Figure 1)

We expected that after cultured in light, those two group would exhibit different enzymatic activity. Because phase could enrich both enzyme and substrate, bacteria in experimental group would perform higher catalytic activity compared to control group.

Figure 1. Illustration of CIB1-G4-GFP-FUS(element 1) and cry2-mcherry-Rluc(element 2)

Result

First of all, we verified that Rluc can indeed work in our system, though much inefficient than wild type enzyme. This was partially because fused protein may affect its activity. On the other hand, co-expressing phase separation system may throw high metabolic pressure on E.coli, reducing number of enzyme per bacterium. (Figure 2)

Figure 2. Verification of enzymatic activity of Rluc working in phase separation system. Though fused protein was of lower enzymatic activity than wild type enzyme, it still works in a stable manner.

Then, we tried to control the entry of enzyme into phase by laser under confocal microscope. After cultured to a proper concentration, E.coli of our experimental group was induced by 0.5 mM IPTG or no IPTG. Utilizing the same parameters as what we used in stimulating our phase separation system, we successfully realized controllable recruitment of Rluc into phase in 0.5 mM group. (Figure 3A) In no IPTG group, we also observed redistribution of Rluc, but in a non-canonical way. It moved to some small aggregates along cell wall, rather than to the end of cell. (Figure 3B) We proposed that it might due to the leakage of protein expression by T7. The amount of FUS might not be enough to form phases like 0.5 mM group, so that they form rather small ones.

Figure 3. Verification of the controllable redistribution of Rluc into phases. A. Before stimulation, mCherry signal distributed in the cell evenly. B. After stimulation, just like element 2, mCherry signal redistributed into phases, representing that Rluc went into phases.

Next, we started to measure the productivity of this reaction in both groups. The productivity exhibits no significant difference in experimental and control group under 0.01 mM IPTG induction. When increasing IPTG concentration, the productivity of experimental group gets higher than control group. (Figure 4) Consistent with our prediction, both groups did not phase separate under 10^-5 M IPTG induction, but experimental group did phase separate under higher concentration of IPTG induction. (Figure 5)

Figure 4. Measurement of Rluc activity regulated by phase separation. Both experimental group and control group exposed to light when cultured. A. Relative velocity of Rluc reaction was measured. Under induction of high concentration of IPTG, experimental group exhibits higher velocity than control group. This is one of the four independent biological replicates. (Data of other three groups not shown) B. The ratio of experimental group to control group is shown here. When there was no phase separation, experimental group exhibits similar reaction velocity as control group; some times a little lower, might due to higher metabolic pressure. As the concentration of protein got higher, phases in cells were formed. In those groups, the this specific reaction was accelerated.

Figure 5. Verification of the distribution of Rluc induced by different concentration of IPTG in E.coli. Both experimental group and control group were exposed to light when cultured. A. The distribution of Rluc in control group induced by 0.01 mM IPTG. MCherry signal distributed in the cell evenly. B. The distribution of Rluc in experimental group induced by 0.01 mM IPTG. MCherry signal also distributed in the cell evenly. C. The distribution of Rluc in control group induced by 0.5 mM IPTG. MCherry signal distributed in the cell evenly. D. The distribution of Rluc in experimental group induced by 0.5 mM IPTG. Rluc went into phases at both ends of the cell. E. The distribution of Rluc in control group induced by 1 mM IPTG. MCherry signal distributed in the cell evenly. F. The distribution of Rluc in experimental group induced by 1 mM IPTG. Rluc went into phases at both ends of the cell.

In summary, we demonstrated our regulation principle using luciferase assay. We propose that enzymes catalyzing substrate whose solubility (or distribution coefficient in phase to the rest of cell) is similar as clz might have same possibility to be regulated by our switch. Since there exists difference in characteristics of biochemical reactions, the appropriate enzyme amount to reach the highest regulation efficiency may differ from one another, which should be determined by further experiments.

