iGEM Tsinghua

Parts(Cry2) Improvement

Background

In our system, phases are formed by CIB1-GCN(4)-FUS-GFP, while the recruitment of downstream proteins fused with mCherry-CRY2 into the phase is presented via the interaction between mCherry-CRY2 and CIB1-GCN(4)-FUS-GFP when stimulated. Therefore, without stimulation of 488nm laser, we anticipate that mCherry-CRY2 is smear in E.coli. Once stimulated, with strong interaction of CRY2-CIB1, mCherry-CRY2 is pulled into the phase. However, CRY2 homo-oligomerization is also stimulated by light 488nm, observed in our control experiment, which might alter the function of our system.

Design

To solve the problem, we introduce mutations on C terminal of CRY2, charges on which is probably reasonable for its oligomerization. According to theoretical postulation, positive charge on C terminal facilitates oligomerization while negative charge inhibits. Upon knowing that, the arginine on 489 is mutated to glutamate (R489E) and aspartate (R489D), or amino acids from 489 to 498 are completely deleted(Δ489-498), making the mutant C terminal more negative than wild type. Mutants and its principle is shown in Table1. CRY2wt and its mutants are all tagged with mCherry, in order to show the distribution of CRY2 in cells. Meanwhile, charge of N terminal is critical for CRY2-CIB1 interaction. We also change the N terminal charge of CRY2 to weak its interaction with CIB1 and increase light threshold of the system.

Mutants	Principle
CRY2(R489E)	Change arginine on 489 to glutamate, making charge of C terminal negative.
CRY2(R489D)	Change arginine on 489 to aspartate, making charge of C terminal negative.
CRY2(Δ489-498)	Delete amino acids from 489-498, making charge of C terminal negative.

Result

After observing under confocal, we found the extent of homo-oligomerization of CRY2 (R489E) is the lowest. Figure 1 shows confocal images of CRY2wt and CRY2(R489E).

Figure 1A Confocal image of CRY2wt (Zoom 1), most E.coli form phases. Figure 1B Confocal image of CRY2 (R489E)(Zoom 1), mCherry in E.coli are smear, showing low extent of oligomerization. All samples are induced with 0.05mM IPTG for 2 h at 16 degrees.

Figure 2 shows homo-oligomerization details of CRY2wt and CRY2(R489E).

Figure 2A Confocal image of CRY2wt(Zoom 3), CRY2wt forms 2 phases on both ends of E.coli. Figure 2B Confocal image of CRY2(R489E)(Zoom 3), CRY2(R489E) is almost smear in E.coli. Although in some cells phase is formed, the phase is much more smaller and darker than the phase formed by CRY2wt. All samples are induced with 0.05mM IPTG for 2 h at 16℃.

We carried out statistical analysis after acquiring merge images (mCherry channel and TD). Three kinds of objects are count in our observation: bacteria with phase separation, bacteria without phase separation, bacteria whose state was unclear (probably because of low expression level for unknown reason or too young to accumulate enough proteins). The examples of captured pictures were given in Figure 3. Then, the total number of bacteria was added up. Finally, we used the ratio of group “phase” to sum to indicate the ability of CRY2 oligomerization under the same condition. The distributions of the data are showed in Figure 4.

Figure 3A example of E.coli with phase separation. Figure 3B example of E.coli without phase separation. Figure 3C example of unclear situation E.coli.

Figure 4 Ratio of E.coli with separation to total number of E.coli

We utilized statistical analysis to assess if the ratio of “phase” showed significant difference between CRY2wt and CRY2(R489E). Student's T test showed that there is a significant difference, as the p-value equals , much less than 0.05.

Significance

In our project, the core objection is to construct a Phase-separation controlled Downstream enzymatic reaction system, so maximizing the impact caused by the different manipulation is necessary to ensure the high efficiency of the system. The smear distribution inhibits the chance of the contact between enzyme and substrate, compared to aggregated distribution in the phase. As a result, the higher possibility of that smear distribution in the bacteria appear, the lower rate will the overall enzymatic reaction be in the macrograph.

As interaction between mCherry-CRY2 and CIB1-GCN(4)-FUS-GFP is the key of phase formation leading to downstream proteins recruitment, excluding any other influence factors that impedes this hetero-oligomerization should be inhibited. Since CRY2 homo-oligomerization decreases the chance for CRY2 to interact with CIB1 presented in the phase, our improvement is valid.

Further improvement

To promote the efficiency of the system, excluding any other influence factors or regulating hetero-oligomerization are both available ways. One of the most instinctive attempts is to regulate the hetero-oligomerization by mutating the interaction site. The CIB1-binding site of CRY2 locates on the N-terminal of the peptide, ordered as MKMDKKTIV…… Obviously, several acid or basic residues consecutively presents, which contribute to the interaction between CIB1 an CRY2. Further mutation can be exerted to these sites.

Future Application

As widely used parts in light-controlled system, CRY2 and CIB1 are in the need of improvement. Our improvement eliminates an impede against the smooth manipulation to light-control systems, thus is potentially valuable in various scientific researches and bioengineering. For instance, it can be used to research on pathways, protein functions, or even testing new Phase Separation Components.