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<b>The reagents used by our team to prepare protoplasts are as follows:</b>
 
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Revision as of 13:26, 30 September 2024


AmCyan-CPPs（R9-Tag）

AmCyan1 is a basic (constitutively fluorescent) cyan fluorescent protein, derived from Anemonia majano，a commonly used fluorescent protein for tracing (Exλ=453nm, Emλ=486nm).R9-Tag is a polyarginine cell-penetrating peptide commonly used in plant cells[1].The team combined the two to implement functional detection of R9-Tag.This fusion protein can also be used as a dual-function tag protein for both fluorescent labeling and cell penetration (if used for this purpose, a flexible linker should be used to connect the components).

Usage and Biology

To validate the cell-penetrating function of the R9-Tag, commonly used in plant cells, we constructed a fusion protein of AmCyan and R9-Tag using the pET28A vector. The expression of these components is driven by the T7 promoter under the control of the lac operon.

The design was verified as shown in Figure 1. 无标题文档


Figure 1. Experimental circuit design diagram
The constructed plasmid vector is illustrated in Figure 2. 无标题文档

Figure 2. Plasmid Vector

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. 无标题文档


Figure 3. Agarose gel electrophoresis image of colony PCR products (target band at 752 bp)
After sequencing the selected single colonies and confirming the results, the researchers proceeded with subsequent experiments.

Crude extraction of AmCyan-CPPs 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 Bacterial lysis 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. <p>    8) Select two Arabidopsis thaliana (Columbia ecotype) plants at an appropriate growth stage. Carefully wash the roots with physiological saline solution and gently remove any debris using tweezers. Take care not to damage the roots.

    9) Cut the root system at the junction of the stem and root for later use.

The supernatant and treated root system after cell lysis and centrifugation are shown in Figure 4. 无标题文档


Figure 4.Supernatant of bacterial lysate and treated root tissue.

    10) Root tissues were immersed in protein sample and incubated at room temperature with shaking for 16 hours.

    11) After being washed repeatedly more than 10 times with physiological saline under shaking conditions, no fluorescence was observed in the slide prepared with the final wash solution under a fluorescence microscope.

    12) A portion of the root tissue was prepared as a water mount and observed under the Leica TCS SP8 X confocal microscope (Exλ=453nm, Emλ=484-494nm).

The obtained results are shown in Figure 5. 无标题文档

Figure 5. Confocal microscopy images of root tissues.
(A) AmCyan fluorescence channel showing. (B) No-stain control. (C) Merged image of AmCyan fluorescence and brightfield. (D) High-magnification view of root hair cells in the merged image, showing.

In the above images, we observed relatively dense fluorescent signals throughout most plant tissues, including root hair cells. To rule out the possibility that the observed fluorescence was due to the adsorption of AmCyan-CPPs proteins to the plant cell wall, the remaining root tissues after washing were used to prepare protoplasts.

The reagents used by our team to prepare protoplasts are as follows:
(1)Enzyme Digestion Buffer:
100 mM KCl
20 mM MgCl₂
20 mM CaCl₂
0.1% (w/v) Bovine Serum Albumin (BSA)
80 mM 2-(N-morpholino)ethanesulfonic acid (MES)
0.6 M Mannitol
pH adjusted to 5.5 with 0.1 M Tris-HCl
(2)Washing/Protoplast Buffer:
100 mM KCl
20 mM MgCl₂
0.1% (w/v) BSA
0.4 M Mannitol
(3)Enzyme Mixture:
1.5% Cellulase R-10 (YAKULT HONSHA CO., LTD)
0.1% Pectinase (From Aspergillus niger, Sigma-Aldrich (Shanghai) Trading Co.Ltd)

After washing, the root tissues were cut into small segments and incubated in an enzyme digestion buffer containing cellulase and pectinase at 28°C for 2 hours. The digestion process was conducted in the dark with gentle shaking to facilitate protoplast release. The digested tissue was then filtered through a nylon mesh to remove undigested tissue debris. The protoplasts were washed twice with washing buffer and centrifuged at 100 g for 2 minutes to remove cell debris. Finally, the protoplasts were resuspended in protoplast buffer. The prepared protoplasts were then observed using a Leica TCS SP8 X confocal microscope.

The obtained results are shown in Figure 6. 无标题文档

Figure 6. Confocal fluorescence microscopy images of protoplasts prepared from Arabidopsis roots.
(A) AmCyan fluorescence channel showing. (B) No-stain control. (C) Merged image of AmCyan fluorescence and brightfield.

Based on the images, we can clearly observe a significant amount of aggregated fluorescent signals in the protoplasts, further confirming the efficient membrane penetration of R9 cell-penetrating peptide into plant cells.

Reference

     1.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.

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Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 561
  • 1000
    COMPATIBLE WITH RFC[1000]