Result Part III Catechol Dioxygenase Assay

Background

To further consolidate our idea that phase separation has the ability of regulating biochemical reactions, we chose catechol dioxygenase (xylE, Part:BBa_J33204) from Pseudomonas putida to test the reaction efficiency before and after phase separation. This enzyme can catalyze catechol, a stable colorless substrate, to a bright yellow compound, 2-HMS, with an absorbance maximum around 377 nm when there is oxygen available. (Figure 1)

Figure 1. Diagram of the reaction catalyzed by catechol dioxygenase (xylE).

Design

CDS of xylE was fused to the C terminal of cry2-mCherry and co-transformed with CIB1-G4-GFP-FUS to render the inducible recruitment of fusion enzyme into the phase of FUS, which made the experimental group. (Figure 2) Cry2, GCN(4) and FUS were deleted from two plasmids to make a control group that cannot induce phase separation. Fusion protein CDS were put after the T7/Anderson promoter and lactose operator on the plasmid.

Figure 2. Diagram of two fusion protein expressed in experimental group of xylE system.

If what our hypothesis suggests is right, the experimental and control group would exhibit different enzymatic activity after culture without special light-proof procedure. Because phase could enrich both enzyme and supposedly highly water-soluble substrate, compared to control group, cell culture in experimental group would perform higher catalytic activity.

Result

As we expected, fusion protein enzyme was relatively weaker in activity than the original enzyme, but it still evinces measurable change in characteristic absorbance of reaction product within minutes after the addition of substrate into the cell culture, with cell expressing other enzyme or blank as control groups.

Next, we have verified their ability to form distinct phase in cells under confocal microscope. After being cultured to a proper concentration, BL21 strain cells of our experimental group was induced in expression of our fusion proteins by adding 0.5 mM IPTG, or no IPTG as some kind of control. Using the same parameters as stimulating our phase separation system, we successfully achieved controllable recruitment of fusion xylE enzyme into phase in 0.5 mM group. (Figure 3A) In no IPTG group, we also observed redistribution of fusion enzyme, but in a slightly different way. It showed a tendency to move towards the cell wall, though no significant aggregated phase was seen. (Figure 3B) We proposed that it might due to the leakage of strong T7 promoter with a single lactose operator.

Figure 3A. Show the co-localization and aggregation of GFP and mCherry signal of Exp-0.5mM-xylE group, indicating the recruitment of fusion enzyme into the phase pre-formed by FUS.

Figure 3B. Show the tendency of GFP and mCherry signal to move towards the cell wall in Exp-0mM-xylE group. Indicating a low concentration of FUS cannot form significant phase.

We also checked the distribution of fluorescence signal in control group. Phase did not form no matter how much IPTG was added. (Figure 3C) Furthermore, in an earlier control group experiment with FUS but without cry2, although CIB1-GCN(4)-FUS-GFP could form phase in the cells, fusion enzyme protein remain diffused in the cytosol anyway. (Figure 3D)

Figure 3C. Show diffused GFP and mCherry signal in cell. Indicating no phase would form without FUS and Cry2.

Figure 3D. Show clustered GFP and diffused mCherry signal in cell. Indicating that fusion protein cannot be recruited into phase without Cry2 element.

Next, we would like to measure efficiency of this reaction under different circumstances, to test the effect of phase-separation on catechol degradation. To more explicitly delineate the influence brought by the concentration of enzyme protein to overall productivity, a series of different IPTG induction group (0, 10-6, 10-5, 10-4 and 10-3 mM) were set here to measure the increase of characteristic absorbance of reaction product after the same time by the addition of same amount of substrate (detailed protocol in Experiment), as the quantification of the fusion xylE enzyme activity.

Figure 4. Show the normalized xylE activity among different IPTG and control groups, with two-way ANOVA (**, p<=0.01; ***, p<=0.001)

Our results are plotted here (Figure 4), in which a global positive correlation between IPTG concentration and enzyme activity could be seen irrespective of experiment or control group. When IPTG level is raised above 10-4 mM, a significant escalation of enzyme activity occurs from the control group to the experiment group with phase-separation, which means that phase-separation could boost the catalysis by xylE under some specific condition.

