Results | Shanghai-HS

Overview

To alleviate the pain experienced by diabetes patients, we have devised a novel CRY2/CIB1 system, which operates via blue-light dependent optogenetics. This innovation aims to enhance the efficiency of light-controlled "off" switches.

In our study, we combined UVR8, a UVB receptor originally identified in plants, with the recently discovered blue-light inhibitors of cryptochromes BIC2 to create the UVR8N-BIC2 fusion protein. Through a series of experiments assessing β-galactosidase activity within a yeast two-hybrid system, we demonstrated the capability to expedite the deactivation process of the CRY2/CIB1 system in the presence of UVB light.

Result

1. Construction of pBridge-BD-CRY2(UVR8-BIC2) plasmid using double digestion

In order to combine UVR8 with BIC2, we construct a fused protein UVR8N-BIC2 (Fig.1). We firstly designed primers and amplified TRP promoter fragment from pBridge vector, UVR8N397 fragment from the plasmid containing UVR8 CDS sequence, and BIC2 fragment from Arabidopsis thaliana genome cDNA by PCR assay. The results showed that we got the 3 DNA fragments successfully (Fig.2A). We also constructed linearized pBridge-BD-CRY2 vector by double enzymatic digestion with NdeI and BstbI (Fig.2A). Then we obtained the DNA fragments of TRP promoter fragment, UVR8N397 fragment and BIC2 fragment and the linearized pBridge-BD-CRY2 vector employing DNA extraction kit. We performed homologous recombination to link the 3 DNA fragments to the linear pBridge-BD-CRY2 vector. Next we transformed the product into E.coli and as shown in figure 2B, we obtained the E.coli with the plasmid. To verify whether the recombinant plasmid presented in the E.col, the colony PCR was performed and the results of the plate suggested the failure of homologous recombination because of the low quality of the linearized pBridge-BD-CRY2 vector (Fig.2B-2C).

Figure 1. pBridge-BD-CRY2(UVR8-BIC2) plasmid map.

Figure 2. Construction and identification of pBridge-BD-CRY2(UVR8-BIC2) plasmid using double digestion.

(A) The identification of DNA fragments by PCR and the linearized vector by NdeI-BstbI double digestion.

(B) Result of transformation with the linearized vector and the three DNA fragments.

(C)Colony PCR results of the plates in (B).

2. Construction of pBridge-BD-CRY2(UVR8-BIC2) plasmid using single digestion

To get the high qualitied linearized vector, we transformed pBridge-BD-CRY2 plasmid into E.coli and cultured the E.coli to obtain more pBridge-BD-CRY2 plasmid. And we identified the quality of the plasmid by DNA agarose gel electrophoresis (Fig.3A). As shown in figure 3A, the plasmid was correct and has high quality. Next, we adapted NdeI single enzyme digestion to linearize the pBridge-BD-CRY2 plasmid and amplified the three DNA fragments (Fig.3B-C). We got the 3 DNA fragments and linear plasmid successfully. Then we transformed the homologous recombination product of the 3 DNA fragments and linear plasmid into E.coli and cultured overnight (Fig 3D). The results of colony PCR showed that the lane 2, 3, 4 of plate 1 and the lane 3 of plate 2 were shown a positive band which is about 1686 bp, suggesting the colony of E.coli contained the recombinant plasmid pBridge-BD-CRY2(UVR8-BIC2) (Fig.3E).

Figure 3. Construction and identification of pBridge-BD-CRY2(UVR8-BIC2) plasmid using single digestion.

(A) The identification of re-extracted pBridge-BD-CRY2 plasmids.

(B) The identification of the linearized pBridge-BD-CRY2 vectors by NdeI single digestion.

(D) Result of transformation with the linearized vector (NdeI digested) and the three DNA fragments in (C).

(E)Colony PCR results of the new plates in (D).

We picked our single colony and sent them to company for DNA sequencing, the final results indicated that there were not genetic mutations in our genes and the recombinant plasmid in lane 4 of plate 1 (Fig.3E) was successfully constructed validated by sanger sequencing (Fig.4).

Figure 4. The sequence data of the recombinant plasmid pBridge-BD-CRY2(UVR8-BIC2).

3. The confirmation of CRY2/CIB1 system in yeast

In order to confirm whether the blue-light dependent CRY2/CIB2 system works, we performed yeast transformation of AD-CIB1 and BD-CRY2. The results showed successful transformation of yeast after 48h culture (Fig.5)

Figure 5. Confirmation of blue-light dependent CRY2/CIB1 system.

Then we did a dot-plate experiment, the results showed that CRY2/CIB1 system could work.

Figure 6. The results of growth of yeast containing the plasmids.

4. Improvement of switch “off” in UVB regulated pBridge-BD-CRY2(UVR8-BIC2) system

We then examined whether UVB exposure could improve the switch “off” in pBridge-BD-CRY2(UVR8-BIC2) system. AD-CIB1 and BD-CRY2(UVR8-BIC2) were transformed into yeast together and cultured on -Trp/-His/-Leu/-Ade solid plate for 72 h. The positive colony was picked to enlarge cultivation in QDO liquid medium under conditions of blue light to allow the β-galactosidase expression. In order to test whether the switch “off” speed of our system was improved under UVB irradiation, we divided the medium with galactosidase expression under blue light condition into four parts to shut down, respectively under dark condition and under dark with UV light condition and under blue light condition and under blue with UV light condition. As shown in Table1 & figure 7 , the activity of β-Gal was low so we believed that pBridge-BD-CRY2(UVR8-BIC2) system did not work under dark and dark with UV condition. Under blue light condition, the activity of β-Gal was increased with time. While under blue with UV light condition, the increasing rate of β-GAL activity in the presence of UV was slower than that in the absence of UV, indicating that The proteins UVB and BIC2 play the role of self-switch off and improve the switch “off” in pBridge-BD-CRY2(UVR8-BIC2) system.

Table1. The data of β-galactosidase (β-GAL) activity assay

Figure 7.. Bar chart and line chart of β-galactosidase (β-GAL) activity assay