Difference between revisions of "Part:BBa K5335028"

Latest revision as of 21:26, 1 October 2024

T7promoter+Spycatcher-VDAL-CPPs+Spycatcher-CPPs+Spytag-His+T7terminator

Our team constructed this composite part using the pET28A vector under the control of the T7 promoter regulated by the lac operon. The circuit includes the engineered SpyCatcher-VDAL-CPPs protein^[1], SpyTag-6His protein, and SpyCatcher-CPPs protein^[2]. Other teams can utilize this composite part to express the plant immune activator VDAL with a 6His tag for cell penetration, while simultaneously co-expressing other proteins fused with SpyTag to obtain target proteins with the R9 CPPs for plant cell entry^[3].

The design was verified as shown in Figure 1.

Experimental Verification

Transformation

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

SDS-PAGE detection of bacterial total 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 1 mL of PBS to obtain a cell suspension.

    5) The cell suspension was mixed with 5X Protein Loading Buffer and heated at 95°C for 10 minutes.E. coli BL21 (DE3) harboring the empty pET28a plasmid was processed in the same manner as a control.

    6) Samples were analyzed by 12% SDS-PAGE, and a protein band corresponding to the expected size of SpyCatcher-VDAL-CPPs + SpyTag-6*His protein (53.7 kDa) was observed.

The results are presented in Figure 4.

    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 relatively large size of the target protein, which was tagged with His using the SpyCatcher-SpyTag (SpyC/SpyT) system, resulted in weaker binding affinity to the Ni-NTA column. To ensure protein purification, a Binding Wash Buffer with a reduced imidazole concentration was used. However, this led to increased contamination by non-specific proteins, although three distinct bands could still be observed near the expected molecular weight.

Western Blot

80μL of each purified sample was mixed with 20 μL of 5x protein loading buffer and incubated at 98°C for 15 minutes. Samples were then loaded onto an SDS-PAGE gel and electrophoresed at 80V for 2 hours. Proteins were transferred onto a nitrocellulose membrane overnight at 4°C. The membrane was blocked and then incubated with a primary antibody (mouse anti-His-tag monoclonal antibody). After washing, the membrane was incubated with a secondary antibody (goat anti-mouse IgG). Following washing, the membrane was developed using a chemiluminescent substrate, and the results were visualized by exposure to film.

The obtained results are shown in Figure 6.

The relatively high level of contaminating proteins in the purified sample, likely due to the His-tag, suggests a potential limitation of the system. However, the presence of three distinct bands near the expected molecular weight confirms the functionality of the SpyCatcher-SpyTag (SpyC/SpyT) system. The lower intensity of the target protein band may be attributed to the relatively large size of the SpyCatcher-VDAL-CPPs fusion protein and its lower expression level.

Reference

     1.Ma A, Zhang D, Wang G, Wang K, Li Z, Gao Y, Li H, Bian C, Cheng J, Han Y, Yang S, Gong Z, Qi J. Verticillium dahliae effector VDAL protects MYB6 from degradation by interacting with PUB25 and PUB26 E3 ligases to enhance Verticillium wilt resistance. Plant Cell. 2021 Dec 3;33(12):3675-3699. doi: 10.1093/plcell/koab221.

     2.Gilbert C, Howarth M, Harwood CR, Ellis T. Extracellular Self-Assembly of Functional and Tunable Protein Conjugates from Bacillus subtilis. ACS Synth Biol. 2017 Jun 16;6(6):957-967. doi: 10.1021/acssynbio.6b00292. Epub 2017 Mar 7.

     3.Soliman A, Laurie J, Bilichak A, Ziemienowicz A. Applications of CPPs in Genome Editing of Plants. Methods Mol Biol. 2022;2383:595-616. doi: 10.1007/978-1-0716-1752-6_39.

Sequence and Features