Although we formerly hypothesized that a phase formed of protein may enrich relatively hydrophobic molecules, and thus is able to boost the reaction with a hydrophobic substrate, catechol is a fairly hydrophilic molecule. It might can be explained that the key factors determining the partition coefficient of different molecule in phase here are not only hydrophobicity, and more research can be done to elucidate the chemical characters of phase in cell.

Result Part III Biliverdin reductase (BLVRA) Assay

Background

To acquire more evidence supporting our idea that phase separation has the ability of regulating biochemical reactions, we cloned the biliverdin reductase (BLVRA) gene of homo sapiens from the cDNA library of human liver cells. BLVRA catalyzes the reaction that transforms biliverdin into bilirubin. It is clear that biliverdin has a better solubility in hydrophobic phase, while bilirubin has a better solubility in hydrophilic phase. Therefore, we suppose that if BLVRA is enriched in the hydrophobic phase, the reaction rate might be increased significantly. In addition, since biliverdin has a maximum absorbance at 400 nm and bilirubin has a maximum absorbance at 450 nm, it is easy to measure the reaction rate with a relatively high accuracy.

Significance

The disease caused by biliverdin reductase defect is called hyperbiliverdinemia. It can manifest as green jaundice, which is a green discoloration of the skin, urine, serum, and other bodily fluids, due to increased biliverdin resulting from inefficient conversion to bilirubin (Gafvels et al, 2009). Hyperbiliverdinemia is a rare genetic disease, but it is such a serious disease that most patients suffer a lot, or even die of it. The babies born with biliverdin reductase defect, known as bronze babies, usually show a series of severe disorders such as biliary atresia. Every one of the disorders might be lethal.

Therefore, if we discover a new means to increase biliverdin reductase activity by phase separation in vivo, it might be possible to develop a cure for hyperbiliverdinemia based on our findings.

Experiment Design

In order to verify that BLVRA can show catalytic activity in E. coli cells, we constructed an expression plasmid, pRSF-BLVRA, that has BLVRA gene in it. We transformed this plasmid into BL21 cells and took the cells to do enzyme activity test. Control group cells were transformed with pRSF empty plasmids.

For further experiments, we fused BLVRA to the C terminal of Cry2-mCherry in plasmid petL8, and co-transformed it with the plasmid pRSF-CIB1-G4-GFP-FUS to make it possible for BLVRA to be recruited into phase. Then, as a control, we deleted the components in both plasmid that may cause phase separation, which were cry2 and FUS. Also, to avoid disturbing the enzymatic activity, BLVRA in both experimental group and control group were linked to the C-terminal of mCherry. In summary, the plasmids transformed into experimental group cells were petL8-Cry2-mCherry-BLVRA and pRSF-CIB1-G4-GFP-FUS, and the plasmids for control group were petL8-mCherry-BLVRA and pRSF-CIB1-G4-GFP.

Our expected result was that after cultured in light, the experimental group and the control group would exhibit different enzymatic activity. This is because the hydrophobic phase can gather both the enzyme and the substrate, so that bacteria in experimental group would perform higher catalytic activity compared to control group.

Results

First of all, the activity of BLVRA expressed in E. coli cells was examined by measuring the OD450 of the cell culture, which represented the concentration of the product, bilirubin. As shown in figure 1, the concentration of product in the experimental group is higher than that in the control group, which demonstrates that BLVRA in E. coli cells is active.

Figure 1 OD450 of cell culture, which shows the concentration of the product

For the second step, we tried to control the entry of enzyme into phase by laser under confocal microscope. After cultured to a proper concentration (which was determined by measuring OD600 of the cell culture), E. coli cells in both the experimental group and the control group were induced by adding 0.5 mM IPTG. Utilizing the same parameters as what we used in stimulating our phase separation system, we successfully realized controllable recruitment of BLVRA into phase in experimental group, as figure 2A shows. In the control group, since the cells did not express phase separation elements Cry2 and FUS, the distribution of BLVRA was in a non-canonical way. It spread evenly in the cells, as figure 2B shows. Therefore, we come to the conclusion that BLVRA can be recruited in the phase.

