Part:BBa_K5335026
VDAL-6*His
VDAL, an Aspf2-like protein derived from Verticillium dahliae, has been shown to activate PTI responses when expressed extracellularly [1] and can also induce endogenous ETI immune responses when expressed intracellularly. To verify the function of the constructed circuit, a pET28a-based expression system was employed, with the T7 promoter under the control of the lac operon. The circuit includes the engineered VDAL-6*His protein. The design was verified as shown in Figure 1.
The constructed plasmid vector is illustrated in Figure 2.
Experimental Verification
Transformation
<p> After chemical transformation using calcium chloride, the constructed plasmid was introduced into E. coli BL21 (DE3). The transformed cells were plated on LB agar plates supplemented with kanamycin and incubated at 37℃ for 16 hours. Colony PCR was performed on individual colonies to verify the presence of the plasmid.
The results of the colony PCR are presented in Figure 3.
SDS-PAGE
Purification and SDS-PAGE analysis of the target protein
1) The single colony confirmed by sequencing was inoculated into a shake flask and cultured at 37°C for 6 hours until the OD600 reached 0.6.
2) IPTG was then added to a final concentration of 1 mM, and the culture was further incubated at 16°C for 18 hours.
3) Cells were harvested by centrifugation at 8000 rpm, 4°C for 10 minutes, washed with PBS, resuspended, and centrifuged again.
4) Finally, the cell pellet was resuspended in 2 ml of Binding Washing Buffer (Sangon Biotech, Shanghai, China) containing 10 mM imidazole and 80 μl of PMSF (Sangon Biotech, Shanghai, China).
5) The cell suspension was then sonicated for 10 minutes with a 1-second pulse followed by a 2-second pause.
6) The cell lysate was centrifuged at 12000 rpm at 4°C for 15 minutes.
7) The supernatant was transferred to a new Eppendorf tube, and the pellet was resuspended in 1 ml of PBS for storage.
8) The supernatant was mixed with an equal volume of Binding Buffer to prepare the sample.
9) The storage solution was slowly drained, and the Ni column was equilibrated with 5 ml of Washing Buffer.
10) The sample was loaded onto the column in two bed volumes, and the first flow-through was reloaded.
11) The column was washed with two bed volumes of Washing Buffer, and the flow-through was collected until the absorbance at 280 nm reached the baseline.
12) The protein was eluted with two bed volumes of Elution Buffer, collecting 2 ml fractions each time, until the absorbance at 280 nm reached the baseline.
13) The purified protein was obtained. (Specific experimental procedures were followed from HyPur T Ni-NTA 6FF (His-Tag) PrePacked Gravity Column Kit, Sangon Biotech, Shanghai, China)
14) The harvested bacterial cell pellet, the supernatant after cell lysis, the purified protein sample, and the E. coli BL21 (DE3) cell suspension containing the empty pET28a plasmid were mixed with 5X Protein Loading Buffer and heated at 95°C for 10 minutes.
15) Samples were separated by 12% SDS-PAGE and stained with Coomassie Brilliant Blue G250 for 12 hours, followed by destaining.
The obtained results are shown in Figure 4.
The first attempt might have resulted in the target protein being included in inclusion bodies rather than appearing in the purified protein sample due to the induction conditions. To address this, the induction method was modified as follows: cells were cultured at 37°C in shake flasks for 6 hours until the OD600 reached 0.6, then IPTG was added to a final concentration of 0.1 mM, and the culture was continued at 16°C for 12 hours. Samples were prepared following the aforementioned protocol.
The obtained results are shown in Figure 5.
The bands labeled 1, 2, 3, and 4 in the figure represent the elution fractions obtained sequentially using Elution Buffer.
DAB-based ROS assay
Protocol
1) Protein Quantification and Preparation: The purified protein sample containing VDAL-6*His was quantified using the Bradford assay to a concentration of 100 μg/mL.