Figure 2 Verification of the controllable redistribution of BLVRA into phases

For the final step, we wanted to measure the reaction rate of both the experimental group and the control group. However, since some unexpected issues happened to our E. coli cells and cell culture, we were not able to finish the experiments and acquire clear results on time. If any further results are achieved in the future, we will present them as a part of the team presentation.

References

[1] Gafvels, M., Holmstrom, P., Somell, A., Sjovall, F., Svensson, J.-O., Stahle, L., Broome, U., Stal, P. A novel mutation in the biliverdin reductase-A gene combined with liver cirrhosis results in hyperbiliverdinaemia (green jaundice). Liver Int. 29: 1116-1124, 2009.

[2] Huffman, C., Chillag, S., Paulman, L., McMahon, C. It's not easy bein' green. Am. J. Med. 122: 820-822, 2009.

[3] Nytofte, N. S., Serrano, M. A., Monte, M. J., Gonzalez-Sanchez, E., Tumer, Z., Ladefoged, K., Briz, O., Marin, J. J. G. A homozygous nonsense mutation (c.214C-A) in the biliverdin reductase alpha gene (BLVRA) results in accumulation of biliverdin during episodes of cholestasis. J. Med. Genet. 48: 219-225, 2011.

[4] Wang, Y. T. et al. Cloning, expression and identification of the biliverdin reductase cDNA of Yanbian yellow cattle. Chin J Vet Sci: 936-940, 2017.

Result Part IV More Possibilities

Except for biochemical reactions, phase separation system can regulate a lot more activities in E.coli. To our observation under microscope, not all phases form on both end of E.coli cell. In fact, many proteins, like Cry2 or FUSLCD, only aggregate at one end of the cell. (Figure 1A 1C) During cell division, the major amount of these proteins would present in only one filial cell, thus result in asymmetric cell division. (Figure 1B) We propose that by recruiting specific material in phase, a uniform E.coli colony could grow into a society with cell differentiation. For example, if proteins targeting specific nucleic acid sequence, like dcas9 (Figure 1D), linked with Cry2 or other oligomerizable protein domains, enrich at one end, specific plasmids would redistribute in the cell. Therefore, filial cells would have different copy number of specific plasmids. As the colony growth, specific plasmid would enrich in some cells while depleted in others.[1][2] Finally, the expression level of reporter gene on that plasmid would differ in different cells in a single colony. Furthermore, if linked with light-responding elements rather than phase separation element, DNA binding proteins could recruit plasmids with a better time-resolution, thus regulate the ratio of cell types in the society more conveniently.

Figure 1. Asymmetric distribution of phase separating proteins. A. GFP signal represents the distribution of FUSLCD in E.coli, which only clustered at one end of E.coli almost in all cells. B. This image shows the distribution of FUS in dividing cell. The bigger phase might be the primary phase in mother cell, while the small one might be the one that came into being in daughter cell. C. MCherry signal represents the distribution of Cry2 in E.coli. It aggregated at one end in about fifty percent of cells. D. Dcas9 aggregate at one end of the dividing cell. The shape of cell is marked by CFP.

In summary, there are many prosperous applications waiting to be developed upon the introduction of this powerful tool in E.coli. To redistribute proteins in E.coli with high time-resolution can achieve regulation of engineered bacteria in a totally different level other than transcriptional and translational regulation. At this level, we could realize our goal in a more swift and more precise manner. What’s more, to manipulate proteins at macro scale could equip cells with new abilities, like differentiation. We hope that PhASE could be a useful tool for future teams in iGEM.

References

[1]Molinari, S., Shis, D., Bhakta, S., Chappell, J., Igoshin, O., & Bennett, M. (2019). A synthetic system for asymmetric cell division in Escherichia coli. Nature Chemical Biology, 15(9), 917-924.

[2]Mushnikov, N., Fomicheva, A., Gomelsky, M., & Bowman, G. (2019). Inducible asymmetric cell division and cell differentiation in a bacterium. Nature Chemical Biology, 15(9), 925-931.