2) Plant Material Preparation: Three healthy Arabidopsis thaliana Columbia wild-type plants of similar size and growth stage were selected. Leaves of comparable size and shape were excised and washed three times with ddH₂O, then blotted dry. Three leaves were grouped together, and a total of four groups were prepared.
3)Treatment: Each group of leaves was incubated for 36 hours at 28°C in one of the following solutions: ddH₂O, 100 μM salicylic acid (SA) solution, 50 μg/mL VDAL-6*His protein solution, or a mixture of 100 μM SA solution and 50 μg/mL VDAL-6*His protein solution.
4)DAB Staining: After incubation, the leaves were washed three times with ddH₂O and blotted dry. Samples were immersed in DAB staining solution and subjected to negative pressure (-0.1 MPa) for 30 minutes, followed by room temperature incubation in the dark for 12 hours until positive sites appeared dark brown. Samples were then rinsed three times with ddH₂O and blotted dry.
5)Decolorization: Samples were immersed in tissue decolorization solution and incubated at 75°C for 30 minutes until the background color was completely removed.
6) The samples were then rinsed three times with distilled water and blotted dry. Subsequently, they were immersed in a tissue preservation solution for 30 minutes before being imaged. (DAB staining kit and procedure were obtained from Beijing Solarbio Science & Technology Co., Ltd.)
7) All leaves were arranged on a white background and flattened to ensure complete expansion. Images were captured under consistent lighting conditions, as shown in Figure 6.
(Note that the ddH₂O and SA groups in this experiment served as negative and positive controls, respectively, and were identical to those used in the BBa_K5335025 element verification.)
Treated with: A. ddH₂O (control), B. salicylic acid (SA) (100 μM), C. VDAL-6*His protein (50 μg/mL), and D. a combination of SA (100 μM) and VDAL-6*His protein (50 μg/mL).
8) The acquired images were imported into ImageJ software and converted to 8-bit grayscale images. Subsequently, the images were inverted. Given that DAB staining results in the deposition of brown-colored precipitates in regions with reactive oxygen species (ROS), with the intensity of the color directly correlating with ROS levels, the inverted images displayed lighter regions corresponding to higher ROS content.
The inverted images are presented in Figure 7.
Treated with: A. ddH₂O (control), B. salicylic acid (SA) (100 μM), C. VDAL-6*His protein (50 μg/mL), and D. a combination of SA (100 μM) and VDAL-6*His protein (50 μg/mL).
9)Using the grayscale measurement tool in ImageJ, the contours of each leaf were outlined, and the average grayscale value for each leaf was calculated. After data normalization and analysis.
The statistical results are presented in Figure 8.
Analysis of the images revealed that VDAL-6*His significantly induced the production of reactive oxygen species (ROS). Furthermore, the co-incubation of SA and VDAL-6*His resulted in a notable interference in their respective not abilities to induce ROS.
Reference
1.Jiang S, Zheng W, Li Z, Tan J, Wu M, Li X, Hong SB, Deng J, Zhu Z, Zang Y. Enhanced Resistance to Sclerotinia sclerotiorum in Brassica rapa by Activating Host Immunity through Exogenous Verticillium dahliae Aspf2-like Protein (VDAL) Treatment. Int J Mol Sci. 2022 Nov 12;23(22):13958. doi: 10.3390/ijms232213958.
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal SpeI site found at 787
Illegal PstI site found at 691 - 12INCOMPATIBLE WITH RFC[12]Illegal SpeI site found at 787
Illegal PstI site found at 691 - 21COMPATIBLE WITH RFC[21]
- 23INCOMPATIBLE WITH RFC[23]Illegal SpeI site found at 787
Illegal PstI site found at 691 - 25INCOMPATIBLE WITH RFC[25]Illegal SpeI site found at 787
Illegal PstI site found at 691 - 1000COMPATIBLE WITH RFC[1000]
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